Patent Application
20210231670
The disclosed processes, methods, and compound are directed to diagnosing and treating conditions related to elevated CBX2 expression, especially in high-grade serous ovarian carcinoma cells and tissues.
Epithelial ovarian cancer is the deadliest gynecologic malignancy and annually accounts for over 220,000 deaths worldwide. In the US, over 22,000 new cases of ovarian cancer are diagnosed each year and over 14,000 women succumb to the disease. The majority of these cases are classified as high-grade serous ovarian carcinoma (HGSOC). HGSOC tends to be diagnosed at a late stage, when cancer has already spread beyond the pelvis, and will recur in the majority of cases. Current evidence suggests that HGSOC originates from transformed secretory fallopian tube epithelium (FTE) cells located on the fimbriated end of the fallopian tube. Precursor lesions, defined by TP53 mutations, include serous tubal intraepithelial carcinoma (STIC), which is focal and displays a cytologic appearance similar to HGSOC. Cells within STIC lesions demonstrate anoikis resistance or anchorage-independent cell survival by exfoliation from the fallopian tube-associated extracellular matrix and dissemination to the ovary and/or peritoneum. Ovarian, fallopian, and primary peritoneal carcinomas differ from other epithelial cancers that metastasize to distant sites predominantly via the circulatory or lymphatic systems (e.g., breast, endometrial) by spreading directly to the ovaries and the abdominal cavity independent of the lymphatic or vascular system. As HGSOC cells spread to the abdominal cavity they promote the production of ascites, a collection of intra-peritoneal fluid containing immune cells, tumor cells, and cytokines, along with other cellular and acellular factors. Notably, the prevalence of ascites is directly correlated to disease stage. For instance, 89% of stage III/IV patients present with some degree of ascites. Tumor cells within ascites are hypothesized to be a subpopulation of cells that contribute to disseminated, recurrent, and chemoresistant disease. However, the genetic drivers of HGSOC dissemination and anchorage-independent survival remain unclear.
A significant proportion of “stem”-like cells have been detected in the ascites fluid associated with HGSOC. One group of transcriptional repressors, the polycomb group (PcG) of proteins, are candidates for producing and maintaining this “sternness” as they have been shown to inhibit cellular differentiation and maintain a stem-like transcriptional program. PcG proteins assemble in two main Polycomb repressive complexes, PRC1 and PRC2. PRC1 and 2 epigenetically repress pro-differentiation and tumor suppressor genes, and are important in several cancer types including prostate, breast, and HGSOC. Epigenetic “readers”, known as chromobox (CBX) proteins, play a critical role in PRC1 repressive activity by recognizing methylated histones through their chromobox domain. In 2014, Clermont et al. initially identified an oncogenic role for CBX2 through a genotranscriptomic meta-analysis in human cancers. In breast and prostate cancers, they reported that CBX2 upregulation and amplification significantly correlated with metastatic progression and lower overall survival. CBX2 depletion reduced cell viability and promoted apoptosis in metastatic prostate cancer, suggesting that CBX2 drives key regulators of cell proliferation and metastasis. Gui et al. evaluated the role of 12 PcG proteins in primary and recurrent ovarian cancer and found that immunohistochemistry (IHC) demonstrated significantly higher levels of CBX2 expression in recurrent tumors compared to primary tumors at presentation (primary ovarian tissue at presentation n=100, recurrent disease at relapse n=50, p<0.001). However, the role of CBX2 in HGSOC progression is unknown.
Disclosed herein are methods of assessing chemoresistance of high grade serous ovarian carcinoma (HGSOC) in a patient suffering from the same, the method comprising the analyzing for Chromobox 2 (CBX2) levels in a patient's biological sample, wherein analyzing comprises analyzing at least one HGSOC cell, and wherein, if the CBX2 levels in the patient's at least one HGSOC cell are higher than in a control sample, the patient's HGSOC is resistant to chemotherapy. In some embodiments, the chemotherapy may comprise cisplatin, and the patient with high CBX2 levels may be further counseled not to receive cisplatin as a HGSOC treatment. Also disclosed are methods of treating or preventing HGSOC in a patient, comprising administering to the patient a therapeutically effective of a compound that inhibits and/or downregulates CBX2, for example by a compound comprising an antibody, siRNA, ribozyme, antisense, aptamer, peptidomimetic, small molecule, or any combinations thereof.
