The present invention relates to diagnosis and anticancer therapy in patients bearing tumors overexpressing Trop-2, by means of drugs directed against components of the signalling network of Trop-2, including but not limited to drugs which inhibit CD9, Akt and molecules of the tetraspanin signalling network. Trop-2 overexpression can also be exploited for the screening in vitro and in vivo of the anticancer activity of new compounds.
The latest generation of targeted anti-cancer therapies make use of tyrosine-kinase inhibitors (TKIs) and/or monoclonal antibodies (mAbs), having dominant oncogenic kinases and/or growth factors receptors as their targets. However, the clinical benefit associated with these agents is generally restricted to subsets of treated patients, in many cases defined by specific molecular changes (mutations, amplifications/deletions, increased/decreased expression) in the tumor. This finding has highlighted the possible importance of tumor genotypes/phenotypes as determinants of sensitivity to specific treatments and the resulting stratification of patients before treatment on the basis of molecular alterations used as biomarkers. These biomarkers are studied in order to be used as “predictive markers”, ie able to predict the response or the resistance to a targeted therapy, with the aim of optimizing the clinical results through an effective personalization of treatment.
Predictive biomarkers are currently used in the clinic as a guide for the choice of very few targeted treatments. Examples include receptors for estrogen (ER) and progesterone, which are used to select patients with breast cancer to be treated with endocrine therapy; amplification of HER-2 for treatment with Trastuzumab; BCR-ABL translocation in chronic myeloid leukemia and mutations of c-kit in gastrointestinal stromal tumors for treatment with Imatinib; EGFR mutations in lung cancer for treatment with Gefitinib/Erlotinib; KRAS mutations in colo-rectal cancer for treatment with Cetuximab.
“Enrichment biomarkers”, i.e. markers that can predict which subgroups of patients may respond to treatment are a vital necessity. In fact they can be used to focus the experimentation towards subgroups in which it could be useful, increasing the likelihood of success and the effectiveness of the trials of new drugs. The combination HER2/Herceptest/Trastuzumab provides an example of successful use of diagnostic tools for targeted therapy. Other cases, although not many, of predictive and enrichment markers support the usefulness of this strategy.
However, in most cases, the currently available biological markers refer to either the therapeutic target itself or an individual component of the main signalling pathway through which the target is believed to act. The common limitation to these strategies is to consider the alteration of a single signalling pathway as the only alteration responsible for the growth of a tumor. At the basis of tumorigenesis, instead, there is a process of multi-stage progression. Multiple lines of evidence, including data obtained from the sequencing of entire exomes and microarray analyses of gene expression profiles of tumor reveal an extremely large number of different mutations and a great complexity of interactions between different signalling pathways in tumors. These investigations classify tumors into differentiated, clinically-relevant subgroups and highlight the importance of hitting multiple signaling pathways or final common pathways in order to obtain an effective treatment.
Genetic alterations which are classically involved in tumorigenesis concern oncogenes, such as PI3K, Ras, BCR-ABL, EGFR, HER-2 (“oncogene addiction”), and are necessary both for triggering and for maintaining the oncogenic condition, and are therefore a logical target for therapy. However, the tumorigenic state also depends on a wide variety of genes and pathways involved in normal cell functions, which present an altered regulation, although they are not intrinsically oncogenic (“non-oncogene addiction” [. This addiction can provide a large number of drug targets with differential toxicity between normal and tumor cells: among them, some are being studied on pre-clinical models. Only some of these targets are being tested in clinical trials or are already in use for therapy (VEGF, for example, for treatment with Bevacizumab or Sorafenib/Sunitinib.
Therefore, in order to improve the effectiveness of existing therapies and to identify new therapeutic targets, the analysis of classical oncogenes must be accompanied the study of normal signalling pathways. These “normal” markers, however, while emerging as important factors in supporting tumorigenesis are still little used for the prediction of response to therapy in man.
Trop-2 (AC: P09758) is a transmembrane glycoprotein able to transduce a cytoplasmic calcium increase signal [Ripani, 1998 #648], is involved in cell-cell adhesion in epithelial tissues, and drives signalling networks that induce tumor growth (Guerra, Trerotola et al. 2013). Trop-2 has structural characteristics which are different from those of the four classical families of adhesion molecules, i.e. integrins, cadherins, selectins, and Ig-CAM. The extracellular domain of Trop-2 contains a cysteine-rich globular portion with an EGF-like domain (GA733 type-I domain (Chong and Speicher 2001)) and a thyroglobulin domain (Linnenbach, Seng et al. 1993; Fornaro, Dell'Arciprete et al. 1995; El Sewedy, Fornaro et al. 1998), which are necessary for the formation of homo-multimers (Balzar, Briaire-de Bruijn et al. 2001). A region without cysteines (“stem”) connects the globular portion of Trop-2 to the hydrophobic transmembrane helix. The intracellular portion of Trop-2 consists of a tail of 26 aa devoid of enzymatic activity, containing a phosphoinositide-binding HIKE (El Sewedy, Fornaro et al. 1998; Ciccarelli, Acciarito et al. 2000). HIKE is found in signal-transducer molecules and can act as a docking site for effector/regulatory molecules/such as G-proteins, PKC, calmodulin (Alberti 1999). HIKE also includes a PKC phosphorylation site (S303) (Basu, Goldenberg et al. 1995).
