Patent Application
20150160222
The present invention generally relates to compositions, methods and kits for the prognosis and treatment of cancer and, in particular, triple negative breast cancer.
Breast cancers are typically classified into several different subtypes: luminal A (ER positive and histologic low grade), luminal B (ER positive and histologic high grade), HER2 overexpressing, basal-like (2 types—BL1 and BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and normal breast-like tumors.
Triple Negative Breast Cancer (TNBC) is a subtype of basal-like breast cancers characterized as estrogen receptor (ER) negative, progesterone receptor (PR) negative and human epidermal growth factor receptor 2 (HER2) negative based on immunohistochemistry (IHC) phenotype. TNBC is characterized by a unique molecular profile, aggressive nature, distinct metastatic patterns and lack of targeted therapies. It is estimated that approximately 170,000 cases of breast cancer worldwide are TNBC, which accounts for ˜10-20% of invasive breast cancers.
Clinical prognosticators for breast cancer include estrogen receptor (ER) status, progesterone receptor (PR) status, HER2 (human EGF receptor 2) status, the Nottingham Prognostic Index (NPI), the Ki67 Index, tumor grade, and clinical stage. In addition to ER, PR, and HER2 receptor status, clinicians use tumor grade and clinical stage to evaluate prognosis, albeit with very limited risk predictive accuracy.
Amplified centrosomes are widely recognized as a hallmark of cancer and, in particular, 80% of human breast tumors harbor supernumerary centrosomes. The presence of more than two centrosomes within a cell can pose a grave conundrum as it may lead to the assembly of a multipolar mitotic spindle and the production of nonviable progeny cells due to lethal levels of chromosomal loss (i.e., death-inducing, high-grade aneuploidy). However, cancer cells harboring extra centrosomes circumvent these catastrophic consequences and survive. This is achieved by centrosome clustering, whereby the excess centrosomes are artfully corralled into two polar foci to enable formation of a pseudo-bipolar mitotic spindle. During a preceding, transient, multipolar state, merotelic kinetochore-microtubule attachments occur, thus engendering low-grade, whole-chromosome missegregation that could be tumor-promoting.
HSET/KifC1, a minus end-directed motor protein that promotes microtubule cross-linking, sliding, bundling and spindle pole focusing, has been recently identified as an essential mediator of supernumerary centrosome clustering in cancer cells. HSET has also been shown to be indispensable for the clustering of acentrosomal microtubule organizing centers (MTOCs) whose production tends to be hyperactivated in cancer cells. By contrast, HSET function appears to be non-essential in healthy somatic cells due to the presence of two centrosomes that shoulder the responsibility of bipolar spindle assembly.
HSET's localization changes dynamically during cell cycle progression; HSET is sequestered in the nucleus in interphase, presumably to avoid untimely microtubule cross-linking. Upon nuclear envelope breakdown at the onset of mitosis, HSET is released into the cytoplasm to resume its activities in bipolar spindle biogenesis. During mitosis, HSET is localized both on the spindle poles and along the spindle length. With mitotic spindle breakdown in telophase, HSET is localized on the minus-end of microtubules near the spindle poles before being shuttled back into the nucleus. HSET transport inside the nucleus is regulated by Ran GTPase via association of the bipartite Nuclear Localization Signal of HSET with nuclear import receptors importin α/β.
Recent studies have focused on the association between HSET and malignancy. HSET is highly overexpressed in brain metastases, and its expression level in lung cancer is associated with increased risk of metastatic dissemination to the brain. Primary breast tumors also overexpress HSET as compared to matched normal breast tissue. Development of docetaxel resistance in breast cancer may be partly mediated by HSET. Its expression is upregulated in docetaxel-resistant breast tumors, and HSET-overexpressing MDA-MB-231 and MDA-MB-468 breast cancer cells (which are TN) exhibit enhanced survival compared to vector controls. In addition, MDA-MB-231 breast cancer cells rely on HSET for efficient clustering of supernumerary centrosomes, a process that not only suppresses potentially fatal spindle multipolarity but also facilitates low-grade chromosome missegregation during cell division. In fact, cells with supernumerary centrosomes rely on HSET-dependent centrosome clustering for their viability. HSET is required for centrosomal and acentrosomal spindle pole focusing in BT-549 breast cancer cells. Due to its intriguing association with malignancy, HSET presents a potential chemotherapeutic target for breast cancer patients, particularly those with triple negative breast cancer (TNBC).
Current treatment guidelines for breast cancer patients in the US (e.g., those of the NIH and NCCN) are based on clinical factors (e.g., age, menopausal status), tumor grade and stage, and the expression of prognostic and predictive markers (e.g., ER, PR, HER2). However, patients with similar clinicopathological features and a similar status with regard to conventional biomarkers may still respond differently to the same treatment, suggesting a need for better risk stratification schemes. To improve personalization of treatment regimens, more molecular biomarkers may be employed. While certain gene expression-based tests (specifically, OncotypeDx and Mammaprint) appear clinically valid for patients with ER+ breast cancer, their clinical utility remains controversial since modifying treatment based on their results may not improve outcomes. Furthermore, genomic tests continue to be expensive and technically challenging. Consequently, the need persists for protein biomarkers, which can be assessed via relatively inexpensive, facile immunohistochemical assays. A novel panel, Mammostrat, successfully stratifies patients who take tamoxifen (because their tumors are ER+) by assessing five proteins, yet there remains a need to stratify ER-negative tumors, including TN breast cancers, which are confoundingly still characterized not by what they are but rather by what they are not. Further, clinical trials using new targeted therapies for triple negative breast cancer have achieved only limited success, perhaps due to the high heterogeneity of TN lesions and the necessity for better molecular stratification of this tumor class.
In light of the foregoing limitations there is a need for new biomarkers for triple negative breast cancer, particularly those of prognostic value, as well as new treatments for patients with triple negative breast cancer.
The present application is based, in part, on work with the protein HSET/KifC1, and features methods of assessing the prognosis or better predicting the outcome for a patient diagnosed with breast cancer. One aspect of the present application relates to a method of assessing the prognosis of a patient diagnosed with triple negative breast cancer, the method comprises the steps of performing an assay on a biological sample comprising breast cancer cells from the patient to determine whether the breast cancer cells express an elevated level of nuclear HSET; and providing an assessment of the prognosis of the patient based on the result of the assay, wherein an elevated level of nuclear HSET in the breast cancer cells indicates a poorer prognosis.
The method of determining whether the cells express an elevated level of nuclear HSET may be carried out by immunohistochemical analysis of a breast cancer sample or an analysis of a nuclear extract from the sample. In one embodiment, the immunohistochemical analysis involves exposing the sample to a monoclonal or polyclonal anti-HSET antibody under conditions sufficient to allow the antibody to specifically bind to HSET.
In another embodiment, the method further includes the step of determining whether the cells in the sample express elevated level(s) of one or more products that are upregulated with HSET, such as Ki67, survivin, phospho-survivin, HIF-1-alpha, and/or aurora kinase B, p-Bcl2, Mad1 or combinations thereof. Alternatively, or in addition, the method may include the step of determining whether the cells in the sample exhibit elevated levels of phosphorylated histone-H3, enhanced Cdk1 activity or both.
In certain embodiments, the method includes the step of identifying the patient as a person of African descent. In some instances, this can be carried out by determining the patient's geographic origin(s) by ancestry analysis of the patient's genomic DNA.
In a further aspect, the method includes administering an inhibitor of HSET to a patient found to express an elevated level of nuclear HSET. In certain embodiments, the inhibitor of HSET is a small molecule drug. The inhibitor of HSET may target the motor domain of HSET and/or may specifically bind to the HSET/microtubule binary complex and inhibit HSET microtubule-stimulated or microtubule-independent ATPase activity. In some embodiments, the inhibitor of HSET is a centrosome declustering agent selected from the group consisting of AZ82, PJ-34, griseofulvin, noscapine, 9-bromonoscapine, reduced bromonoscapine, N-(3-bromobenzyl) noscapine, aminonoscapine and CW069. In other embodiments, the inhibitor of HSET is an siRNA or an expression vector carrying an shRNA.
In some embodiments, the patient may be administered an HSET inhibitor in combination with an inhibitor of a product that is upregulated with HSET, such as Ki67, survivin, phospho-survivin, HIF1α, aurora kinase B, Mad1 and/or p-Bcl2.
In other embodiments, the patient may be administered an HSET inhibitor in combination with a PARP inhibitor, an inhibitor of the Ras/MAPK pathway, an inhibitor of the PI3K/AKT/mTOR pathway, an inhibitor of FoxM1 or Plk1 or Prc1, or a combination thereof.
In a further aspect, a kit for determining elevated expression of HSET includes an HSET binding agent along with one or more secondary binding agents specifically binding to one or more gene product(s) upregulated with HSET, such as Ki67, survivin, phospho-survivin, HIF-1-alpha, aurora kinase B, Mad1, p-Bcl2, FoxM1, Plk1 and Prc1. The kit may further include one or more reagents for staining of nuclei, and/or one or more reagents for preparation of a nuclear fraction or extract.