Also disclosed are methods of determining a treatment for a patient with, or at risk of developing a cancer, the method comprising obtaining a first sample from the patient comprising one or more cancerous or pre-cancerous cells, obtaining a second sample from the patient that is similar to the first sample but does not comprise cancerous or pre-cancerous cells, processing the first sample and the second sample to analyze at least one biomarker related to CBX2, quantifying the amount of biomarker in the first sample and the second sample, wherein if the amount of CBX2-related biomarker in the first sample is greater than the amount of CBX2-related biomarker in the second sample, the patient is identified as having aggressive or chemoresistant cancer, wherein the first sample is derived from ovarian, uterine, or fallopian tissue. In some cases, the first sample may include one or more HGSOC cells, the biomarker may comprise CBX2 protein or fragment thereof, or at least on nucleic acid, such as an mRNA sequence from the CBX2 gene, and the amount of biomarker may be quantified by immunoblot or mass spectrometry. In some cases, a second biomarker may also be obtained from each sample, wherein the second biomarker is not CBX2. In many embodiments, chemoresistance may be to a platinum-based chemotherapeutic agent, such as cisplantin, carboplatin, or oxaplatin.
Also disclosed herein are methods of determining whether a tumor is likely to metastasize. The method comprising steps of measuring expression levels of at least one CBX2-associated biomarker from a sample of the tumor tissue, measuring expression levels of at least one CBX2-associated biomarker from a sample of non-tumor tissue, wherein if the expression level of the at least one CBX2-associated biomarker from the tumor sample is greater than the expression level of the at least one CBX2-associated biomarker from the non-tumor sample, the tumor is determined to be likely to metastasize. In many cases, the first sample is derived from tissue of Müllerian origin, and the first and second samples may be derived from ovarian or fallopian tissue. In some cases, the first sample may include one or more HGSOC cells, the biomarker may comprise CBX2 protein or fragment thereof, or at least on nucleic acid, such as an mRNA sequence from the CBX2 gene, and the amount of biomarker may be quantified by immunoblot or mass spectrometry. In some cases, a second biomarker may also be obtained from each sample, wherein the second biomarker is not CBX2.
Kits for diagnosing a chemoresistant cancer in a patient are also disclosed herein. IN many embodiments the kits comprise: a quantitation reagent comprising one or more detectors specific for at least one CBX2-associated biomarker from at least one biological sample; a detection reagent; instructions for using the kit to diagnose a patient as having ovarian cancer when the expression levels of the CBX2-associated biomarker in the biological sample from the patient is higher than the expression level of the same biomarkers in a control subject or control biological sample. In many cases, the first sample is derived from tissue of Müllerian origin, and the first and second samples may be derived from ovarian or fallopian tissue. In some cases, the first sample may include one or more HGSOC cells, the biomarker may comprise CBX2 protein or fragment thereof, or at least on nucleic acid, such as an mRNA sequence from the CBX2 gene, and the amount of biomarker may be quantified by immunoblot or mass spectrometry. In some cases, a second biomarker may also be obtained from each sample, wherein the second biomarker is not CBX2.
Methods of inhibiting or reducing proliferation in a cancer cell are also disclosed herein. The methods may comprise steps of: contacting the cancer cell with a compound or molecule that inhibits CBX2 expression; allowing the compound to reduce the amount of CBX2 protein in the cell; and thereby reducing or inhibiting proliferation of the cell compared to a control cell that is not contacted with the compound. In many cases, these methods may be practiced on cells in-vitro, or in-vivo, and the cells may be mammalian cells, such as human cells. In most embodiments, the compound may be a nucleic acid, such as a short hairpin ribonucleic acid, or the compound may comprise two or more amino acids. The methods may further comprise as a step of contacting the cell with one or more chemotherapeutic agents which may be performed before the reducing step.
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Disclosed herein are studies showing that CBX2 is overexpressed in primary HGSOC tumors and that CBX2 protein is upregulated in HGSOC cells grown in an anchorage-independent fashion (forced suspension). In a primary human HGSOC tumor microarray high CBX2 expression was observed in a majority of specimens. Also shown is that the loss of CBX2 inhibits proliferation, reduces stemness, and increases cisplatin sensitivity. Also disclosed are methods of diagnosing advanced cases of HGSOC, HGSOC that may be resistant to one or more anti-cancer therapies, for example platinum-based chemotherapy, such as cisplatin therapy. Also disclosed are compounds and methods for treating a subject having advanced or chemoresistant HGSOC, wherein the compounds and methods reduce CBX2 expression in HGSOC cells and/or reduce dissemination of primary tumors.