Trop-2 expression has been associated with tumor growth in experimental models (Klein, Hartmann et al. 1990; Basu, Goldenberg et al. 1995; Wang, Day et al. 2008; Cubas, Zhang et al. 2010; Goldstein, Stoyanova et al. 2010). In human tumors, overexpression of Trop-2 is a negative prognostic factor in tumors of the pancreas, stomach, oral cavity, ovary, lung and colo-rectal cancer (Trerotola, Cantanelli et al. 2013). Moreover Trop-2 is also a marker of progenitor cells in prostate cancer (Goldstein, Stoyanova et al. 2010; Trerotola, Rathore et al. 2010) .
The Authors of the present invention have previously demonstrated that Trop-2 is expressed at high levels by the majority of tumors and tumor cell lines in man (Trerotola, Cantanelli et al. 2013). In particular, the Authors demonstrated that Trop-2 is overexpressed in cancer cells even when compared to tissues of origin that already express it (S. Alberti “Anti-Trop-2 monoclonal antibodies and uses thereof in the treatment and diagnosis of tumors”—PCT/IT2009/000035), indicating that the increased expression confers a selective advantage to tumor cells (Trerotola, Cantanelli et al. 2013).
The Authors of the present invention have previously demonstrated the function of stimulus of Trop-2 in the development of tumors, both in vitro and in vivo. Overexpression of Trop-2 stimulates the growth of both immortalized and tumor cells, with an increase in the proportion of cells in the S phase of the cell cycle. On the other hand, the use of small-interfering (si) RNAs directed against Trop-2 abolishes the proliferation of breast cancer and colon cancer cells lines which express Trop-2. Similar results have also been obtained in experimental tumors: the expression of Trop-2 increases tumor growth dramatically, and this growth is directly proportional to the levels of expression.
The integrity of the cytoplasmic tail of Trop-2 is necessary in order to have the growth stimulus: deletion of the entire tail or the HIKE domain, point mutations of serine 303 (phosphorylated by da PKCα) and 322, replacement of the four glutamic acid residues, which confer a negative charge all along one side of the a-helix , with lysine positive residues, all cause loss of Trop-2 stimulatory activity (
The stimulation of cell growth, both in vitro and in vivo, has been obtained through the overexpression of a normal Trop-2. Consistent with this, the Authors of the present invention have not identified mutations or structural alterations of the TROP2 gene in tumors, either in the coding region and in the 5 ′ and 3′ untranslated regions (UTR), or in the regions proximal to the promoter (Trerotola, Cantanelli et al. 2013).
These results indicate that an increase in Trop-2 expression, in the absence of mutations, is both necessary and sufficient to stimulate the growth of cancer cells (Trerotola, Cantanelli et al. 2013). Therefore Trop-2 is a “non-oncogene”, to the overexpression of which the tumor cell is addicted for growth (Trerotola, Cantanelli et al. 2013). This growth stimulation is independent from the histotype (carcinoma, sarcoma) and from the species (human, murine), indicating that the signalling network through which Trop-2 acts (Guerra, Trerotola et al. 2013) is highly conserved (Trerotola, Cantanelli et al. 2013) (S. Alberti “Anti-Trop-2 monoclonal antibodies and uses thereof in the treatment and diagnosis of tumors”—PCT/IT2009/000035).
This signalling network will now be further defined by the Authors, in order to identify effector molecules activated specifically and differentially in tumors that express Trop-2, which be used as targets for Trop-2-dependent anticancer therapy.
Electron microscopy analyses carried out by the Authors show that Trop-2 is localized in aggregates in macrovilli at the apical surface of the cell, in villi that extend between cells and in discrete regions of the membrane (
It is known that tetraspanin binding to their partners is cholesterol-dependent (Hakomori 2002): further confirmation of the fact that Trop-2 is an integral component of the tetraspanin platforms is provided by the ability of methyl-β-cyclodextrin and digitonin, which are respectively able to remove and precipitate cholesterol from cell membranes (Hakomori 2002, Charrin, 2003 #17269), to induce the release of Trop-2 in the culture medium and the precipitation of Trop-2 from lysates of cell membranes in BRIJ (Le Naour, Andre et al. 2006) (
The tetraspanin networks provide support for diverse cell surface receptors which activate lipids, protein kinases and intracellular effectors (Hemler 2003; Le Naour, Andre et al. 2006). Accordingly, the Authors of the present invention had already shown that Trop-2 is able to modulate the expression of trans-membrane tyrosine-kinase receptors (PDGFR, Met, Ret, VEGFR), tyrosine and serine/threonine kinases, phosphatases , regulatory molecules of cell cycle and apoptosis (Guerra, Trerotola et al. 2013) (S. Alberti “Anti-Trop-2 monoclonal antibodies and uses thereof in the treatment and diagnosis of tumors”—PCT/IT2009/000035). These results are here extended with new proteomic analyses (
These data have thus enabled the Authors of the present invention to demonstrate that PKCs are key mediators in the signalling network of Trop-2. Altogether, Trop-2 modulates 7 PKC substrates and 55 substrates/regulatory molecules of PKC (
On the basis on these indications, the Authors of the present invention have shown that CD9 microdomains function as connectors between PKCα and Trop-2: PKCα co-immunoprecipitates with CD81 and CD151 tetraspanins and to a greater extent with CD9 (
Bioinformatic analyses of this signalling network has then led the Authors to identify the Akt signalling pathway as key pathway in the stimulation of cell growth by Trop-2, through the GSK3 and S6K arms, but not that of mTOR (
The interaction of Trop-2 with tetraspanin membrane complexes and in particular with CD9 activates a complex downstream signalling network that has the Akt signalling pathway as key component, and stimulates cell proliferation.