The following detailed description is presented to enable any person skilled in the art to make and use the invention. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that these specific details are not required to practice the invention. Descriptions of specific applications are provided only as representative examples. The present invention is not intended to be limited to the embodiments shown, but is to be accorded the broadest possible scope consistent with the principles and features disclosed herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “small molecule drug” includes a plurality of such small molecule drugs, reference to “the small molecule drug” is a reference to one or more small molecule drugs, including equivalents thereof known to those skilled in the art, and so forth.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein, the term “triple negative breast cancer” (TNBC) refers to breast cancers in which the tumor cells score negative (i.e., using conventional histopathology methods) for estrogen receptor (ER) and progesterone receptor (PR), both of which are nuclear receptors (i.e., they are predominantly located at cell nuclei), and are not amplified for epidermal growth factor receptor type 2 (HER2 or ErbB2), a receptor normally located on the cell surface. The tumor cells should be considered negative for expression of ER and PR if less than 5% of the tumor cell nuclei are stained for ER and PR expression using standard immunohistochemical techniques. Tumor cells are considered highly amplified for HER2 if, when tested with a HercepTest™Kit (Code K5204, Dako North America, Inc., Carpinteria, Calif.), a semi-quantitative immunohistochemical assay using a polyclonal anti-HER2 primary antibody, they yield a test result score of 3+, or, they test HER2 positive by fluorescence in-situ hybridization (FISH). As used herein, tumor cells are considered negative for HER2 overexpression if they yield a test result score of 0 or 1+, or 2+, or if they are HER2 FISH negative.
The term “patient” includes a human or other mammalian animal that receives either prophylactic or therapeutic treatment.
The term “gene product,” refers to the transcription product of a gene, such as mRNA, and the translation product of a gene, such as protein.
The term “therapeutic agent” includes any substance, molecule, element, compound, entity, or a combination thereof having a therapeutic effect in a triple negative breast cancer patient. It includes, but is not limited to, e.g., proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and the like. A therapeutic agent can be a natural product, a synthetic compound, a chemical compound or a combination of two or more substances.
The term “inhibitor of HSET” means any agent or compound that reduces, or decreases, or lessens the expression or activity of HSET kinesin, wherein the term “expression” should be understood to mean expression of HSET mRNA or expression of HSET protein in a cell and wherein the term “activity” should be understood to mean the enzymatic activity or associated biological properties of HSET, including, but not limited to, ATPase activity and microtubule binding activity.
The term “an effective amount” refers to an amount of a therapeutic agent sufficient to effect treatment in a patient with triple negative breast cancer. In this context, “treating” should be understood to mean encompass treatment resulting in a decrease in tumor size; a decrease in rate of tumor growth; stasis of tumor size; inhibition of tumor metastases formation; a decrease in the number of metastases; improved progression-free survival (PFS) (e.g., calculated as the number of days from diagnosis to the first local recurrence or metastasis if one occurred); improved overall survival (OS) (e.g., calculated based on the number of days from diagnosis to death or last follow-up if death was not recorded); a decrease in invasiveness of the cancer; a decrease in the rate of progression of the tumor from one stage to the next; inhibition of tumor growth in a triple negative patient; regression of established tumors; decrease in the angiogenesis induced by the cancer; inhibition of growth and proliferation of cancer cells; and combinations thereof.
One aspect of the present application relates to a method of assessing the prognosis of a patient diagnosed with cancer, the method comprises the steps of (a) performing an assay on a biological sample comprising cancer cells from the patient to determine whether the cancer cells express an elevated level of nuclear HSET; and providing an assessment of the prognosis of the patient based on the result of step (a), wherein an elevated level of nuclear HSET in the cancer cells indicates a poorer prognosis. Specifically, high levels of nuclear HSET expression indicate a poor prognosis and poor overall survival, particularly without appropriate and aggressive treatment. In some embodiments, the cancer is breast cancer. In other embodiments, the cancer is triple negative breast cancer. In other embodiments, the cancer is ovarian cancer. In yet other embodiments, the cancer is colon cancer, head and neck cancer, bladder cancer and glioma. In other embodiments, the cancer is vaginal cancer, cervical cancer, uterine cancer, prostate cancer, anal cancer, stomach cancer, pancreatic cancer, insulinoma, adenocarcinoma, adenosquamous carcinoma, neuroendocrine tumor, lung cancer, esophageal cancer, oral cancer, brain cancer, medulloblastoma, neuroectodermal tumor, pituitary cancer, or bone cancer.
Although the patient can undergo tests to determine the stage or grade of their cancer, the present methods can provide a prognosis, allow for a more accurate prediction of outcome, and inform the treatment regime in the absence of staging or grading. The methods can be repeated at intervals throughout a course of treatment (e.g., at the beginning and end of a treatment regime or about every 4-6 months) as an indicator of the patient's responsiveness to a treatment. Thus, the methods are also useful in modifying a prognosis or updating an expected outcome over time.
Patients amenable to the prognostic and therapeutic methods described herein are patients who have been diagnosed as having breast cancer, which is determined to be triple negative. In one embodiment, the method includes identifying the patient as a person of African descent, such as an African American. As further demonstrated in the Examples below, nuclear HSET expression was significantly associated with the proliferation marker Ki67; clinicopathological factors (e.g., tumor grade, tumor stage, and tumor size); with the Nottingham prognostic index (NPI); and with triple negative status. Its expression was also highly associated with race, with African American women being 1.6 times as likely to present with nuclear localization compared to European American women, after adjusting for triple negative status. In multivariate analysis, increased nuclear HSET expression was associated with worse overall, progression-free, and metastasis-free survival (HR=1.37, 1.30, and 1.34, respectively, with p<0.05 for all). Within the African American subset, increased expression of nuclear HSET was associated with even worse overall survival (HR=1.56, p=0.006), progression-free survival (HR=1.44, p=0.012), and metastasis-free survival (HR=1.44, p=0.015) in multivariate analysis. Intriguingly, survival outcomes were significantly associated with nuclear but not cytoplasmic HSET.
A sample from a triple negative breast cancer patient can be obtained from breast cancer cells within the patient (e.g., a tumor) or a fluid sample therefrom. The cells can be obtained by a variety of methods. For example, the sample can be obtained by any procedure in which tumor cells are dislodged from the tumor (e.g., the tumor cells may be obtained from a tumor biopsy removed during a mastectomy, from an aspirate of the tumor, from a lavage or other procedure in which tumor cells are dissociated from the tumor, or from a portion of the tumor that has been surgically removed). In the event breast cancer cells break free from the tumor and circulate, they can be detected in a fluid sample from the patient (e.g., blood, serum, or plasma).
Most samples will utilize at least a dozen cells, and likely at least a few hundred cells (e.g., about 200-500 cells) or more. Once obtained, the sample may be treated according to the requirements of the impending test. For example, tissue to be analyzed by immunohistochemistry can be fixed and embedded for sectioning. Alternatively, whole cell extracts, nuclear extracts or fractions thereof can be processed from the tissues or cells for expression analysis by conventional techniques.
The step of determining whether a given patient's cells express an elevated level of nuclear HSET can be carried out by an immunohistochemical analysis of the sample or an analysis of a nuclear fraction or extract from the sample. For the immunohistochemical analysis, the sample can be directly exposed to a binding agent (e.g., an antibody such as a rabbit polyclonal anti-HSET antibody for a time and under conditions sufficient to allow the binding agent to specifically bind nuclear HSET. Alternatively, a nuclear extract of the sample may be prepared and analyzed for binding of nuclear HSET to the binding agent.
HSET binding agents and those for binding other co-regulated proteins can be prepared using methods known in the art. For example, an intact protein (i.e., full length HSET or a co-regulated protein) or an antigenic fragment thereof can be injected into a laboratory animal (such as a rodent or rabbit), from which antibody-containing blood is later collected. The antibodies generated can be further developed to generate, for example, monoclonal, chimeric, single chain and humanized antibodies, as well as biologically active fragments thereof (e.g., an Fab fragment) may prepared from any suitable immunoglobulin class (e.g., an IgG) according to established methodologies known in the art.
HSET binding antibodies useful in the present methods may be directed to any suitable epitope. For example, HSET binding antibodies may target the N-terminus of HSET (e.g., an epitope constituting residues 1-304; residues 1-152; or residues 151-218) or they may target the C-terminal region (e.g., residues 625-673;
In addition to analyzing HSET expression, the present methods can include a step of determining whether the cells express other products (e.g., proteins or RNAs) that are upregulated with HSET. Exemplary products include Npap60L, cellular apoptosis susceptibility protein (CAS), protein regulator of cytokinesis 1(Prc1), Ki67, survivin, phospho-survivin, HIF-1-alpha, aurora kinase B, Mad1, p-Bcl2 FoxM1, Plk 1, Auror A and KPNA2. Any combination of these markers may be evaluated to determine whether their expression levels are elevated relative to normal breast tissue controls.
Npap60 is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. Whereas Npap60S stabilizes the binding of importin α to classical nuclear localization signal (NLS)-cargo and suppresses nuclear import of NLS-cargo, Npap60L promotes the release of NLS-cargo from importin α and accelerates the nuclear import of NLS-cargo. Cellular apoptosis susceptibility protein (CAS), also known as exportin 2 promotes the dissociation of the Npap60/importin α complex. It is believed that regulation of nucleoporin complexation and dissociation plays a role in determining nuclear expression levels of HSET, as well prognosis in AA TNBC patients.
In a specific embodiment, the method further comprises the step of determining expression levels of Npap60L and CAS from the patient's biological samples and determining an Npap60L to CAS expression level ratio, wherein a ratio of <0.7 indicates a poorer prognosis for the patient compared to a patient with triple negative breast cancer with an Npap60L to CAS expression level ratio of >0.7.