High grade serous ovarian carcinoma (HGSOC) is often diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in other cancers, but little is known about its role in HGSOC. Applicants hypothesized that CBX2 upregulation promotes HGSOC via induction of a stem-like transcriptional profile and inhibition of anoikis. Examination of Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) established that increased CBX2 expression conveyed chemoresistance and worse disease-free and overall survival. In primary HGSOC tumors, Applicants observed CBX2 expression was significantly elevated compared to benign counterparts. In HGSOC cell lines, forced suspension promoted CBX2 expression. Subsequently, CBX2 knockdown inhibited anchorage-independent proliferation and potentiated anoikis-dependent apoptosis. Furthermore, CBX2 knockdown re-sensitized cells to platinum-based chemotherapy. Forced suspension promoted increased ALDH activity and ALDH3A1 expression and CBX2 knockdown led to a decrease in both ALDH activity and ALDH3A1 expression. Investigation of CBX2 expression on a HGSOC tissue microarray revealed CBX2 expression was apparent in both primary and metastatic tissues. CBX2 is an important regulator of stem-ness, anoikis escape, HGSOC dissemination, and chemoresistance and potentially serves as a novel therapeutic target.
Applicants have discovered that CBX2 is upregulated in HGSOC, high CBX2 expression portends poorer survival, and increased CBX2 expression correlates with platinum resistance. Applicants have also discovered that CBX2 is overexpressed in HGSOC primary tumors, as well as in cell lines that have escaped anoikis in suspension culture. Here, Applicants demonstrate that the loss of CBX2 is associated with decreased proliferation of HGSOC cells in multiple culture conditions, an increase in chemotherapy sensitivity, and a reduction in stem-like cells. Lastly, utilizing a HGSOC tissue microarray of advanced stage primary patient tumors Applicants found CBX2 protein expression was expressed in a majority of tumors.
CBX2 was found to be highly expressed in primary HGSOC tumors from seven patients. Clinically, three of these patients had more extensive peritoneal disease (Table 2) suggesting that CBX2 could serve as a predictive marker of advanced disease, reinforcing the potential clinical significance of CBX2. Moreover, examination of five patients with matched primary tumor, ascites-associated tumor cells, and distant metastasis revealed three of the five patients had an increase in CBX2 expression in distant metastasis/ascites-associated tumor cells compared to primary tumors. This highlights that CBX2 is potentially important in driving HGSOC progression, however there are indeed other contributing factors. Notably we observed that high expression of CBX2 correlated to a loss of an active tumor suppressor, FOXO3. This suggests that the downregulation of FOXO3 independent of CBX2 could drive tumor progression. As we have attempted to elucidate the mechanism behind CBX2-dependent activity we see common themes of increased proliferation and stem-like differentiation, which appeared to lead to a more aggressive and chemoresistant phenotype. The loss of CBX2 led to the loss of stemness measured through ALDH activity, ALDH3A1 expression, and SOX4 expression suggesting that reduced stemness is a major driver of these phenotypes. In addition, we linked the anoikis-induced autophagy, EMT, and apoptosis response to CBX2 expression. Further investigations will functionally evaluate the relationship between CBX2 and these key survival processes.
CBX2 is a subunit of the polycomb repressor complex (PRC1), which has been shown to play a role in ovarian cancer. The enzymatic subunit or “writer” of PRC1, BMI-1, is considered to play a role in malignant transformation of multiple cancers, including ovarian cancer. In ovarian cancer, BMI-1 has been demonstrated to be associated with stem-ness and tumor initiation and serves as an independent predictor of poor outcome. Moreover, silencing of BMI-1 can lead to improved sensitivity to chemotherapy. This understanding of BMI-1 directly correlates and aligns with our CBX2 findings. Taken together with observations in other types of cancer, it seems likely that the PRC1 and specifically, CBX2, are novel therapeutic targets not only for HGSOC, but potentially for breast and prostate cancers.
CBX2 is considered to be an epigenetic “reader”. The existing literature suggests that targeting epigenetic “readers” is an effective strategy for targeted therapy. Bromodomain (acetyl-histone “reader”) inhibitors have the potential to suppress ALDH activity in ovarian cancer, providing evidence that targeting of an epigenetic reader may be able to alter the stem-like phenotype of a cell. One key example is JQ-1, a potent and selective inhibitor of the bromodomain and extra-terminal domain (BET) family of proteins, including BRD2, BRD3, and BRD4. Preliminary clinical data suggests that BET inhibitors may have therapeutic potential in human cancers. Furthermore, a Phase I clinical trial of an oral BET inhibitor demonstrated that this small molecule inhibitor was well tolerated in vivo, a critical development which paves the way for future targeting of epigenetic readers. This highlights that a “reader” or chromobox domain inhibitor could prove to be more effective with fewer adverse effects compared to inhibitors of epigenetic “writer” enzymes.
Disclosed herein, Applicants describe the role of CBX2 in promoting HGSOC disease progression. Mechanistically, CBX2 protects HGSOC against apoptosis and promotes a more stem-like phenotype. CBX2 is an epigenetic reader and is therefore targetable with a small molecule inhibitor. This work expands our understanding of the progression of HGSOC and identifies a novel therapeutic target.