The Authors then studied the effect of selective inhibition of CD9 and Akt in proliferating cells expressing Trop-2. The downregulation of CD9, mediated by siRNA (
The Authors of the present invention have identified a novel signalling network that stimulates tumor growth and is specifically activated by Trop-2 through interaction with CD9 and activation of the Akt signaling pathway. Trop-2 overexpression dictates sensitivity to Akt and CD9 inhibitors, which are able to selectively stop the growth of the cells that express Trop-2. These results pave the way to an original mode of personalized anticancer therapy.
Therefore it is a specific embodiment of the present invention a method to predict in vitro the outcome of an anticancer therapy with drugs inhibiting the activity of components of the Trop-2 signaling network, wherein said components are selected from the group consisting of CD9, Akt and molecules of the tetraspanin signalling network, said method comprising or consisting of determining the expression levels of Trop-2 protein or of the corresponding mRNA in a biological sample, an effective outcome occurring when an increase of the expression levels of Trop-2 protein or of the corresponding mRNA as compared to the levels in the corresponding normal tissues is detected.
Molecules of the tetraspanin signalling network are molecules that belong to the Trop-2 network, as identified by the Authors of the invention, and that are also part of the tetraspanin signaling network, and in particular the epidermal growth factor receptor (EGFR), the tyrosine-protein kinase Met, the serine/threonine-protein kinase c-RAF, the proto-oncogene tyrosine-protein kinase Src, the small GTP binding protein CDC42, the tyrosine-protein kinase JAK2, the cAMP-dependent protein kinase catalytic subunit alpha (PKA C-alpha), the tyrosine-protein phosphatase non-receptor type 11 (SHP2), the insulin receptor substrate 1 (IRS1), the serine/threonine-protein kinase PAK 1, the mitogen-activated protein kinase 8 (JNK).
In one embodiment, positivity for the biological marker Trop-2 means an increase of Trop-2 mRNA or protein equal to or greater than 10% in the tumor as compared to the corresponding normal tissues of the same subject. In one embodiment the said positivity is used to select patients on which to employ anticancer drugs for therapeutic purposes, including clinical trials, the said drugs being directed against components of the signalling network of Trop-2, including but not limited to CD9, Akt and molecules of the tetraspanin signalling network. Thus the present invention teaches a novel biological marker, the said marker characterized by Trop-2 overexpression. This is a predictive marker of response to specific therapy, and a marker of enrichment that can identify subgroups of patients which are potentially responsive, and can be recruited in clinical trials of drugs directed against molecules driven by Trop-2, especially but not limited to CD9 and Akt. The said biomarker can be used alone or in combination with other biomarkers.
In another embodiment the present invention provides a method for in vitro screening of candidate drugs for the treatment or prevention of cancer, or the inhibition of cancer cell growth, wherein such drugs are directed against components of the Trop-2 signaling network, wherein said components are selected from the group consisting of CD9, Akt and molecules of the tetraspanin signalling network , i.e molecules that belong to the Trop-2 network, as identified by the Authors of the invention, and that are also part of the tetraspanin signaling network, and in particular the epidermal growth factor receptor (EGFR), the tyrosine-protein kinase Met, the serine/threonine-protein kinase c-RAF, the proto-oncogene tyrosine-protein kinase Src, the small GTP binding protein CDC42, the tyrosine-protein kinase JAK2, the cAMP-dependent protein kinase catalytic subunit alpha (PKA C-alpha), the tyrosine-protein phosphatase non-receptor type 11 (SHP2), the insulin receptor substrate 1 (IRS1), the serine/threonine-protein kinase PAK 1, the mitogen-activated protein kinase 8 (JNK). This method comprises the following steps: the compound to be tested, in sterile saline solution, is administered to cells expressing Trop-2 and not expressing Trop-2; in parallel the sterile saline solution alone is administered to cells expressing Trop-2 and not expressing Trop-2. The non-expressing cells in contact with the compound allow to control the specificity of action of the compound itsels, while cells expressing and not expressing Trop-2 in contact with the sterile saline solution alone allow to control the activity of the compound itself. Subsequently the biological activity of the compound is measured, i.e. the proliferation of the cells expressing Trop-2 in comparison with the cells not expressing Trop-2 subjected to the treatment and with the cells expressing and not expressing Trop-2, not subjected to the treatment, i.e. in contact with the saline solution alone. Finally the compound is selected that is able to reduce cell proliferation by at least 10% in the cells expressing Trop-2 and subjected to the treatment, in comparison with the proliferation of cells not expressing Trop-2 and subjected to treatment and to cells not subjected to treatment.