In another embodiment, the method further comprises the step of performing an assay from the patient's breast cancer cells to determine whether the breast cancer cells express an elevated level of Prc1, FoxM1, plk1, KPNA2 and/or Aurora A, wherein an elevated level of nuclear HSET and Prc1, FoxM1, plk1, KPNA2 and/or Aurora A indicates a poorer prognosis for the patient compared to a patient with triple negative breast cancer expressing lower levels of nuclear HSET and Prc1, FoxM1, plk1, KPNA2 and/or Aurora A. In some embodiments, the breast cancer cells expressing elevated lavels of nuclear HSET and nuclear Prc1, FoxM1, plk1, KPNA2 and/or Aurora A indicates a poorer prognosis. Prc1 is a non-motor-microtubule-associated protein that appears to be co-regulated and co-localized with HSET.
Alternatively, or in addition, a TNBC patient's samples may be evaluated to determine whether the patient's breast cancer cells exhibit increased Cdk1 activity and/or increased levels of phosphorylated histone-H3 relative to normal breast tissue controls.
In certain embodiments, rather than testing for nuclear HSET expression, expression levels of HSET mRNAs and other co-regulated gene products are determined by RT-PCR as a prognostic gene expression signature in patients with triple negative breast cancer.
Expression levels, including percent increases in expression level over controls, may be determined at the protein level (e.g., by immunohistochemistry, Western blot, antibody microarray, ELISA, etc.) or at the mRNA level (e.g., by RT-PCR, QT-PCR, oligonucleotide array, etc.). Preferred methodologies for determining protein expression levels (and ratios therefrom) include the use of immunohistochemistry, ELISAs, antibody microarrays and combinations thereof. Preferred methodologies for determining mRNA expression levels (and ratios therefrom) include quantitative reverse transcriptase PCR (QT-PCR), quantitative real-time RT-PCR, oligonucleotide microarrays and combinations thereof.
Elevated expression levels of HSET proteins, HSET mRNAs and/or co-regulated proteins or mRNAs may represent increase(s) of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% relative to normal breast tissue controls. In other embodiments, elevated expression levels may represent increase(s) of 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold increases relative to normal breast tissue controls. Similarly, increased Cdk1 activity and/or increased levels of phosphorylated histone-H3 may represent increase(s) of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (activity or phosphorylation) relative to normal breast tissue controls or may represent increase(s) of 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold increases relative to normal breast tissue controls.
In certain embodiments, ancestry analysis may be performed by SNP analysis using ancestry informative markers (AIMs) to identify a patient's geographic origin(s). AIM markers can reveal the geographic origin of regions of a genome in, for example, about 1 million by region size chunks. Reference genomes are available for each geographic region to which samples are compared to identify the geographic origin(s) based on markers present in the patient's genome from up to at least 500 years ago (before much of the recent intercontinental travel) and can be used to identify those of African descent. In certain embodiments, ancestry analysis can be carried out commercially (e.g., 23andme and family tree DNA analysis companies).
High levels of nuclear HSET expression indicate a poor prognosis and poor overall survival, particularly without appropriate and aggressive treatment. Accordingly, where the cells from a triple negative breast cancer patient is found to express an elevated level of nuclear HSET or total HSET mRNA, the patient may be further treated with one or more therapeutic agents.
In one embodiment, the patient is administered an inhibitor of HSET. The inhibitor of HSET can be a small molecule drug or a nucleic acid-based therapeutic, such as an siRNA, an shRNA-encoded expression vector or an antisense oligonucleotide, whereby the inhibitor inhibits the activity and/or expression of HSET in the targeted cell. Alternatively, or in addition, the patient may be administered an inhibitor of a protein that is upregulated with HSET. HSET co-regulated product targets include, but are not limited to Npap60L, CAS, Prc1, Ki67, survivin, phospho-survivin, HIF-1-alpha, aurora kinase B, p-Bcl2, Mad1, Plk1, FoxM1, KPNA2, Aurora A and combinations thereof. In other embodiments, the patient is administered one or more agents that block the nuclear accumulation of HSET during interphase.
siRNAs are double-stranded RNAs that can be engineered to induce sequence-specific post-transcriptional gene silencing of mRNAs. Synthetically produced siRNAs structurally mimic the types of siRNAs normally processed in cells by the enzyme Dicer. siRNAs may be administered directly in their double-stranded form or they may be expressed from an expression vector is engineered to transcribe a short double-stranded hairpin-like RNA (shRNA) that is processed into a targeted siRNA inside the cell. Suitable expression vectors include viral vectors, plasmid vectors and the like and may be delivered to cells using two primary delivery schemes: viral-based delivery systems using viral vectors and non-viral based delivery systems using, for example, plasmid vectors. Exemplary viral vectors may include or be derived from an adenovirus, adeno-associated virus, herpesvirus, retrovirus, vaccinia virus, poliovirus, poxvirus, HIV virus, lentivirus, retrovirus, Sindbis and other RNA viruses and the like.
As used herein, the term “oligonucleotide” refers to a single stranded nucleic acid containing between about 15 to about 100 nucleotides. An antisense oligonucleotide comprises comprise a DNA backbone, RNA backbone, or chemical derivative thereof, which is designed to bind via complementary binding to an mRNA sense strand of a target gene (such as HSET) so as to promote RNase H activity, thereby leading to degradation of the mRNA. Preferably, the antisense oligonucleotide is chemically or structurally modified to promote nuclease stability and/or increased binding. The single stranded antisense oligonucleotide may be synthetically produced or it may be expressed from a suitable expression vector. In addition, the antisense oligonucleotide may be modified with nonconventional chemical or backbone additions or substitutions, including but not limited to peptide nucleic acids (PNAs), locked nucleic acids (LNAs), morpholino backboned nucleic acids, methylphosphonates, duplex stabilizing stilbene or pyrenyl caps, phosphorothioates, phosphoroamidates, phosphotriesters, and the like.
In certain embodiments, the small molecule drug targets the motor domain of HSET and/or specifically binds to the HSET/microtubule binary complex so as to inhibit HSET's microtubule-stimulated and/or microtubule-independent ATPase activities. In a specific embodiment, the small molecule drug is AZ82 (shown below) or a therapeutically effective derivative, salt, enantiomer, or analog thereof.
AZ82 binds specifically to the KIFC1/microtubule (MT) binary complex and inhibits the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a Ki of 0.043 μM. Treatment with AZ82 causes centrosome declustering in BT-549 breast cancer cells with amplified centrosomes.
Other small molecule HSET antagonists and/or centrosome declustering agents include, but are not limited to griseofulvin; noscapine, noscapine derivatives, such as brominated noscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN), N-(3-brormobenzyl) noscapine, aminonoscapine and water-soluble derivatives thereof; CW069; the phenanthridene-derived poly(ADP-ribose) polymerase inhibitor, PJ-34; N2-(3-pyridylmethyl)-5-nitro-2-furamide, N2-(2-thienylmethyl)-5-nitro-2-furamide, N2-benzyl-5-nitro-2-furamide, an anthracine compound as described in U.S. Patent Application Publication 2008/0051463; a 5-nitrofuran-2-carboxamide derivative as described in U.S. Provisional Application 61/619,780; and derivatives and analogs therefrom.
In certain embodiments, the patient may be additionally administered a poly(ADP-ribose) polymerase (PARP) inhibitor, an inhibitor of the Ras/MAPK pathway, an inhibitor of the PI3K/AKT/mTOR pathway, an inhibitor of FoxM1, Hif1α, surviving, Aurora, Plk1 or a combination thereof. Exemplary PARP inhibitors include, but are not limited to olaparib, iniparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338 and combinations thereof. Exemplary Ras/MAPK pathway agents include, but are not limited to MAP/ERK kinase (MEK) inhibitors, such as trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026, R04987655, R05068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, GDC-0973 and combinations thereof. Exemplary PI3K/AKT/mTOR pathway inhibitors include, but are not limited to everolimus, temsirolimus, GSK2126458, BEZ235, PIK90, PI103 and combinations thereof.
Alternatively, or in addition to administering an HSET-targeted therapeutic, a patient expressing high levels of nuclear HSET may be additionally treated with adjuvant chemotherapeutic agents to further reduce the risk of adverse events, such as metastasis, disease relapse, and poor survival. Adjuvant chemotherapies may include administration of cyclophosphamide, taxanes, such as docetaxel and paclitaxel; anthracyclines, such as epirubicin and doxorubicin; gemcitabine, cisplatin, fluorouracil, ixabepilone, capecitabine, epidermal growth factor receptor-targeting agents, and combinations thereof.
The appropriate dosage (“therapeutically effective amount”) of the therapeutic agent(s) will depend, for example, on the severity and course of the breast cancer, the mode of administration, the bioavailability of the therapeutic agent(s), previous therap(ies), the age and weight of the patient, the patient's clinical history and response to the therapeutic agent(s), the type of the therapeutic agent used, discretion of the attending physician, etc. The therapeutic agent(s) are suitably administered to the patent at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards. The therapeutic agent(s) may be administered as the sole treatment or in combination with other drugs or therapies useful in treating the breast cancer. When used with other drugs, the therapeutic agent(s) may be used at a lower dose to reduce toxicities and/or side effects.