Increased CBX2 protein in HGSOC (compared to fallopian tube epithelium, and other histotypes) is associated with poorer prognosis for the patient, repression of the FOXO3 tumor suppressor, and chemoresistance of HGSOC tumors and cells. CBX2 expression may be determined by various methods. In some embodiments, CBX2 protein expression may be assessed by densitometry of western immunoblot study. In many embodiments, increased expression may refer to CBX2 intensity that is greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, or 900%, and less than about 1000%, 900%, 800%, 700%, 600%, 500%, 400%, 300%, 200%, 160%, 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% compared to CBX2 in non-HGSOC cells. In other embodiments, relative CBX2 protein expression may be determined by other methods for quantitation of protein expression that are well-known to those of skill in the art. In many embodiments, CBX2 protein may be identified by an antibody specific to CBX2, for example anti-CBX2 antibody from Thermo Fisher Scientific (Cat # PA5-30996). Relative quantitation of CBX2 may be adjusted or standardized by comparing CBX2 amounts to various control proteins, for example actin. CBX2 expression may be quantified by various methods. In some embodiments, CBX2 protein is analyzed and/or quantified by flow cytometry or mass spectrometry.
Reducing CBX2 expression, for example by various knockdown or repression methods, significantly decreases viability of HGSOC cells. In many embodiments, repression or knockdown leads to decreased growth rate and/or anoikis (anchorage-independent cell death) in HGSOC cells.
Suppression of CBX2 expression can promote cell death in HGSOC cells and/or reduce their growth rate. Suppression may be accomplished by various methods. In some embodiments, CBX2 mRNA transcripts may be targeted by one or more compounds including, but not limited to, siRNA, ribozyme, antisense, aptamer, or other small molecule. In some embodiments, CBX2 repression or knockdown may be accomplished by targeting CBX2 protein with one or more of, but not limited to, an antibody, peptide, peptidomimetic, small molecule, or other compound. In one embodiment, CBX2 transcripts may be targeted by one or more short hairpin RNA (shRNA) molecules, or other types of RNAi well-known to those of skill in the art. In some embodiments, the shRNA molecules may comprise a sequence selected from GCCAAGGAAGCTCACTGCCAT (shCBX2 #1; SEQ ID NO:19) or ACGGAAAGGAACAGGAAGCAT (shCBX2 #2; SEQ ID NO:20). In many embodiments, HGSOC cells may be induced to reduce their growth rate or enter anoikis by reducing the amount of CBX2 transcripts by greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, and less than about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% compared to HGSOC cells that have not been treated to reduce their CBX2 expression.
Patients having HGSOC cells with enhanced expression of CBX2 indicates that chemotherapy may be less effective. In some embodiments, the chemotherapy is a platinum-based compound, for example selected from one or more of cisplatin, carboplatin, and oxaplatin. In some embodiments, patients whose HGSOC tumor cells express high levels of CBX2 require an increased dosage of chemotherapeutic drug of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or 400%, and less than about 500%, 400%, 300%, 200%, 1500%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%, compared to patients with tumors having lower CBX2 expression.
Repression of CBX2 expression can increase sensitivity of HGSOC cells to chemotherapy. In some embodiments, various therapies may be used to reduce expression of CBX2 in HGSOC cells and tumors. This may allow the use of various chemotherapeutic compounds to treat a patient positive for HGSOC and/or may allow the use of lower doses of chemotherapy to achieve a beneficial therapeutic effect, such as a reduction in tumor growth, lower cell growth, an increase in cancer cell death, or reduction in adverse side effects such as fatigue, hair loss, bruising, bleeding; infection; low red blood cell counts; nausea, etc.
CBX2 expression correlates to mRNA expression of genes associated with apoptosis, autophagy, and epithelial to mesenchymal transition (EMT). In many embodiments, the genes may include one or more of MYLK, NOG, and TNFSF10
Increased CBX expression results in driving cells toward a stem-like phenotype. In many embodiments, a stem-like phenotype may be evaluated by measuring aldehyde dehydrogenase (ALDH), where stemness is associated with increased ALDH activity. In many embodiments, CBX2 expression correlates with ALDH3A1 expression, and knockdown of CBX2 expression decreases ALDH3A1 expression, as well as the stem cell-associated transcription factor, SOX4.
Biomarker may refer to any measurable indicator of the state of a cell, tissue, organ, or subject. In some embodiments, the biomarker is a molecule associated with a cell, for example a protein, peptide, or mRNA transcript. In some embodiments, the protein may be full-length or truncated protein, or a peptide fragment of a full-length protein. In some embodiments, a biomarker may be a nucleic acid, such as an mRNA transcript. In many embodiments, the biomarker may be an mRNA or protein associated with a given gene, for example CBX2, SOX4 MYLK, NOG, and TNFSF10. In most embodiments, the biomarker is a mammalian biomarker, for example human biomarker.