The present invention provides also a method for the in vivo screening of candidate drugs for the treatment or prevention of cancer, or the inhibition of cancer cell growth, where the said drugs are directed against components of the signalling network of Trop-2, including but not limited to CD9, Akt and molecules of the tetraspanin signalling network. This method comprises the following steps: the compound to be tested, in sterile saline solution, is administered to tumors which are positive (i.e. overexpressing) or negative (i.e. not overexpressing) for the biological marker Trop-2, in preclinical and clinical models; in parallel the sterile saline solution alone is administered to tumors which overexpress or not Trop-2. The non-overexpressing tumors in contact with the compound allow to control the specificity of action of the compound itsels, while tumors overexpressing or not Trop-2 in contact with the sterile saline solution alone allow to control the activity of the compound itself. Subsequently the biological activity of the compound is measured, i.e. the growth of the tumors overexpressing Trop-2 in comparison with the tumors not overexpressing Trop-2 subjected to the treatment and with the tumors overexpressing or not Trop-2, not subjected to the treatment, i.e. in contact with the saline solution alone. Finally the compound is selected that is able to reduce tumor growth by at least 10% in the tumors overexpressing Trop-2 and subjected to the treatment, in comparison with tumor growth of treated and untreated controls.
In addition, the present invention concerns a compound inhibiting the activity of components of the Trop-2 signaling network, wherein said components are selected from the group consisting of CD9, Akt and molecules of the tetraspanin signalling network, for use in the treatment of tumors expressing levels of Trop-2 protein or corresponding mRNA higher than normal tissue. As mentioned above molecules of the tetraspanin signalling network are molecules that belong to the Trop-2 network, as identified by the Authors of the invention, and that are also part of the tetraspanin signaling network, and in particular the epidermal growth factor receptor (EGFR), the tyrosine-protein kinase Met, the serine/threonine-protein kinase c-RAF, the proto-oncogene tyrosine-protein kinase Src, the small GTP binding protein CDC42, the tyrosine-protein kinase JAK2, the cAMP-dependent protein kinase catalytic subunit alpha (PKA C-alpha), the tyrosine-protein phosphatase non-receptor type 11 (SHP2), the insulin receptor substrate 1 (IRS1), the serine/threonine-protein kinase PAK 1, the mitogen-activated protein kinase 8 (JNK).
Some already known inhibitory molecules are: MK-2206, A6730, Perifosine, GSK690693, GSK2110183, GDC-0068, AT7867, ARQ092, AZD5363, A-674563, PHT-427, PF-04691502, SureCN1078972, 842148-40-7, AC1NX3D3, MLS002702033, SureCN10005574, SureCN1559590, SureCN570829, SH-5, SH-6, Honokiol, Miltefosine, Triciribine phosphate (Akt inhibitors), K00598a, DAPH 2, SureCN238877 (EGFR inhibitors), SureCN4269573 (inhibitor of EGFR, c-RAF, Src), dasatinib (Src inhibitor), SureCN1518805, 1,9-Pyrazoloanthrone, AS-601245, aminopyridine deriv. 2 (JNK inhibitors).
Optionally the above compound can be used in combination with other therapies, such as chemotherapy, alkylating agents (such as mechlorethamine chlorambucil, cyclophosphamide, ifosfamide, melphalan, nitrosoureas, busulfan, dacarbazine (DTIC), temozolomide, thiotepa and altretamine), antimetabolites (such as 5-fluorouracil (5-FU),6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine), anti-tumor antibiotics (such as anthracyclines, actinomycin-D, bleomycin, mitomycin-C), topoisomerase inhibitors (such as topotecan, irinotecan, etoposide, teniposide, mitoxantrone), mitotic inhibitors (such as paclitaxel, docetaxel, ixabepilone, vinblastine, vincristine, vinorelbine, estramustine), corticosteroids (such as prednisone, methylprednisolone, dexamethasone), differentiating agents (such as retinoids, tretinoin/ATRA, bexarotene and arsenic trioxide), hormone therapy, targeted kinase inhibitors, the proteosome inhibitor bortezomib.
The compound according to the present invention for use according to the invention can be chosen from the group consisting of oligonuclotides, engineered molecules corresponding to CD9, Akt and molecules of the tetraspanin signalling network which act as “dominant negative” molecules, monoclonal antibodies, pharmacological inhibitors, small-molecule chemical compounds or combination thereof.
As mentioned above, the inhibition of the activity of the above molecules can be obtained, as a non-limiting example, by means of corresponding engineered molecules, which act as “dominant negatives”, i.e. molecules similar to the normal ones but devoid of biological activity; these dominant negative molecules replace the normal ones and abolish their function in the cellular processes. The inhibition of biological activity can also be obtained with specific monoclonal antibodies that bind to catalytic and/or regulatory sites of the normal molecules and inhibit their activity. The inhibition of biological activity can also be obtained with pharmacological inhibitors. One of the main examples are MK-2206 and A6730, pharmacological inhibitors of Akt, for which Trop-2 expression constitutes indication for anti-cancer use according to the present invention.