The therapeutic agent(s) may be administered to the patient with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical and/or inhalation routes. As a general proposition, the therapeutically effective amount(s) of the above described therapeutic agent(s) will be in the range of about 1 ng/kg body weight/day to about 100 mg/kg body weight/day whether by one or more administrations. In a particular embodiments, each therapeutic agent is administered in the range of from about 1 ng/kg body weight/day to about 10 mg/kg body weight/day, about 1 ng/kg body weight/day to about 1 mg/kg body weight/day, about 1 ng/kg body weight/day to about 100 μg/kg body weight/day, about 1 ng/kg body weight/day to about 10 μg/kg body weight/day, about 1 ng/kg body weight/day to about 1 μg/kg body weight/day, about 1 ng/kg body weight/day to about 100 ng/kg body weight/day, about 1 ng/kg body weight/day to about 10 ng/kg body weight/day, about 10 ng/kg body weight/day to about 100 mg/kg body weight/day, about 10 ng/kg body weight/day to about 10 mg/kg body weight/day, about 10 ng/kg body weight/day to about 1 mg/kg body weight/day, about 10 ng/kg body weight/day to about 100 μg/kg body weight/day, about 10 ng/kg body weight/day to about 10 μg/kg body weight/day, about 10 ng/kg body weight/day to about 1 μg/kg body weight/day, 10 ng/kg body weight/day to about 100 ng/kg body weight/day, about 100 ng/kg body weight/day to about 100 mg/kg body weight/day, about 100 ng/kg body weight/day to about 10 mg/kg body weight/day, about 100 ng/kg body weight/day to about 1 mg/kg body weight/day, about 100 ng/kg body weight/day to about 100 μg/kg body weight/day, about 100 ng/kg body weight/day to about 10 μg/kg body weight/day, about 100 ng/kg body weight/day to about 1 μg/kg body weight/day, about 1 μg/kg body weight/day to about 100 mg/kg body weight/day, about 1 μg/kg body weight/day to about 10 mg/kg body weight/day, about 1 μg/kg body weight/day to about 1 mg/kg body weight/day, about 1 μg/kg body weight/day to about 100 μg/kg body weight/day, about 1 μg/kg body weight/day to about 10 μg/kg body weight/day, about 10 μg/kg body weight/day to about 100 mg/kg body weight/day, about 10 μg/kg body weight/day to about 10 mg/kg body weight/day, about 10 μg/kg body weight/day to about 1 mg/kg body weight/day, about 10 μg/kg body weight/day to about 100 μg/kg body weight/day, about 100 μg/kg body weight/day to about 100 mg/kg body weight/day, about 100 μg/kg body weight/day to about 10 mg/kg body weight/day, about 100 μg/kg body weight/day to about 1 mg/kg body weight/day, about 1 mg/kg body weight/day to about 100 mg/kg body weight/day, about 1 mg/kg body weight/day to about 10 mg/kg body weight/day, about 10 mg/kg body weight/day to about 100 mg/kg body weight/day.
In certain embodiments, the therapeutic agent(s) are administered at a dose of 500 μg to 20 g every three days, or 10 μg to 400 mg/kg body weight every three days. In other embodiments, each therapeutic agent is administered in the range of about 10 ng to about 100 ng per individual administration, about 10 ng to about 1 μg per individual administration, about 10 ng to about 10 μg per individual administration, about 10 ng to about 100 μg per individual administration, about 10 ng to about 1 mg per individual administration, about 10 ng to about 10 mg per individual administration, about 10 ng to about 100 mg per individual administration, about 10 ng to about 1000 mg per injection, about 10 ng to about 10,000 mg per individual administration, about 100 ng to about 1 μg per individual administration, about 100 ng to about 10 μg per individual administration, about 100 ng to about 100 μg per individual administration, about 100 ng to about 1 mg per individual administration, about 100 ng to about 10 mg per individual administration, about 100 ng to about 100 mg per individual administration, about 100 ng to about 1000 mg per injection, about 100 ng to about 10,000 mg per individual administration, about 1 μg to about 10 μg per individual administration, about 1 μg to about 100 μs per individual administration, about 1 μg to about 1 mg per individual administration, about 1 μg to about 10 mg per individual administration, about 1 μg to about 100 mg per individual administration, about 1 μg to about 1000 mg per injection, about 1 μg to about 10,000 mg per individual administration, about 10 μg to about 100 μs per individual administration, about 10 μg to about 1 mg per individual administration, about 10 μg to about 10 mg per individual administration, about 10 μg to about 100 mg per individual administration, about 10 μg to about 1000 mg per injection, about 10 μg to about 10,000 mg per individual administration, about 100 μg to about 1 mg per individual administration, about 100 μg to about 10 mg per individual administration, about 100 μg to about 100 mg per individual administration, about 100 μg to about 1000 mg per injection, about 100 μg to about 10,000 mg per individual administration, about 1 mg to about 10 mg per individual administration, about 1 mg to about 100 mg per individual administration, about 1 mg to about 1000 mg per injection, about 1 mg to about 10,000 mg per individual administration, about 10 mg to about 100 mg per individual administration, about 10 mg to about 1000 mg per injection, about 10 mg to about 10,000 mg per individual administration, about 100 mg to about 1000 mg per injection, about 100 mg to about 10,000 mg per individual administration and about 1000 mg to about 10,000 mg per individual administration. The therapeutic agent(s) may be administered daily, or every 2, 3, 4, 5, 6 and 7 days, or every 1, 2, 3 or 4 weeks.
In other particular embodiments, the therapeutic agent(s) are administered at a dose of about 0.0006 mg/day, 0.001 mg/day, 0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day, 0.06 mg/day, 0.1 mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6 mg/day, 10 mg/day, 30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600 mg/day, 1000 mg/day, 2000 mg/day, 5000 mg/day or 10,000 mg/day. As expected, the dosage(s) will be dependent on the condition, size, age and condition of the patient.
Another aspect of the present application relates to a method for treating TNBC patients with high nuclear HSET accumulation by increasing the Npap60L-to-Npap60S ratio in these patients. In some embodiments, the method comprises the step of administering to a TNBC patient with high nuclear HSET accumulation an effective amount of an agent that increases the Npap60L-to-Npap60S ratio in the breast tissue of the patient.
Another aspect of the present application relates to a method for treating TNBC patients with high nuclear HSET accumulation by inhibiting the expression or activity of Prc1 in these patients. In some embodiments, the method comprises the step of administering to a TNBC patient with high nuclear HSET accumulation an effective amount of an agent that inhibits the expression or activity of Prc1 in the breast tissue of the patient.
Another aspect of the present application relates to a method for treating TNBC patients with high nuclear HSET accumulation by inhibiting the expression or activity of FoxM1 and/or Plk1 in these patients. In some embodiments, the method comprises the step of administering to a TNBC patient with high nuclear HSET accumulation an effective amount of an agent that inhibits the expression or activity of FoxM1 and/or Plk1 in the breast tissue of the patient.
Another aspect of the present application relates to a method for treating TNBC patients with high nuclear HSET accumulation by inhibiting the expression or activity of Aurora A and/or KPNA2 in these patients. In some embodiments, the method comprises the step of administering to a TNBC patient with high nuclear HSET accumulation an effective amount of an agent that inhibits the expression or activity of Aurora A and/or KPNA2 in the breast tissue of the patient.
Another aspect of the present application relates to a kit for determining elevated expression of HSET. In some embodiments, the kit includes an HSET binding agent along with one or more secondary binding agents specifically binding to one or more gene product(s) upregulated before, during or after (e.g., subsequent to and as a result of) HSET elevation. In some embodiments, the HSET binding agent and/or the one or more secondary binding agents are antibodies. In some embodiments, the one or more gene product(s) are selected from the group consisting of gene products of Npap60L, CAS, Prc1, Ki67, survivin, phospho-survivin, HIF1α, aurora kinase B, Mad1, p-Bcl2, FoxM1, Plk1, Aurora A and KPNA2. In some embodiments, the kit further includes one or more reagents for preparation of a nuclear fraction or extract. In some embodiments, the kit further includes one or more reagents for immunohistochemistry. In some embodiments, the one or more reagents for immunohistochemistry include reagents for staining the nuclei. In some embodiments, the kit further includes instructions for using the reagents for the detection of HSET and/or the one or more gene products.
Approval from the Emory Institutional Review Board (IRB) was obtained for all aspects of these studies. Archival paraffin-embedded tissue samples were collected during patient care and diagnostics. Since no direct patient interaction occurred, a formal consent was not required for testing of these samples. Surgical pathology files from Emory University and Grady Memorial Hospitals (Atlanta, Ga.) between the years 2003-2008 were searched for African American and European American breast carcinoma samples with clinicopathological, demographic, and outcome information (e.g., tumor grade, stage, and size; ER, PR, and HER2 status; Ki67 staining; age; ethnicity; overall, progression-free, and metastasis-free survival).
Samples from 193 breast carcinoma patients were obtained, the characteristics of which are provided in Table 1:
Tissue microarrays (TMAs) were constructed from cores (2 each, 1 mm in diameter) of breast tumors along with normal breast tissue (controls), all of which had been previously fixed with formalin and embedded in paraffin. Five micron sections were taken from the TMAs for immunohistochemistry. The TMAs were processed for immunostaining by performing antigen retrieval in citrate buffer (pH 6.0) in a pressure-cooker (15 psi) for 3 minutes. Immunostaining for HSET at a 1:1000 dilution was performed using a rabbit polyclonal antibody.
HSET staining intensity was assessed for both the cell nucleus and cytoplasm by an experienced pathologist who was blinded to patient and tissue characteristics. Nuclear and cytoplasmic staining were assessed semi-quantitatively by assigning a relative intensity score (0=none, 1=low, 2=moderate, or 3=high). The percentage of cell nuclei or cytoplasms demonstrating any HSET positivity (i.e., a score of 1, 2, or 3) was also determined. The average percentage was taken from the two cores that represented each sample and used for subsequent calculations. The product of the relative intensity and percent positivity was recorded as the weighted index (WI) for both the nucleus and cytoplasm. The sum of the nucleus WI and cytoplasm WI was recorded as the total WI. The HSET WI for the nucleus had an average of 63.91, median of 45, and a range of 0-240. The HSET WI for the cytoplasm was generally higher and had an average of 126.00, a median of 100, and a range of 0-300. These and other statistics regarding HSET staining can be found in Table 1.