Subject includes any mammal, including mice, rats, guinea pigs, rabbits, dogs, cats, cows, horses, monkeys, and humans. A patient is a subject undergoing treatment or observation for a condition or disease, such as cancer. Cancer includes conditions or diseases of mammals characterized by uncontrolled cellular growth, hyperproliferative growth, hyperplasic growth, neoplastic growth, cancerous growth or oncogenic processes. Cancer may also refer to tumors, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
Chemotherapeutic agents include synthetic, natural, semisynthetic compounds and molecules useful in treating, killing, suppressing, or controlling cells that are displaying uncontrolled growth. Chemotherapeutic agents may reduce the proliferation of such cells and/or induce their death (programmed or non-programmed cell death). Chemotherapy refers to administration of such agents to a patient in need thereof. Chemoresistance may refer to the ability of a cell to avoid death or a decrease in proliferation in the presence of a chemotherapeutic agent.
Biological sample may refer to any sample removed, extracted, or derived from a patient or subject. In many embodiments a biological sample includes tissues, cells, protein, nucleic acids, etc. A biological sample may be processed for viewing or analysis, such as protein or nucleic acid quantitation. A control sample or reference sample may be a sample obtained from a similar subject, tissue, or cell that does not have, or is not expected to have, a disease or condition that is being assayed. In some embodiments, a reference sample may include samples from two or more subjects.
Therapeutically effective, as used herein refers to a therapeutic treatment, for example administration of a chemotherapeutic agent, that is adequate to accomplish a desired, expected, or intended result. When used in the context of treating a patient or subject, it may refer to an amount of agent which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease or condition, and/or ameliorates at least one consequence of the disease or condition.
Inhibition or repression, as used herein means to lessen by some amount. For example, inhibition of gene expression by a compound or agent may cause the amount of protein or transcript in a given cell to be reduced relative to a cell that has not been contacted with the compound or agent.
Antibody may refer to any natural, synthetic, human, non-human, or humanized protein that may bind to an epitope on a target antigen. Antibodies may be mono or bi-specific. Antibodies may be multi or single chain proteins. In some embodiments the antibodies may include one or more Fc domains, Fv domains and/or CDRs. Antibodies may refer to peptides or peptide mimetics that bind to a target protein.
RNA interference (RNAi) includes any method of targeting expression of one or more genes by degrading mRNA transcribed from the gene. RNAi include siRNA, miRNA, shRNA, ribozyme, aptamers and the like, which typically contain some amount of sequence complementary to the gene transcript. RNAi techniques and methods are well known to those of skill in the art.
Peptide, polypeptide, and protein fragment may refer to natural or synthesized compounds and molecules containing natural or synthetic amino acids, amino acid equivalents and/or other non-amino groups. Peptides may be modified by replacement of one or more amino acids with related compounds, and/or modified by removing or adding elements, compounds, or molecules to one or more side chains or functional groups. The peptides can be linear or cyclic. Peptidemimetic refers to a compound or molecule that mimics a peptide in the peptide's ability to assume a three-dimensional structure on its own, or as a result of binding or contacting an epitope.
Metastasize may refer to the ability of a cancer cell to travel from the primary tumor, proliferate, and establish a second tumor. In some embodiments, a metastatic cell may be able to proliferate in suspension without attaching or adhering to a substrate, for example connective or other solid non-cancerous tissue.