The present invention concerns also oligonucleotides (RNA or DNA, double o single stranded) able to inhibit the growth of tumors in patients, these tumors being positive for the biological marker Trop-2, by inhibiting the expression of components of the signalling pathway of Trop-2, including but not limited to CD9, Akt and molecules of the tetraspanin signalling network. As an example, an oligonucleotide able to inhibit CD9 expression consists in silencing RNAs (siRNAs) targeted against CD9, wherein the target sequence in the sense strand is contained in the mRNA of CD9 (GenBank accession number: NM_007657.3), including but not limited to the following sequences:
These silencing target sequences can also be fused to other sequences in order to obtain oligonucleotides that are transcribed into hairpin RNAs when inserted into appropriate vectors, having the following sequence as an example, where the target sequences in sense and antisense orientation are underlined:
As an example, a method to inhibit AKT expression consists in siRNAs targeted against AKT, wherein the target sequence in the sense strand is contained in the mRNA of AKT (NM_009652.3), including but not limited to the following sequences:
The present invention can be applied to the clinic for the diagnosis of tumors that overexpress Trop-2 and for the cure of patients bearing such tumors, where Trop-2 overexpression constitutes a biological marker that guides the therapeutic choice toward compounds and/or treatments targeted against components of the signalling network of Trop-2, including but not limited to CD9, Akt and molecules of the tetraspanin signalling network. Another application of the present invention is in the screening of new drugs and treatments for cancer therapy, using cells and tumors that overexpress Trop-2 and are sensitive in vitro and in vivo to substances and/or treatments directed against targets identified in the molecules belonging to the signalling network of Trop-2, in particular CD9, Akt and molecules of the tetraspanin signalling network.
TROP2 mRNA increase in tumor cells compared to the corresponding control cells can be quantified in cells in culture and biopsies consisting of fresh or frozen cells and tissues. The mRNA can be extracted and purified by one skilled in the art by applying available techniques. Kits and instrumentation for extraction and purification can be acquired from commercial sources. The detection and quantification of TROP2 mRNA by cDNA synthesis and PCR or real-time quantitative PCR, as described in the present invention, can be carried out according to techniques known in the art. Reagents and instrumentation for real-time quantitative RT-PCR are available on the market.
Trop-2 protein increase in tumor cells compared to the corresponding control cells can be quantified in cells in culture and biopsies consisting of fresh, frozen or formalin-fixed cells and tissues, by means of immunohistochemistry, ELISA assays, Western blotting or other equivalent techniques known in the art. Trop-2-reactive antibodies, kits and equipment for the execution of these tests and measurements are available on the market.
The immortalized murine cell line MTE 4-14 (Naquet, Lepesant et al. 1989) can be transfected according to techniques known in the art. MTE 4-14 cells transfected with an expression vector for Trop-2 and with the empty vector can be grown in vitro and used for the screening of compounds targeted against components of the signalling network of Trop-2, including inhibitors of CD9, Akt and molecules of the tetraspanin signalling network. Cell proliferation can be measured by direct counting or staining for cellular constituents or metabolic assays, according to techniques known in the art.
Preclinical models to be used for the in vivo screening of compounds directed against components of the signalling network of Trop-2, including inhibitors of CD9, Akt and molecules of the tetraspanin signalling network, consist of immunocompromised mice which are subcutaneously injected with cells overexpressing or not overexpressing Trop-2 (xenografts), and similar models, according to methods known in the art.
The present invention will be now described by way of illustration and example, according, but not limited, to, some of its preferred embodiments, with particular reference to the detailed examples and figures of the attached drawings.
Cell Cultures
The human MCF-7 and OVCA-432 cell lines were grown in RPMI 1640 medium (GibcoBRL, Paisley, Scotland). The human 293 and KM12SC and the murine immortalised MTE 4-14 (Naquet, Lepesant et al. 1989) and L cell lines were maintained in DMEM. All media were supplemented with 10% fetal bovine serum (GibcoBRL), with 100 IU/ml penicillin and 100 μg/ml streptomycin (Euroclone, Milano, Italy).
DNA Transfection
Cells were transfected with DNA in Lipofectamine 2000 or LTX (Invitrogen, Carlsbad, Calif.) following the manufacturer instructions. Stable transfectants were selected in G-418-containing medium.
Immunofluorescence Analysis.
Cells plated on glass coverslips were stained with the indicated antibodies conjugated with Alexa Fluor-488/546/633, or with the same unconjugated antibodies detected by a secondary antibody conjugated with Alexa Fluor-488/546/633. Cells were either stained alive, before fixation, in medium with 10% FBS, at 37° C. for 5 minutes, or stained after fixation, by incubation with the relevant antibodies in PBS at room temperature for 30 minutes. Cells were fixed with 4% paraformaldehyde in PBS for 20 min. Permeabilisation and blocking of non-specific interactions were performed with 10% FCS, 0.1% saponin. The slides were then washed in PBS and mounted to be analyzed by LSM-510 META confocal microscope (Zeiss, Oberkochen, Germany).
Cell Footprints
“Cell footprints” are membrane remnants on tissue culture dishes after detachment of cells with EDTA (Hakomori 2002). Cell treatment and immunofluorescence analyses were conducted essentially as described (Hakomori 2002).