All statistical analyses were conducted using SAS Version 9.3 with p<0.05 considered statistically significant. Optimal cut points using maximum log-rank test statistic method for HSET WI with respect to survival outcomes were not found, which supported treating HSET WI as a continuous variable in the model. Furthermore, there is no published threshold for a hazardous level of HSET expression in breast cancer. Consequently, HSET was initially treated as a continuous variable. In subsequent analyses of African American patients only, optimal cut points for HSET nucleus WI with respect to survival outcomes were found; therefore it was categorized based on those cut points for the subgroup analysis.
Overall survival was defined as the number of days from diagnosis to death or last follow-up if death was not recorded. Progression-free survival was defined as the number of days from diagnosis to the first local recurrence, metastasis, or death, whichever occurred first, or the last follow-up if the patient did not experience an event. Metastasis-free survival was defined as the number of days from diagnosis to the first metastasis, or death, whichever occurred first, or the last follow-up if the patient did not experience an event. Covariates included TN status, tumor size, grade, stage, positive lymph nodes, age at diagnosis, Nottingham Prognostic Index (NPI) and Ki67 WI. NPI was calculated from grade, positive lymph nodes, and 0.2× tumor size.
Descriptive statistics were reported for all variables. The unadjusted association of all covariates with continuous HSET nucleus was assessed using ANOVA and the Kruskal-Wallis test for categorical covariates and Pearson and Spearman correlation coefficients for numerical covariates. Since the distribution of HSET nucleus WI was right skewed, it was square root transformed for the purpose of ANOVA. The association of race with HSET was additionally assessed adjusting for TN status. A general linear model was used to predict HSET.
The unadjusted association of each covariate with overall, progression-free, and metastasis-free survival was assessed using Cox proportional hazards models. Additionally, Cox models were fit including nuclear HSET WI. Main effects models were fit including race and HSET. Additionally, the covariates, TN status, stage, age, and NPI, were entered into the model subject to a backward variable selection method with an alpha=0.20 removal criteria. NPI was used in place of tumor size, grade, and positive lymph nodes. Ki67 was not included due to the high number of missing values. Subgroup analysis was also repeated on TN patients and African American (AA) patients. Among AA patients, TN status was forced into the models instead of race. Unadjusted Kaplan-Meier survival curves were produced for each outcome stratified by HSET group for African Americans. Survival differences between the groups were assessed using the log-rank test.
I. Data Collection:
One channel microarray data for various cancers were collected from Gene Expression Omnibus (GEO) database (Edgar R et al., Nucleic Acids Res., 2002, 30:207-210). The list of the GSE ID's is given in Table 2.
II. Data Pre-Processing:
One channel microarray data was processed using Robust Multiarray (RMA) normalization (Gautier L et al., Bioinforatics, 2004, 20: 307-315) and was further used for gene expression analysis.
III. Analysis of HSET Gene Expression:
Log2 n transformed HSET expression levels were analyzed in glioblastoma, leukemia, lung, breast, colon and cervical tumor samples as compared to their corresponding normal tissues.
All paraffin-embedded tissue slides were commercially obtained (from Accumax, and US Biomax). A subset of well-annotated tissue microarrays (TMAs) (193 biospecimens) with information on clinical outcomes, were obtained from Dr. Gabriela Oprea, Grady Memorial Hospital. The Emory Institutional Review Board (IRB) approval was obtained for all aspects of the study.
HeLa-HSET-GFP cells were generously provided by Claire Walczak (Indiana University). HeLa and HeLa-HSET-GFP, MDA-MB-231 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Briefly, cells were seeded onto 100-mm plates 1 day prior to transfection. Plasmid DNA (5 μg) and 15 μl of DharmaFECT 4 transfection reagent (Thermo Scientific, PA, USA) were used for each transfection. HSET-pEGFP plasmid was generously provided by Claire Walczak. Cells overexpressing HSET were selected in the medium containing G418 (400 μg/ml). The G418-resistant colonies were collected and examined for HSET expression. SMARTpool: ON-TARGETplus KIFC1 siRNA (Dharmacon, PA, USA) was used to knockdown HSET in MDA-MB-231 cells.
Cells were cultured to ˜70% confluence and protein lysates were collected following transfection or otherwise. Fresh frozen tissue sections were first sonicated and lysates were then prepared. The immune-reactive bands corresponding to respective primary antibodies were visualized by the Pierce ECL chemiluminescence detection kit (Thermo Scientific). β-actin was used as loading control. For immunofluorescence staining, cells grown on glass coverslips were fixed with cold (−20° C.) methanol for 10 min and blocked by incubating with 2% bovine serum albumin/PBS.0.05% Triton X-100 at 37° C. for 1 h. Specific primary antibodies were incubated with coverslips for 1 h at 37° C. at the recommended dilution. The cells were washed with 2% bovine serum albumin/PBS for 10 min at room temperature before incubating with a 1:2000 dilution of Alexa 488- or 555-conjugated secondary antibodies. Cells were mounted with Prolong Gold antifade reagent that contains 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Polyclonal rabbit anti-HSET antibody was provided by Claire Walczak. Antibodies against α-tubulin and β-actin were from Sigma (St. Louis, Mo., USA). Antibodies against γ-tubulin, α-tubulin and ƒ1-actin were from Sigma (St. Louis, Mo., USA). Anti-Mad2 antibody was from BD Biosciences (Pharmingen, San Jose, Calif., USA). Antibodies against p-Bcl2 and cleaved caspase-3 were from Cell Signaling (Danvers, Mass., USA). Alexa 488- or 555-conjugated secondary antibodies were from Invitrogen (Carlsbad, Calif., USA). Anti-Mad1 antibody was a generous gift from Andrea Musacchio. Anti-Ki67 antibody was from Abcam (Cambridge, Mass., USA). Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, Calif., USA).
To examine cdk1 kinase activity, anti-cdk1 antibody was used to selectively immunoprecipitate cdk1-containing complexes from HeLa and HeLa-HSET-GFP cell lysates. The resulting immunoprecipitate was incubated with pure histone-H3 protein in the presence of p32-labelled ATP and kinase buffer. The kinase assay reaction allowed immunoprecipitated cdk1 to phosphorylate histone-H3 in vitro, the extent of which was measured by immunoblotting using phosphohistone-H3 antibody from Cell Signaling (MA, USA). Histone-H3 protein was from Millipore (MA, USA) and ATP was from Cell Signaling.
The slide samples from tumor cell lines or tumor tissue were hybridized by 2-color FISH with an HSET-specific BAC probe (RPCI-11 602P21, green) and a chromosome 6 centromere (CH514-7B4, red) (BACPAC). The HSET and centromere 6 probes were labeled with Cy3-dUTP (red) and FITC-dUTP (green), respectively, and hybridized with nuclei from cell lines or tumor tissue samples. Plasmids for production of a particular FISH probe were combined in equimolar amounts (55-70 pM). Nick translation was performed on 2 μg of this substrate by using Nick translation kit (Abbott Molecular, IL, USA). The translation product was denatured for 3 mins at 95° C. followed by fast cooling on ice and confirmed in 1.5% agarose gel electrophoresis as a smear of fragments ranging between 100 and 300 bp. A 2 min denaturation at 76° C. was followed by overnight (12-16 h) incubation at 37° C. Hybridization of the FISH probes was carried out in LSI/WCP hybridization buffer (Abbott Molecular, IL, USA). The slides were counterstained with DAPI (Invitrogen, NY, USA) and the Zeiss LSM 700 confocal microscope was used to capture FISH images. Results were expressed as a ratio of the number of copies of the HSET gene to the number of chromosome 6-centromeric markers.
Trypsinized cells were resuspended in PBS at 106 cells/ml. Cells were then fixed by addition of ice-cold 70% ethanol. Ethanol-fixed cells were kept overnight at 4° C. before staining. Cells were pelleted and washed twice with PBS. Cell pellets were incubated for an hour at room temperature with mouse anti-MPM-2 antibody (Millipore, Mass., USA), followed by 1 h incubation with Alexa-488 anti-mouse secondary antibody (Life Technologies, NY, USA). Finally cells were washed, pelleted and resuspended in propidium iodide-containing isotonic buffer (0.1 mg/mL) and 0.5% Triton X-100. Cell cycle distribution was determined by flow cytometry using an LSR Fortessa Flow cytometer (BD Biosciences, CA, USA) and analyzed using Flowjo software (Tree Star, OR, USA).
Cells were cultured to ˜70% confluence followed by centrifuging and pellet was resuspended in 1 mL culture medium. 0.1 mL of 0.4% Trypan Blue solution was then added to 1 mL of cell suspension. The hemocytometer was loaded with 10 μL of the solution and examined immediately under a microscope. Live (white) and dead (blue) cells were counted and the percent cell viability was calculated using the following formula: percent viable cells=[1.00−(Number of live cells Number of total cells)]×100.