We examined CBX2 expression in HGSOC in several publicly available datasets (Gene Expression Omnibus; GEO Dataset and The Cancer Genome Atlas; TCGA). High expression of CBX2 in TCGA HGSOC samples conveyed both a significantly worse disease-free survival (DFS; 11.7 vs. 17.6 months, Log-rank test p-value 0.00316) and overall survival (OS; 34 vs. 44.8 months, Log-rank test p-value 0.00116) (
Ovarian surface and FTE are proposed to be the precursor cells for HGSOC; more recent data strongly support FTE as the predominant site of origin. Comparing CBX2 expression in ovarian surface epithelium or FTE to CBX2 expression in HGSOC, we observed that CBX2 was significantly higher in HGSOC (
HGSOC is unique compared to other solid types in its tendency to directly seed and disseminate throughout the peritoneal cavity, which requires an escape from anoikis, an anchorage-independent cell death. To determine whether CBX2 plays a role in HGSOC tumor cell's ability to survive without anchorage, or in a suspended setting, we examined the role of CBX2 on HGSOC growth in suspension. A forced suspension setting was achieved by plating cells on polyHEMA-coated tissue culture dishes (
The high grade serous ovarian cancer cell lines OVCAR4, PEO1, and OVCAR8 were grown in adherent and suspended settings (
The panels of
To further elucidate the role of CBX2 in HGSOC, we evaluated the impact of CBX2 modulation in PEO1, OVCAR4, and OVCAR8 HGSOC cell lines. One of the two independent small hairpin RNAs (shRNA) specific for CBX2 or a control (shControl) were transduced into OVCAR4, PEO1, and OVCAR8 cells. CBX2 knockdown was confirmed via quantitative PCR (qPCR) and immunoblots, with approximately 60% knockdown in the presence of shCBX2#1 and 30% knockdown in the presence of shCBX2#2 (
In order to correlate the CBX2 in vitro findings to clinically relevant specimens, we utilized a tissue microarray (TMA) of HGSOC that recapitulated tumor progression. The TMA contained 24 primary tumors, with matched lymph node or distant metastases (Table 3). IHC was optimized and performed using a previously published CBX2 antibody, predicted to preferentially stain the nucleus (
In parallel the TMA was stained with PAX8, a marker for Müllerian origin (
A majority of HGSOC patients treated with platinum-based (i.e., carboplatin) chemotherapy develop chemoresistance. A comparison of carboplatin sensitive HGSOC tumors to platinum resistant tumors demonstrates an increase in CBX2 in platinum resistant tumors (GSE1926) (
For example, CBX2 knockdown OVCAR4 cells had an 1050 of 12.68 μM in shCBX2#1 and 15.37 μM in shCBX2#2 compared to 38.67 μM for the control with intact CBX2 (
Panels of
The panels in
Chemoresistance and dissemination of ovarian cancer cells are associated with changes in apoptosis, epithelial to mesenchymal transition (EMT), and autophagy. The PcG is an epigenetic complex that is responsible for transcriptional reprogramming through histone modification thus CBX2 could regulate a variety of genes. Utilizing HGSOC TOGA data we generated a list of potential CBX2 target genes through examination of mRNA correlations (Spearman r>0.15, 5838 genes). Utilizing published gene sets for EMT, autophagy, stemness, and apoptosis we cross-referenced the CBX2-associated genes (Table 4). CBX2-associated genes accounted for 18.8-28.4% of genes in the respective pathways (
The panels of
Several reports have demonstrated cells that survive in anchorage-independent conditions possess stem-like characteristics. We hypothesize that the polycomb repressor complex could be involved by inhibiting cellular differentiation and thus maintaining stemness. Applicants examined the CBX2-associated genes from TOGA against a published stemness gene set. We observed 25.2% of stemness-related genes overlapped with CBX2 genes. We next evaluated stemness by measuring aldehyde dehydrogenase (ALDH) activity both in the setting of suspension growth and CBX2 knockdown. We first determined whether placing OVCAR4 and OVCAR8 cells in suspension increased ALDH activity. We observed that in OVCAR4 cells ALDH activity was significantly increased in cells grown in suspension for 7 days (
The panels of
Next, we evaluated whether CBX2 knockdown impacted suspension-induced ALDH activity, we found that culturing OVCAR4 and OVCAR8 shCBX2 cells in suspension significantly inhibited ALDH activity compared to shControl cells (
The panels of
OVCAR4, PEO1, and OVCAR8 human high grade serous ovarian cancer cell lines were authenticated using small tandem repeat (STR) analysis (The University of Arizona Genetics Core) and routinely tested for mycoplasma with MycoLookOut (Sigma, St. Louis, Mo.). OVCAR8 and OVCAR4 cells were obtained from the Gynecologic Tumor and Fluid Bank (University of Colorado, Aurora, Colo.). PEO1 purchased from American Type Culture Collection. Cells were cultured in RPMI-1640 medium supplemented with 1% penicillin—streptomycin and 10% fetal bovine serum. The cell lines were maintained in 5% CO2 at 37° C.
Gene Expression Omnibus (GEO), hosted by the National Center for Biotechnology Information (NCBI), was queried for relevant databases. GSE18521, GSE10971, GSE1926, and GSE73064 were examined for relative CBX2 expression. The Cancer Genome Atlas (TCGA) Ovarian Serous Cystadenocarcinoma (Nature 2011) database was accessed via the cBIOPortal (www.cbioportal.org/) to evaluate the role of CBX2 in HGSOC disease-free and overall survival. Note: patient numbers for overall survival (n=485) and disease-free survival (n=396) differ due to the availability of data with TCGA. The database was queried for HGSOC identifying a total of 557 tumors with mRNA expression data and CBX2 upregulation was defined as CBX2 mRNA expression >1.5 standard deviation. RPPA data was assessed from the HGSOC Provisional dataset via the cBIOPortal.