Co-Immunoprecipitation Assays
Cells were lysed in BRIJ buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM CaCl2, 1% BRIJ, protease inhibitors). Cell lysates were cleared by incubation with Protein-G Sepharose (GE Healthcare, Piscataway, N.J.) at 4° C. for 1 h. Cleared lysates were incubated with primary antibodies against putative Trop-2 interactors at 4° C. for 2 h, followed by incubation with Protein G Sepharose (at 4° C., 1 h). Protein complexes were eluted from the resin with 0.1 M glycine, pH 2.5, and probed by Western blotting with specific antibodies as indicated.
Depletion and Precipitation of Cholesterol from Cell Membranes.
To deplete cell membranes of cholesterol, intact cells were washed three times in PBS to remove serum and incubated in DMEM with 10 mM methyl-β-cyclodextrin at 37° C. for 45 min. Control cells were processed in parallel in the absence of methyl-β-cyclodextrin. Samples were centrifuged at 2000 g to remove cells and cell debris. Proteins in the supernatant were precipitated in trichloroacetic acid and analysed by Western blotting.
Cells were also harvested by scraping and lysed in buffer with 1% BRIJ. The supernatant was mixed with either 1/10 (vol/vol) methanol (control) or 1/10 (vol/vol) 10% digitonin in methanol, and incubated on ice for 10 min. Cell membranes were recovered by centrifugation at 12,000 g for 15 min. The pellet was washed in cold acetone, resuspended in Laemmli buffer and analysed by Western blotting.
Immuno-Electron Microscopy.
For immuno-gold electron microscopy (EM), cells were fixed in 4% formaldehyde, 0.05% glutaraldehyde, 0.15 M HEPES, pH 7.3, for 10 min at 37° C., and post-fixed for 50 min in 4% paraformaldehyde, 0.15 M HEPES, pH 7.3 at room temperature, as described (Polishchuk, Polishchuk et al. 2000). Cationised gold, protein A-gold, and gold-conjugated anti-rabbit antibodies (10 nm colloidal gold particles) were from British BioCell (Cardiff, UK). Immunoperoxidase EM was performed as described (Brown and Farquhar 1989). Estimates of minimal labelled areas were performed assuming an average length of antibody molecules of 11 nm.
In Vitro Cell-Growth Assays.
MTE 4-14 cells transfected with the empty vector or with Trop-2 were seeded at 1.5-3×103 cells/well in 96-well plates (five replica wells per data point). Cell numbers were quantified by the MTT colorimetric assay (Roche Molecular Biochemicals, Mannheim, Germany) or by staining with crystal violet. Cell numbers were normalised against a reference standard curve of serially diluted cell samples.
Antibodies.
Rabbit polyclonal anti-Trop-2 antisera were generated by subcutaneous immunisation with recombinant Trop-2, that had been produced in bacteria (Fornaro, Dell'Arciprete et al. 1995). Trop-2-reactive antibodies were purified by binding to recombinant Trop-2 immobilized onto NHS-Sepharose columns (Pharmaci, Uppsala, Sweden), and eluted with 0.2 M glycine, pH 2.5. The goat anti-Trop-2 polyclonal antibody AF650 was obtained from R&D Systems, Inc. (Minneapolis, Minn.). Additional antibodies directed against murine molecules were: rat monoclonal anti-CD9 (sc-18869); rabbit monoclonal anti-Akt (ser473) (D9E) (Cell Signal Technology, MA); goat polyclonal anti-CD151 (sc-18753), anti-phospho-PKCα (S657) (sc-12356), anti-Akt (sc-1618); rabbit polyclonal anti-caveolin-1 (sc-894), anti-PKCα (sc-208), anti-phospho-Akt (Thr 308) (sc-16646-R) (Santa Cruz Biotechnology, Santa Cruz, Calif.); hamster polyclonal anti-CD81 (GTX75430) (GeneText, Irvine, Calif.). Mouse monoclonal antibodies directed against corresponding human molecules were: anti-CD9 (14-0098), anti-CD98 (14-0982) (eBioscience, San Duego, Calif.), anti-CD151 (H00000977-M02) (Abnova, Taiwan, China) and anti-CD81 (TS81) (generously supplied by Dr. F. Lanza). Secondary Alexa Fluor-488/546/633 conjugated antibodies were from Invitrogen.
Pharmacological Inhibitors.
Cells were treated with 50-100-200-400 nM A6730_SIGMA (Sigma Technical Company, St Louis, Mo. 63178-9916) or with 400 nM MK-2206 (Selleck Chemicals, Houston, Tex.) to inhibit Akt. Stock solutions were prepared in DMSO and diluited in ethanol or water, with the final concentration of DMSO and ethanol never exceeding 0.1% and 1% respectively. Control cells received vehicle alone. A second treatment was performed 24 hours after seeding.