Asynchronous proliferating HeLa and HeLa-HSET-GFP cells were grown on coverslips to a confluency of ˜70% and then incorporated with 10 μM BrdU for 1 h followed by fixation with 70% ethanol at room temperature and immersion in 0.07 N NaOH for 2 minutes (which was then neutralized with PBS, pH 8.5). Coverslips were then incubated in 2% bovine serum albumin/PBS.0.05% Triton X-100 at 37° C. for 1 h followed by immunostaining using a 1:1000 dilution of Anti-BrdU-FITC antibody (BD Biosciences, San Jose, Calif., USA). BrdU positive cells, indicative of cell proliferation, were captured on a Zeiss Axioplan-2 fluorescence microscope (20×).
MDA-MB-231 cells were transiently transfected with CV, HSET-pEGFP plasmid or HSET SMARTpool siRNA as described above, and lysates were collected. Cell lysates were clarified by centrifugation at 10,000 rpm, and the supernatants (500 μg of protein) were subjected to immunoprecipitation with 4 μL of anti-HSET or anti-survivin antibodies. After overnight incubation at 4° C., protein A-agarose beads were added and left at 4° C. overnight. Immunocomplexes were then subjected to Western blot analysis as described previously. Western blot analysis with anti-ubiquitin antibody (Life Sensors, PA, 1:500) was performed by first incubating the PVDF membrane with 0.5% glutaraldehyde/PBS pH 7.0 for 20 min and then probing for the antibody.
HeLa cells were grown to 60-70% confluence and then transiently transfected with CV, HSET-pEGFP plasmid or HSET SMARTpool siRNA as described above. After 48 h, the cell clock dye (Biocolor, UK) (pre-warmed at 37° C.) was added (150 μl per well in 12-well plate) and the cells were incubated at 37° C. for 1 h. Dye was the washed twice with pre-warmed DMEM medium. Fresh medium was added and the cells were imaged in bright field (to assess different phases of cell cycle) and fluorescent (red for PI) channel. Cell clock dye is a redox dye, which is readily taken up by live cells. In G1 phase, the dye in its reduced form is yellow in color, while in the intermediate state it is green (S and G2 phase) before turning dark blue in the fully oxidized form (mitosis).
To probe whether HSET may serve as a prognostic biomarker in breast cancer, the relationship between HSET expression and disease progression was investigated in a dataset of 193 breast cancer patients. Since HSET was treated as a continuous variable, hazard ratios (HR) for nuclear, cytoplasmic, and total HSET were calculated based on the respective standard deviations for these values (Table 1). HSET expression was evaluated using a WI (the product of the staining intensity and the proportion of positive cells). Both the expression level in the nucleus and cytoplasm, along with their sum, were assessed. In univariate analysis, nuclear HSET expression at the time of diagnosis was found to be significantly associated with worse progression- and metastasis-free survival (HR=1.23, p=0.046 and HR=1.27, p=0.025, respectively), with a borderline-significant trend noticed for overall survival (HR=1.25, p=0.052), as shown in Table 3:
No significant or borderline-significant associations were found for cytoplasmic or total HSET expression with overall, progression-free, or metastasis-free survival (Table 2). African American race was also associated with worse overall survival (HR=2.66, p=0.023), although TN status was not (p=0.030).
Given the association of nuclear HSET with survival outcomes, additional prognostic indicators were examined for a further association with nuclear HSET expression. As the distribution of the nuclear HSET WI was right-skewed, it was square root transformed in order to perform ANOVA. Nuclear HSET was found to be significantly and positively associated with TN status, tumor size, tumor grade, and NPI (p<0.001 for all) along with tumor stage (p=0.013), as shown in Table 4.
In univariate analysis of all patients (Table 3), nuclear HSET expression was elevated in African American (AA) patients as compared to European American (EA) patients, although this association did not reach statistical significance (p=0.1). Importantly, nuclear HSET was highly significantly associated with TN status, tumor size ≧2 cm, grade, NPI, and Ki67 WI (p<0.001) and pronouncedly associated with stage (p=0.014). Ki67 is utilized as a proliferation marker in breast cancer and may be associated with worse survival, although no significant associations between Ki67 and overall, progression-free, or metastasis-free survival were found by univariate analysis (p>0.40 for all).
Having identified strong association of higher nuclear HSET with poorer prognostic indicators (such as TN status, tumor size, grade, NPI, and Ki67 WI and progression- and metastasis-free survival) by univariate analysis, multivariate analyses were carried out to evaluate whether the relationship between nuclear HSET expression and disease outcomes was retained after adjusting for standard prognostic indicators and possible confounding factors, such as NPI stage, age, and ethnicity. The associations of nuclear HSET with worse overall, progression-free, and metastasis-free survival retained significance and were in fact found to be stronger in multivariate analysis (HR=1.37, p=0.030; HR=1.30, p=0.044; and HR=1.34, p=0.035, respectively), as shown in Table 5:
This finding confirms that nuclear HSET is not merely associated with these standard negative prognostic indicators such as Ki67, but is rather independently associated with worse outcomes. In the overall survival multivariate model, the hazard for AA patients was significantly greater than for EA patients (HR=2.95, p=0.031).
Interrelationships between AA ethnicity, disease progression and mortality, and HSET expression were examined. Although AA patients are more likely to be diagnosed with TN receptor status, this does not appear to translate into worse clinical outcomes. Thus interrelationships between TN status, ethnicity, and HSET expression were analyzed. Within non-TN patients, there was no difference in HSET expression (nuclear, cytoplasmic, or total) between African American and European American patients (p>0.40 for all), as shown in Table 6:
However, within TN patients, African American women demonstrated higher nuclear HSET expression (square-root transformed data; p=0.011), whereas EA patients demonstrated higher cytoplasmic HSET expression (p=0.023). Altogether, these data suggest that nuclear HSET is exclusively associated with African American ethnicity within TN patients.
Nuclear HSET expression is a negative prognostic indicator within African American patients in both univariate and multivariate analyses: Given the marked associations between nuclear HSET expression, ethnicity, and disease progression, an evaluation was undertaken to determine whether HSET has a prognostic value that extends beyond that TN status in AA patients. Thus, the prognostic value of HSET within the AA cohort alone (n=149) was examined. Within the set of AA patients, only nuclear HSET expression (and not cytoplasmic or total) was found to be associated with survival outcomes in univariate analysis. Without adjusting for other factors, higher nuclear HSET expression was associated with worse overall survival (HR=1.41, p=0.008), progression-free survival (HR=1.33, p=0.018), and metastasis-free survival (HR=1.37, p=0.011), as shown in Table 7:
These associations were found to be even stronger in multivariate analysis, with higher nuclear HSET predicting worse overall survival (HR=1.56, p=0.006) when adjusting for NPI, stage, TN status, and age; progression-free survival (HR=1.44, p=0.012) when adjusting for stage, TN status, and age; and metastasis-free survival (HR=1.44, p=0.015) when adjusting for stage and TN status (Table 6). Consequently, HSET appears to be a much better prognostic indicator for AA breast cancer patients than the EA population of breast carcinoma patients even after adjusting for age along with TN status and other tumor characteristics. Nuclear HSET was a better prognostic indicator than TN status, which was not a statistically significant prognostic indicator for overall, progression-free, or metastasis-free survival in univariate analysis, and a more significant indicator than tumor size.
To evaluate the impact of putative declustering drugs on cell cycle progression and hypodiploidy (<2N DNA content, which may indicate apoptotic cells), MDA-MB-231 (231), PC3, and HeLa cells were treated with different concentrations of declustering drugs, stained with propidium iodide, labeled with anti-MPM2 antibody, and then assessed by flow cytometry at multiple time points over 48 h. The chosen cell lines displayed different levels of endogenous centrosome amplification (CA). 231 cells (mutant p53) exhibit high levels of CA (˜20-45%) compared with PC3 (p53 null) and HeLa (wild-type but E6-inactivated p53), which have low basal levels of CA. Consistent with previous reports, the data showed that all drugs induced sustained MA (at least 2× mitotic cells compared with untreated control cultures) at the concentrations indicated. The duration, highest degree, and rapidity of onset of MA varied between drugs, drug concentrations, and cell lines (
In general, no consistent associations between the extent, duration, or timing of MA within drugs or across cell lines was found (see
Given that brominated noscapine (RBN) increases the expression of Plk4, a mediator of CA, other declustering drugs were investigated to determine their effect on expression of PLK4 along with two other mediators of CA, Cyclin E and Aurora A. All of the drugs studied were found to increase expression of PLK4, Cyclin E and Aurora A compared with untreated cultures (
When analyzing correlations between the upregulation of key molecular markers of CA and the extents of drug-induced CA, no significant correlations between the degree of CA (
To better understand the “potency” of drug-induced CA with time, the average fold change in CA over controls over 24 h was assessed (
Notably, average fold-increases in CA were generally more frequent in interphase cells when compared to mitotic cells (
As shown in Table 8, compared to HeLa and PC3 cells, 231 cells (which exhibit the greatest endogenous CA among controls, approximately 20-30% on average) were most susceptible to declustering drugs in general:
Table 8. Peak subG1 Percents Over 48 h for Each Cancer and Non-Malignant Cell Line by Drug and Concentration
This is corroborated by the fact that 231 cells exhibited the greatest peak subG1 fraction across cell lines and drugs (25× control after treatment of 231 cells with 25 μM RBN, vs. 9× for HeLa and 8× for PC3, both treated with 10 μM RBN). Within drugs and across cell lines, BN was most effective in 231 cells, (the maximum subG1 fraction was 9.3× control, vs. 4.4× for HeLa and 9.2× for PC3, all treated with 25 μM BN), as was PJ (the maximum subG1 fraction was 10.4× control after treatment with 25 μM PJ, vs. 7.9× control in PC3 cells treated with 25 μM PJ and 4.8× control in HeLa cells treated with 10 μM PJ) (Table 8). GF was most effective in PC3 cells (the maximum subG1 fraction 16.3× control after treatment with 50 μM GF, vs. 6.4× for 231 cells treated with 25 μM GF and 4.3× for HeLa cells treated with 50 μM GF, although these cells do not have substantial endogenous CA (approximately 3% interphase and 4% mitotic CA on average. Altogether, it appears that certain declustering drugs (namely, RBN, BN, and PJ) may be more effective against cancer cell lines with endogenous CA, whereas the efficacy of other agents (namely, GF and Nos) may depend less on endogenous CA.