The adherent environment was created using standard tissue culture dishes. For suspension, tissue culture dishes were covered with 6 mg/ml poly-2-hydroxyethyl methacrylate (Poly-HEMA, Sigma) in 95% ethanol. The plates were incubated under sterile conditions to allow ethanol evaporation, followed by 30 min of ultraviolet light for sterilization. OVCAR4, PEO1, and OVCAR8 cells were cultured in each of these environments for 7 days.
PEO1 and OVCAR4 cells were plated in 96-well plates in both adherent (4000 cells per well) and suspension (6000 cells per well) environments, as described above. These cells were treated over 24 h with increasing concentrations of Cisplatin (0.5-100 μM). 1640 RPMI media and media with 0.9% NaCl were used for control and vehicle control, respectively. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay (Promega, Madison, Wis.). Means of at least five wells are reported and experiments were independently repeated in triplicate. Representative dose-response curves are shown.
RNA was isolated using RNAeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. NanoDrop spectrophotometry was performed to confirm the concentration of extracted RNA. RT-qPCR was performed using the Luna Universal One-step RT-qPCR kit (New England BioLabs, Ipswich, Mass.) on a BioRad CFX96 or Applied Biosystems QuantStudio 6 Flex thermocycler using primers for specific target transcripts; 18s rRNA was examined as a housekeeping gene (Table 6).
6. shRNA Knockdown
CBX2-specific shRNA were obtained from the University of Colorado Functional Genomics Facility (CBX2 #1: TRCN 0000020327 and CBX2 #2: TRCN 0000020328). An empty pLKO.1-puro was utilized as shControl (shCtrl). Plasmid isolation was performed using Plasmid Midi-Prep Kit (Qiagen). Twenty-four hours after seeding, cells were transfected with a total of 12 μg of DNA, including lentiviral packaging plasmids and the shRNA, in addition to 36 μg of polyethyenimine (PEI), for a 1:3 ratio of DNA to PEI. Cells were incubated overnight and transitioned to Dulbecco's Modified Eagle Media (DMEM) the following morning. Forty-eight hours after medium change, lentivirus was harvested. PEO1, OVCAR4, and OVCAR8 cells were seeded into six-well plates. When cells reached 80% confluence, they were transduced with lentivirus encoding CBX2-specific shRNAs or an shRNA control. A control well was maintained without virus to confirm puromycin selection. A 48-h puromycin selection was performed immediately following transduction. After medium change, cells were allowed to recover and then subjected to functional assays.
Cell lysis was performed using radioimmunoprecipitation assay (RIPA) buffer (150 mM sodium chloride, Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS [sodium dodecyl sulfate], 50 mM Tris, pH 8.0) supplemented with complete EDTA-free protease inhibitor cocktail (Roche), as well as NaF and NaV. Protein was quantified using bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, Mass.) and spectrophotometry. An 8% SDS polyacrylamide gel resolving gel was created with a 4% stacking gel. Twenty to thirty micrograms of total protein were loaded per well. Proteins were transferred to PVDF membrane using a Bio-Rad TransBlot Turbo. Western Blot analysis was performed using a rabbit primary antibody specific to CBX2 (Thermo Fisher Scientific, Cat # PA5-30996, 1:1000) and a mouse primary antibody against actin (Abcam, Cat # ab6276). CBX2 previously validated in Clermont, P. L. et al. Genotranscriptomic meta-analysis of the Polycomb gene CBX2 in human cancers: initial evidence of an oncogenic role. Br. J. Cancer 111, 1663-1672 (2014). Primary antibody incubation was performed overnight at 4° C. Secondary goat anti-rabbit green (LI-COR Biosciences, Lincoln, Nebr., Cat #926-32211) and goat anti-mouse red (LI-COR, Cat #926-68070) antibodies were applied the following morning for 1 h at room temperature. Bands were visualized using the LI-COR Odyssey Imaging System.
For the Gaussia luciferase (gLuc) assay, the BioLux Gaussia Luciferase Assay kit (New England BioLabs) was utilized. OVCAR4 and PEO1 cells were grown in a 96-well plate, starting with 2000 cells per well. Media were collected every 24 h and stored in at −20°. For the assay and luminometer readings, the media were thawed and placed in a new 96-well plate. The assay was performed following the manufacturer's protocol and the relative light units were obtained using luminometry (GloMax) and charted with Prism software. Colony formation assays were performed in parallel using crystal violet staining. Briefly, cells were fixed (10% methanol/10% acetic acid/PBS), stained with crystal violet (0.4%) and washed with de-ionized water. Crystal violet was dissolved and absorbance was measured using a spectrophotometer (SpectraMaxM2e, Molecular Devices, San Jose, Calif.) at 590 nm and SoftMaxPro software. For the spheroid assay, 4000 cells were plated from a single cell suspension onto growth factor reduced Matrigel (Corning, Corning, N.Y.) and allowed to incubate for 12 days. Microscopic images were obtained and the diameter of each spheroid was measured in ImageJ (NIH). At least 50 spheroids were measured for each cell type and the diameters were averaged and graphed using Prism software.