In Vivo Models: Xenografts in Athymic Nude Mice
MTE 4-14 immortalized cell line transfected with Trop-2 (Alberti, Nutini et al. 1994), or Colo205 colon cancer cells, endogenously expressing Trop-2, were injected subcutaneously (4×106 cells/injection) into nude mice groups (8 weeks females, CD1-Foxn1 nu/nu mice (Charles River Laboratories, Calco, Lecco, Italy). MTE 4-14 cells transfected with Trop-2 were co-injected with Matrigel 1:3 vol/vol (growth factor reduced matrix, BD Biosciences, Franklin Lakes, N.J. USA). A stock solution of MK-2206 in DMSO was diluted at the time of use in apyrogenic saline solution, to a final concentration of 0.7 mg/ml, and the drug was administered intraperitoneally at a dose of 0.35 mg/mouse every day, starting from the time in which the tumors became palpable. Control group mice were treated with the vehicle alone. Mice were visually inspected every 5-7 days and the major and minor diameters of the experimental tumors were measured. The tumor volume was calculated using the formula for the volume of ellipsoid (Dxd2/2). MTE 4-14 cells transfected with the empty vector were not tumorigenic in this model. Procedures involving animals and their care were conducted in compliance with institutional guidelines, national laws and international protocols (D.L. No.116, G.U., Suppl. 40, Feb. 18, 1992; No. 8, G.U., July, 1994; UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia; EEC Council Directive 86/609, OJ L 358. 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 1996).
Antibody-Mediated Co-Capping.
Cells were detached from culture plates using trypsin, and incubated with the primary anti-Trop-2 antibody (T16) (Alberti, Miotti et al. 1992) for 20 min on ice. After washing, cells were incubated with the secondary Alexa Fluor 488-conjugated antibody for 10 min at 37° C., to cross-link target molecules and to induce capping of the antigen-antibody complex (Levy and Shoham 2005). The ‘capped’ cells were fixed with 1% paraformaldehyde for 10 min at room temperature. The paraformaldehyde was quenched with FBS, and cells were stained with antibodies against the membrane proteins of interest.
Western Blot Analysis.
Western blotting was performed as previously described (El Sewedy, Fornaro et al. 1998). In brief, cell lysates were analyzed by electrophoresis on denaturing polyacrylamide gel (SDS-PAGE) and transfered onto nitrocellulose filters. Filters were incubated with the relevant primary antibodies (Abcam, Cambridge, UK; Santa Cruz) in TBS with 5% non-fat dried milk. Hybridized filters were washed in TBS, 0.1% Tween-20. Antibody binding was revealed by chemiluminescence (ECL, Amersham, Aylesbury, UK), using horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Calbiochem, La Jolla, Calif.). Signal intensity was quantified with NIH-image 1.62, using as reference a Kodak gray-scale standards power curve (www.kodak.com).
Plasmids
The pEYFP expression vector was obtained from Clontech (Palo Alto, Calif.). A corresponding vector devoid of the coding sequence of EYFP was used to express wild-type or mutagenized Trop-2 cDNAs. The coding sequence of the wild-type human TROP2 was amplified by PCR with the forward primer 5′ GCGATTCTCG AGTCCGGTCC GCGTTCC 3′ (SEQ ID NO:13) and with the reverse primer 5′ GCGCCGGTAC CAAGCTCGGT TCCTTTC 3′(SEQ ID NO:14) and subcloned into the pEYFP-N1 vector. The expression vectors pCMV-SPORT6 and CD316 pCMV-SPORT6 were obtained from Imagenes (Berlin, Germany). The chimeric proteins between CD9 (EcoRI/BamHI), CD316 (EcoRI/AgeI) and mCherry were constructed following standard procedures.
siRNA.
Four design strategies were used, following the procedures according to: Tuschl criteria (Elbashir, Harborth et al. 2001): Invitrogen (rnaidesigner.invitrogen.com/rnaiexpress/), Whitehead Institute web site (jura.wi.mit.edu/bioc/siRNAext/) (Semizarov, Frost et al. 2003); Sonnhammer (sonnhammer.cgb.ki.se/siSearch/) (Chalk, Wahlestedt et al. 2004). The siRNAs that were chosen were the ones identified by more than one of the above methods, or considered optimal by at least one of them . The coding sequences for hairpin RNAs (shRNAs) obtained from the corresponding siRNAs (Elbashir, Harborth et al. 2001), specific for the genes of interest, have been subcloned in the pSUPER vector (Brummelkamp, Bernards et al. 2002), under the control of the promoter for the RNA polymerase-III H1 gene. CD9 inhibition was obtained by means of anti-CD9 siRNA with target sense sequence 5′ GGTAGCAAGT GCATCAAAT 3′ (SEQ ID NO:1), and antisense sequence 5′ ATTTGATGCA CTTGCTACC 3′(SEQ ID NO:2), as an example included in the following sequences for the transcription of hairpin RNA, where the target sequences in sense and antisense orientation are underlined:
The inhibition of Akt has been obtained by means of anti-AKT siRNAs with target sense sequence 5′ GCACCTTTAT TGGCTACAA 3′ (SEQ ID NO:5) and antisense sequence 5′ TTGTAGCCAA TAAAGGTGC 3′ (SEQ ID NO:6), as an example included in the following sequences for the transcription of hairpin RNA:
The corresponding constructs were transiently transfected into MTE 4-14 cells, and their effect on cell proliferation was evaluated. The levels of transcripts after silencing were quantified by real-time RT-PCR. siRNAs directed against irrelevant targets were used as negative controls, chosen after thorough verification of the absence of spurious effects on cell growth. The corresponding sequences are given below; for each siRNA the corresponding gene is indicated, with the identification code according to the RefSeq database (GenBank). siRna directed against murine Co-029/Tspan8 (NM_146010.2), used as negative control for human cells, having the target sequence sense 5′CTTTCAAACC TGAGTATAA 3′ (SEQ ID NO:15), antisense 5′ TTATACTCAG GTTTGAAAG 3′ (SEQ ID NO:16), as an example included in the following sequences for the transcription of hairpin RNA:
Mutagenesis of the Cytoplasmic Domain of Trop-2
The mutants of the cytoplasmic domain of Trop-2 were obtained by PCR with the following primers:
Real-Time Quantitative RT-PCR.