The above data indicate that RBN, BN and PJ appear to be most effective in 231 cells. To test whether higher susceptibility of 231 cells to these three drugs is related to the extent of drug-induced CA in these cell lines, the average fold-increase in CA (compared to untreated controls) induced by RBN, BN and PJ in 231 cells was evaluated and compared to the average fold-increases in CA induced by these drugs in PC3 and HeLa cells (
Upon treatment with RBN, BN, and PJ, the final total centrosomal burden (the percent of cells with CA, regardless of cell cycle stage) is much higher in 231 cells as compared to HeLa and PC3 cells (
To determine whether the CA-inducing activity of declustering drugs is restricted to cancer cells, two non-malignant cell lines, mammary fibrocystic (MCF10A) cells and adult human dermal fibroblasts were treated with these drugs. Neither one of RBN, GF or PJ induced CA or cell death (Table 8) in these cell lines. Specifically, an analysis of the CA phenotypes produced by declustering drug treatment of MCF10A and HDFs showed that neither concentrations of Nos or BN significantly increased CA over control levels in interphase or mitotic MCF10A cells at any time point assessed over 24 h. However, both concentrations of RBN significantly increased the peak extent of CA in interphase and mitotic MCF10A cells (p<0.001 for all,
Importantly, a therapeutic window exists for several of these agents at the concentrations and in the cell lines tested compared to cancer cells. Nos, BN, and PJ did not cause a significant increase in peak subG1 percent compared to controls (Table 8). RBN and GF did increase peak SubG1 in MCF10A cells compared to controls (p<0.01 for all). However, 10 μM RBN induced a smaller peak subG1 in MCF10A cells as compared to 231 cells (p<0.001), although the same was not true for PC3 and HeLa cells (Table 8). By contrast, increasing the dose of RBN to 25 μM, which caused slightly increased toxicity to MCF10A cells, resulted in much greater increases in toxicity to 231 and PC3 cells (p<0.001). These data suggest that for RBN, even in in vitro cell cultures, a therapeutic window exists and can be exploited to selectively target cancer cell lines. Interestingly, previous work has demonstrated cancer selectivity of RBN in nude mice carrying human ovarian cancer xenografts. In those previous experiments, RBN inhibited tumor progression by inducing apoptosis in tumor cells, but toxicity was not detected in normal tissues. All cancer cell lines were found to be more susceptible to 25 μM GF than MCF10A cells (p<0.001). When the concentration was increased to 50 μM, however, MCF10A and PC3 cells were equally susceptible to the GF, although 231 and HeLa cells remained more susceptible (p<0.001).
In HDFs, all the drugs tested increased peak subG1 over controls in a significant fashion (p<0.01 for all) (Table 8). Nevertheless, for Nos and PJ, both concentrations caused more death in all cancer cell lines vs. HDFs (p<0.001 for all). For BN, the same was true for 231 and PC3 cells (p<0.05 for all) but not HeLa cells, in which there was no significant difference. For GF, both concentrations caused more death in 231 and HeLa cells (p<0.001 for all) but not PC3 cells, in which there was no significant difference. For RBN, both concentrations caused more death in 231 cells and 25 μM RBN caused more death in PC3 cells as compared to HDFs, (p<0.001 for all), but the same was not true for both concentrations in HeLa or 10 μM RBN. Thus, it appears that there may be clinically relevant therapeutic windows for these drugs depending on the type of cancer and the drug dosage.
Altogether, although centrosome declustering drugs induced MA, significant differences existed in the (i) extents and durations of MA, (ii) the size of the subG1 population, (iii) the rapidity of the onset of MA and hypodiploidy, and (iv) the extent to which hypodiploidy was accompanied by caspase-dependent apoptosis (
The declustering drugs were further evaluated to determine the extent to which they induce MP. MP was considered low grade if there were only 3 or 4 spindle poles and high grade if there were ≧5 poles. All of the declustering drugs, at one or both concentrations, induced spindle MP in at least one cell type above control levels (
The declustering agents were further evaluated to determine the extents to which they induced declustering. This analysis shows that the extent of total declustering (the percentage of cells with amplified centrosomes in which no centrosomes were clustered) induced by all these drug regimens was the lowest in 231 cells, which have higher endogenous CA (
Thus, it appears that the drugs tested largely induce spindle MP in a declustering-independent manner. Declustering drugs may therefore prove effective in cancers regardless of the extent of CA present.
Associations between drug-induced spindle MP, centrosome declustering, and drug efficacy (subG1 extent) were probed in order to identify the phenotypes that contributed most to cell death. Beta regression (a statistical methodology more appropriate for proportions data than linear regression when very low or high percentages are observed) was used to analyze correlates of peak subG1. For this technique, pseudoR2 (the squared correlation of linear predictor and link-transformed response) is reported rather than R2 as in linear regression, and it indicates the goodness-of-fit of the model.
By this analysis, peak MP was found to significantly correlate with peak subG1 (P=0.00840, pseudoR2=0.321) across all drugs and cell lines, suggesting that generation of spindle MP is a shared mechanism whereby declustering drugs trigger cell death. Importantly, no significant associations between CA and spindle MP were found, consistent with the result that declustering drugs appear to induce spindle MP by disrupting spindle pole and/or centrosome integrity, which in some cases may also decluster centrosomes if an excess is present. Within 231 cells, an even stronger, positive correlation with a very good fit between peak high-grade MP and peak subG1 was found (
In HeLa cells, peak MP (any grade) positively correlated with peak subG1 (P=0.0055; pseudoR2=0.575;
In PC3 cells, no association between peak MP and peak subG1 across drugs was found. However, when analyzing the correlation between the average fold increase in CA induction with peak subG1 percent, an interesting trend emerged. Specifically, in PC3 cells, the proportion variable (peak subG1) always lay within the 30-70% range and the other variable (fold increase in CA) was continuous; therefore a linear regression was implemented for analysis. This analysis showed that the average fold increase in CA in interphase positively correlated with peak subG1 (P=0.057; R2=0.619;
MCF10A cells and human dermal fibroblasts were further evaluated to study the impact of treatment with declustering drugs on spindle MP and subG1 induction in non-transformed cells. In both of these cell types, peak MP positively correlated with peak subG1 (R2=0.82 with P=0.003 and R2=0.89 with P<0.001, respectively), suggesting that MP is also toxic to normal cells.