Stemness was evaluated using the Aldefluor Kit (Cat #01700, Stemcell Technologies, Vancouver, Canada). OVCAR4 and OVCAR8 cells grown in adherent and suspension settings for 7 days were collected and prepared following manufacturer's protocol. An exception to the manufacturer's protocol was the reduction of Aldefluor reagent by 50%, using 2.5 μl per 1 ml of cell suspension. Flow cytometry was performed on a Gallios 561 Cytometer (BD Biosciences) with analysis at 488 nm
The University of Colorado has an Institutional Review Board approved protocol (COMIRB #07-935) in place to collect tissue from gynecologic patients with both malignant and benign disease processes. All participants are counseled regarding the potential uses of their tissue and sign a consent form approved by the Colorado Multiple Institutional Review Board. The tissues are processed, aliquoted, and stored at −80° C.
Patient-derived xenograft tissue samples of HGSOC were fixed in formalin and embedded in paraffin. The University of Colorado Cancer Center Histology Core performed serial sectioning of the tissue at 5 micron thickness. For histopathologic examination, sections were de-paraffinized using xylene and hydrated in graded alcohol solutions. Antigen retrieval was performed using citrate buffer (pH 6.0) and boiling in pressurized steamer to 110° C. for 30 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol for 20 min, followed by washing in TBS. A hydrophobic barrier was drawn around each section and tissues were blocked in 1% BSA in TBS for 30 min. TMA slides were single stained. Rabbit anti-CBX2 (Thermo Scientific, Cat # PA5-30996) was diluted to 1:50 in 1% BSA in TBS, applied to all sections, and incubated overnight at 4° C. Rabbit anti-PAX8 (Proteintech, Cat #10336-1-AP) was diluted to 1:200 in 1% BSA in TBS, applied to all sections, and incubated overnight at 4° C. An isotype control (Rabbit IgG) was incubated in parallel. The secondary antibody, anti-rabbit Dako Envision+System HRP Labeled Polymer (Dako Ref#K4003) was applied to the sections and allowed to incubate for 60 min at room temperature. Slides were subsequently washed with TBS and developed under the microscope using Liquid 3,3′-diaminobenzidine tetrahydrochloride (DAB)+Substrate Chromagen System (Agilent, Santa Clara, Calif.; Ref#K3468). Slides were counterstained with hematoxylin
A previously constructed TMA comprised of matched primary, lymph node, and peritoneal metastases samples in duplicate from 24 patients with high grade serous carcinoma treated at the University of Colorado (COMIRB #14-0427), was stained with the CBX2 and PAX8 antibodies. With the aid of the University of Colorado Histology Core, the stained slides were scanned using Aperio imaging technology and annotated to highlight tumor based on PAX8 staining using ImageScope software. The TMA was then analyzed and scored by the University of Colorado Histology Core. Subsequently, two board-certified pathologists (M.D.P. and A.A.B.) manually reviewed and scored the CBX2 stain. Each sample was given a score for intensity (0, 1+, 2+, 3+) and percentage of cells staining (continuous variable). Only distinct nuclear staining of tumor cells was considered positive. From this data, H-scores were generated.
Following 72 h of growth in adherent and suspended states, OVCAR4 and PEO1 cells were harvested and washed in PBS. Alexa 488 Conjugated AnnexinV and Propidium Iodide (PI) (Thermo Fisher Scientific) staining were performed following manufacturer's protocol.
Prism Graph Pad Prism software (v7) was utilized to generate graphs. Statistical tests include unpaired two-sided t-tests (comparing two groups), Log-rank (survival) or ANOVA (comparing greater than two groups) unless noted. A significance threshold was set at p<0.05, which was used for sample size determination. All experiments were performed in technical triplicates and biological triplicates unless noted. FloJo software (BD Biosciences, San Jose, Calif.) was used for analyzing flow cytometry data.
While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the detailed description is to be regarded as illustrative in nature and not restrictive.
All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each was included at its citation, in its entirety. In case of conflict between reference and specification, the present specification, including definitions, will control.
Although the present disclosure has been described with a certain degree of particularity, it is understood the disclosure has been made by way of example, and changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims.
This application claims benefit of priority pursuant to 35 U.S.C. § 119(e) of U.S. provisional patent application No. 62/685,107 entitled “Methods of Evaluating Treatment Outcome in High Grade Serous Ovarian Cancer,” filed on 14 Jun. 2018, which is hereby incorporated by reference in its entirety.
This invention was made with government support under grant number CA194318 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/037289 | 6/14/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62685107 | Jun 2018 | US |