Total RNA from the indicated cell lines was extracted in Trizol (Invitrogen) following the manufacturer's instructions. One μg of total RNA was used for each reverse transcription (RT) reaction, using the ImProm-II Reverse Transcriptase (Promega) according to standard protocols. Real-time quantitative PCR reactions were performed using an ABI-PRISM 7900HT Sequence Detection System (PE Applied Biosystems, Foster City, Calif.) with specific primers (Hs99999905; CD9: Mm00514275_g1; B2M: Mm00437762_ml) (Applied Biosystems) and the corresponding TaqMan® (Roche) fluorescent probes, according to the manufacturer instructions (Giulietti, Overbergh et al. 2001). Each sample was assayed in triplicate and the 2−ΔΔCT method was used to calculate relative changes in gene expression (Livak and Schmittgen 2001). A more accurate base of 1.834 was used (Guerra, Trerotola et al. 2008), as 1.1 cycles are required to double the amplified material. The GAPDH and B2M housekeeping genes were used as an internal control. For each cDNA the ΔCT (CT, target gene-CT, GAPDH/B2M) was calculated: using least-squares linear regression analysis. As amplification efficiency was linear over the range of RNA amounts used, amplification curves were used to calculate cross-over point values for siRNA-treated samples. Each sample was routinely assessed for genomic DNA contamination by using non-retrotranscribed RNA as a templates for PCR reactions.
2D Gel and Mass-Spectrometry.
A vision over the entire genome was obtained by 2D-PAGE performed as described (Swiss Institute of Bioinformatics (SIB)—http://us.expasy.org/ch2d/protocols/). The mass spectrometry analyses were performed at the University of York (www.york.ac.uk/depts/biol/tf/proteomics/).
Pathway Analysis and Identification of Trop-2 Signaling Network
The proteome of Trop-2 was analyzed using the SNOW (snow.bioinfo.cipf.es/cgi-bin/snow.cgi) and Ingenuity Pathways Analysis (IPA, Ingenuity Systems, www.ingenuity.com) bioinformatics tools. SNOW (snow.bioinfo.cipf.es/cgi-bin/snow.cgi) builds networks of protein-protein interaction, mapping the protein over a reference interactome. The reference interactome is a set of pair-wise direct protein interactions, where the nodes are the proteins themselves and the connecting edges are the interaction events. A Trop-2-dependent interactome was built using the HPRD (Human Protein Reference Database), IntAct, BIND (Biomolecular Interaction Network Database), DIP (Database of Interacting Proteins) and MINT (Molecular Interaction Database) databases. These were used to build a minimal connected network (MCN). Topological networks were evaluated on the basis of the node connections degree (i.e. number of edges of each node); the betweenness (i.e. a measure of centrality of nodes and of their distribution); the clustering coefficient distribution (i.e. the connectivity of the neighbourhood of each node); the number of components of the network (i.e. the different groups of nodes that are generated in a network analysis); the bicomponents (i.e. the number of node groups connected to another node group by a single edge) and the articulation points (i.e. the edges that join bicomponents in a network). MCNs were identified using the Dijkstra's algorithm applying a stringent option that permits the introduction of only one external connector protein between any pair of input proteins analyzed. In this way it was possible to obtain a single network that combines 114 of the 165 proteins modulated by Trop-2 which were subjected to the analysis, with 22 connectors. Statistical significance was verified versus networks generated over the entire reference interactome and versus networks with random composition. For the analysis using the Ingenuity Pathways Analysis software, protein expression values, expressed as ratios between the normalized mean signal intensities between Trop-2 and control transfectants, were converted to fold-change values, where the negative inverse (−1/x) was taken for values between 0 and 1. As the data-set was filtered, no threshold cut-offs were imposed. All the molecules (Swiss-Prot identifiers) were mapped onto the Ingenuity Knowledge Base as focus points. The Ingenuity Knowledge Base organises protein interactions into an ontology format, as extracted by manual curation from published experimental results. Networks of focus molecules were generated by maximising specific connectivities, i.e. actual interconnectedness between molecules versus that with all molecules in the Knowledge Base. Networks were ranked by scores (negative log of the p-value calculated by right-tailed Fisher's exact test) that takes into account the number of eligible molecules in the network and its final size as well as the input data-set size and the total number of molecules in the Ingenuity Knowledge Base included in networks. The higher the score, the lower the chance of stochastic interaction networks. Focus molecules were then mapped onto the Ingenuity Canonical Pathways.
Statistical Analysis
The Two-way ANOVA test was used to compare the tumor growth curves (Rossi, Di Lena et al. 2008).
Number | Date | Country | Kind |
---|---|---|---|
CH2012A000008 | May 2012 | IT | national |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/IT2013/000139 | 5/16/2013 | WO | 00 |