Given the crucial requirement of centrosome clustering mechanisms for the viability of cancer cells with extra centrosomes, the abundance of the clustering protein HSET in various cancers harboring extra centrosomes was investigated. Upregulating HSET expression may provide a means to permit clustering of extra centrosomes and may facilitate maintenance of low-grade aneuploidy so as to foster cell viability and allow malignant transformation and tumor evolution to proceed. An in silico gene expression analysis using publically available microarray data was employed to determine the expression level of HSET in various cancer tissue types. One-channel microarray data for glioblastoma, leukemia, lung and breast cancer patients with their normal sample pairs were collected from Gene Expression Omnibus (GEO) database. Each of these samples was then Robust Multiarray (RMA) normalized, and their logarithm to base 2-transformed HSET gene expression values were plotted to determine the difference as shown in
The in silico analyses of microarray data showed that breast cancers display significantly higher HSET expression (˜5-fold) than corresponding normal tissue. Further, given the pronounced occurrence of amplified centrosomes and centrosome clustering in aggressive breast cancer, HSET was further evaluated to determine whether a role in tumor progression was wholly dependent on its known function of clustering supernumerary centrosomes. Accordingly, 16 fresh-frozen human tumor samples were immunoblotted along with their paired adjacent normal tissues for HSET. An enhanced expression of HSET was observed in 10 tumor samples compared to their normal adjacent tissues; seven representative normal/tumor sample pairs are shown in
Since higher HSET protein levels could arise either from an upregulation of transcription from the endogenous locus and/or an amplification of the locus encoding HSET, the copy numbers of the locus encoding HSET gene in normal and breast tumor tissues were determined using fluorescence in situ hybridization (FISH) to directly evaluate the HSET copy number per cell in paraffin-embedded breast tumor tissues. Two bacterial artificial chromosome (BAC) probes were hybridized to primary breast tumor tissues, one from the HSET locus on chromosome 6 (RPCI-11 602P21, green) and one from the chromosome 6 centromere (CH514-7B4, red). Amplification of HSET was visualized as an increase in the number of HSET signals relative to the number of control centromere signals. HSET amplification was scored by FISH in four breast tumor tissues; among these, three tumors exhibited HSET amplifications. No amplification of the HSET locus was observed in the normal adjacent tissues in these samples. Various types of copy number changes associated with HSET were observed as shown in
An immunohistochemical staining approach was employed to determine whether HSET overexpression correlates with breast cancer progression and aggressiveness. A total of 60 clinical specimens representing 10 cases each of normal breast, ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive breast carcinoma (low-grade) and invasive breast carcinoma (high-grade) were stained. Consistent with the immunoblotting data, this immunohistochemical analysis showed that HSET is selectively overexpressed in human breast cancers with negligible or absent expression in normal breast epithelia (
Having established a significant correlation between HSET expression and tumor differentiation, it was of interest to investigate a possible association of nuclear HSET WI with progression-free survival (PFS) and overall survival (OS) in breast cancer patients, whereby PFS was calculated as the number of days from diagnosis to the first local recurrence or metastasis if one occurred or the last follow-up if the patient did not progress, and OS was calculated based on the number of days from diagnosis to death or last follow-up if death was not recorded. Nuclear HSET WI was also categorized into high and low groups based on the median. Irrespective of the receptor status (for n=163 patients), those with higher nuclear HSET WI (shown as HSET WI positive in
Since elevated HSET expression exhibits a strong correlation with the development and progression of cancer, it was of interest to determine whether high HSET levels had any impact on the kinetics of cancer cell proliferation in vitro. To this end, HeLa cells stably transfected with HSET-GFP were evaluated to examine and compare the levels of various cell proliferation markers in HeLa-HSET-GFP and HeLa cells. Immunoblot analysis revealed that Ki67 levels were substantially elevated in HeLa-HSET-GFP cells compared with wild-type HeLa cells (
Colony formation assays with HeLa cells transiently transfected with control vector (CV), HSET-GFP plasmid (OE) or HSET-GFP siRNA (KD) were also performed. HSET overexpressing (OE) cells were able to form a significantly greater number of colonies as compared with cells transfected with control vector (CV). Much fewer colonies were observed following transfection with the HSET knockdown plasmid, HSET-GFP siRNA (KD). Similar proliferation effects were confirmed by colony formation assay in another breast cancer cell line, MDA-MB-231, following transient transfection with HSET OE and KD (
Since HSET overexpression enhances cellular proliferation in HeLa cells, changes in the cell cycle kinetics was investigated in cells stably overexpressing HSET (HeLa-HSET-GFP cells) as compared with the parental ones. To this end, HeLa and HeLa-HSET-GFP cells were synchronized using a single thymidine block (19 h) followed by flow cytometric analysis of cell cycle profiles of HeLa-HSET-GFP and HeLa cells upon their release from a G1/S block. DNA content was analyzed with propidium iodide (PI) staining, in which the G2/M population was represented by double the intensity of PI (4N) compared with the G1 cell population (2N). Anti-MPM-2 antibody tagged with Alexa-488 secondary antibody was used to detect a mitosis-specific marker (MPM-2), in order to distinguish between 4N DNA-bearing G2 and M populations. Close interval cell cycle profiling revealed that HeLa-HSET-GFP cells demonstrated faster cell cycle progression kinetics; in other words, the duration of one complete cell cycle was reduced in HSET-transfected cells (10.5 h) as compared with wild-type cells (13 h), with a stark shortening of the G2 and M phases (
In view of the significant contribution of the G1 phase to cell cycle duration, the effect of HSET overexpression (OE) and HSET knockdown (KD) on G1 phase kinetics was investigated. Upon gradual lowering of the serum concentration from 10% to 0% over 24 h and an additional 12 h serum starvation, HeLa cells transiently transfected with control vector (CV), HSET overexpressing plasmid (OE) and HSET knockdown vector (KD) were replenished with serum-containing medium and stained with a cell-clock dye (a redox dye that changes color corresponding to distinct cell cycle phases) in a Cell Clock™ Assay (Biocolor, UK). Yellow cells in the culture represent G1 phase cells, and their color changes to light green in S phase. This allowed for monitoring of the proportion of G1 (yellow-colored) cells from 0 h (50-70% G1enrichment) to 9 h after serum replenishment in all three cases (CV, OE and KD). Negligible differences in the proportion of G1 cells among all three conditions was observed (
Faster kinetic progression of HeLa-HSET-GFP cells (through G2 and M) compared with HeLa cells raises the possibility that G2/M or spindle assembly checkpoint (SAC) functions may be compromised in HeLa-HSET-GFP cells. Mad1 is a critical component of the SAC along with Mad2, and an imbalance in the Mad1/Mad2 protein ratio results in a damaged SAC permitting premature anaphase entry and chromosome instability. Interestingly, HeLa-HSET-GFP cells were found to express markedly higher levels of Mad1 with a distinct nuclear envelope localization compared with parental HeLa cells (
The data from the HeLa-HSET-GFP cells demonstrates that HSET overexpression (OE) can accelerate the kinetics of G2 and M phases (
While the rate of cellular proliferation dictates the number of tumor cells and tumor growth, cell survival and/or apoptosis pathways also have a significant bearing on overall tumor growth. Having found that HSET overexpression (OE) can enhance the kinetics of cell proliferation in tumors, it was of interest to determine whether elevated HSET levels have any impact on the status of pro-survival signaling in HeLa cells. Immunoblots showed enhanced survival signaling as evidenced by notably high survivin and p-Bcl2 levels in HeLa-HSET-GFP cells (
To further explore the physiological role of HSET in cell survival signaling, the ability of MDA-MB-231 cells to resist UV-induced apoptosis was examined. Briefly, MDA-MB-231 cells were transiently transfected with a control vector (CV), HSET overexpression (OE) construct or an HSET knockdown (KD) construct expressing HSET siRNA (˜70% transfection efficiency) 24 h prior to UV irradiation. Following a 10 min exposure to UV-C at 25 J/m2, cells were placed in the incubator for apoptosis induction for 5 h. Lysates were then collected for determining HSET and cleaved caspase-3 (an early marker for apoptosis induction) protein levels, and cell viability was determined using a Trypan Blue assay. Western blot analysis revealed significantly higher cleaved caspase-3 induction in cells with HSET KD, whereas cells with HSET OE showed slightly lower cleaved caspase-3 levels as compared with cells transfected with control vector (CV) (
Given the extensive upregulation of survivin protein expression upon HSET OE and significant reduction upon HSET depletion, it was of interest to determine if HSET occurs in the same protein complex as survivin and whether HSET overexpression has any effect on the APC/C-dependent proteolysis of survivin. Accordingly, co-immunoprecipitation analysis was undertaken to determine if HSET and survivin co-immunoprecipitate with each other. HSET was immunoprecipitated from whole cell lysates of MDA-MB-231 cells carrying (i) a control vector (CV), (ii) an HSET OE plasmid or (iii) an HSET siRNA-bearing construct (KD). Immunoblots of these immunoprecipiates probed for survivin confirmed that the anti-HSET antibody was able to pull down survivin in all the three cases, with an increased survivin pull down in cell lysates overexpressing HSET (
Since the role of survivin in prosurvival signaling is regulated by its degradation via ubiquitination, it was of interest to test the hypothesis that increased HSET binding to survivin protects survivin from ubiquitination and its APC/C-dependent degradation. In MDA-MB-231 cells transiently transfected with control vector (CV), HSET-GFP plasmid (OE) or HSET siRNA plasmid (KD), anti-survivin antibody was used to pull down survivin immunoprecipitates, which were then immunoblotted for survivin and ubiquitin. Intriguingly, reduced polyubiquitin signals in HSET overexpressing cells were observed, even though survivin was extensively overexpressed in those cells (
A key driver of metastasis in people of African descent with TNBC may be a low Npap60L-to-Npap60S ratio owing to which more HSET accumulates in nuclei of African American (AA) TNBCs wherein it activates expression of metastasis-related genes. Table 9 shows differences in Npap60L expression relative to CAS expression in AA TNBC patients compared to European American (EA) TNBC patients, suggesting that the Npap60L-importin-α-KifC1 pathway may be targeted to inhibit metastasis in AA TNBCs. In fact, HSET and Npap60L were co-immunoprecipitated together from breast cancer tissue, indicating that they are both present in the same protein complex in breast cancer cells (data not shown).
Protein regulator of cytokinesis 1 (Prc1), is a non-motor microtubule-associated protein that has been shown by several groups to be a first degree neighbor of HSET in interactomes. Over 90% of TNBCs overexpress Prc1 (˜10.5-fold greater than adjacent normal breast tissue). In addition, Prc1 is included in the MammaPrint 70-gene signature, which predicts distant metastasis in breast cancer. Higher Prc1 is independently associated with worse distant metastasis-free survival across breast cancer patients, a trend that was upheld in TNBC patients. Prc1 was also elevated in MDA-MB-231 TNBC cells that migrated faster in a transwell assay as compared with those that did not. Table 10 shows Prc1 expression in AA TNBC patients and EA TNBC patients. The significantly higher Prc1 expression in AA TNBC patients suggests that Prc1 might be collaborating with KifC1 to drive more aggressive tumor phenotypes in AA TNBCs. It was also found that HSET and Prc1 colocalize extensively in the nucleus in MDA-MB-231 cells, that HSET and Prc1 mostly localize to the nucleus during interphase and that HSET co-immunoprecipitates with Prc1 (data not shown).
The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present application, and is not intended to detail all those obvious modifications and variations of it that will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present application, which is defined by the following claims. The claims are intended to cover the components and steps in any sequence that is effective to meet the objectives there intended, unless the context specifically indicates the contrary. All of the references and patent disclosures cited in the specification are expressly incorporated by reference in their entirety herein.
This application claims the benefit of U.S. Provisional Application Ser. No. 61/912,467, filed Dec. 5, 2013. The entirety of the aforementioned application is incorporated herein by reference.
This invention was made with government support from the National Cancer Institute at the National Institute of Health (NIH-NCI 1RO1CA169127-01). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61912467 | Dec 2013 | US |
