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Purpose: We determined the gene copy numbers for MET, for its transcriptional activator MACC1 and for its ligand hepatocyte growth factor (HGF) in liver metastases from colorectal carcinoma (mCRC). We correlated copy numbers with mRNA levels and explored whether gain and/or overexpression of MET and MACC1 predict response to anti-Met therapies. Finally, we assessed whether their genomic or transcriptional deregulation correlates with pathologic and molecular parameters of aggressive disease.Experimental Design: One hundred three mCRCs were analyzed. Copy numbers and mRNA were determined by quantitative PCR (qPCR). Thirty nine samples were implanted and expanded in NOD (nonobese diabetic)/SCID (severe combined immunodeficient) mice to generate cohorts that were treated with the Met inhibitor JNJ-38877605. In silico analysis of MACC1 targets relied on genome-wide mapping of promoter regions and on expression data from two CRC datasets.Results: No focal, high-grade amplifications of MET, MACC1, or HGF were detected. Chromosome 7 polysomy and gain of the p-arm were observed in 21% and 8% of cases, respectively, and significantly correlated with higher expression of both Met and MACC1. Met inhibition in patient-derived xenografts did not modify tumor growth. Copy number gain and overexpression of MACC1 correlated with unfavorable pathologic features better than overexpression of Met. Bioinformatic analysis of putative MACC1 targets identified elements besides Met, whose overexpression cosegregated with aggressive forms of colorectal cancer.Conclusions: Experiments in patient-derived xenografts suggest that mCRCs do not rely on Met genomic gain and/or overexpression for growth. On the basis of pathologic correlations and bioinformatic analysis, MACC1 could contribute to CRC progression through mechanisms other than or additional to Met transcriptional upregulation. Clin Cancer Res; 17(10); 3146–56. ©2011 AACR.

Purpose: Advanced ovarian cancers are initially responsive to combinatorial chemotherapy with platinum drugs and taxanes but, in most cases, develop drug resistance. We recently showed that, in vitro, hepatocyte growth factor (HGF) enhances death of human ovarian cancer cell lines treated with cisplatin (CDDP) and paclitaxel. The present study addresses whether in vivo HGF makes ovarian carcinoma cells more responsive to these chemotherapeutics.Experimental Design: Using Lentiviral vectors carrying the HGF transgene, we transduced SK-OV-3 and NIH:OVCAR-3 ovarian carcinoma cell lines to obtain stable autocrine and paracrine HGF receptor activation. In vitro, we assayed growth, motility, invasiveness, and the response to CDDP and paclitaxel of the HGF-secreting bulk unselected cell populations. In vivo, we tested the cytotoxic effects of the drugs versus s.c. tumors formed by the wild-type and HGF-secreting cells in immunocompromised mice. Tumor-bearing mice were treated with CDDP (i.p.) and paclitaxel (i.v.), combined in different schedules and doses.Results:In vitro, HGF-secreting cells did not show altered proliferation rates and survival but were strongly sensitized to the death triggered by CDDP and paclitaxel, alone or in combination. In vivo, we found a therapeutic window in which autocrine/paracrine HGF made tumors sensitive to low doses of the drugs, which were ineffective on their own.Conclusions: These data provide the proof-of-concept that in vivo gene therapy with HGF might be competent in sensitizing ovarian cancer cells to conventional chemotherapy.

Supplementary Table S1 from Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations

Supplementary Figure S1 from Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations

Supplementary Materials and Methods from Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations

Supplementary Figure 1 from Caveolin-1 Reduces Osteosarcoma Metastases by Inhibiting c-Src Activity and Met Signaling

Companion animal spontaneous tumors are suitable models for human cancer, primarily because both animal population and the tumors are genetically heterogeneous. Feline mammary carcinoma (FMC) is a highly aggressive, mainly hormone receptor–negative cancer, which has been proposed as a model for poor prognosis human breast cancer. We have identified and studied the feline orthologue of the HER2 gene, which is both an important prognostic marker and therapeutic target in human cancer. Feline HER2 (f-HER2) gene kinase domain is 92% similar to the human HER2 kinase. F-HER2–specific mRNA was found 3- to 18-fold increased in 3 of 3 FMC cell lines, in 1 of 4 mammary adenomas and 6 of 11 FMC samples using quantitative reverse transcription-PCR. Western blot showed that an anti-human HER2 antibody recognized a protein comigrating with the human p185HER2 in FMC cell lines. The same antibodies strongly stained 13 of 36 FMC archival samples. These data show that feline HER2 overexpression qualifies FMC as homologous to the subset of HER2 overexpressing, poor prognosis human breast carcinomas and as a suitable model to test innovative approaches to therapy of aggressive tumors.

Supplementary Figure 2 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Figures S1 & S2 from MET Overexpression Turns Human Primary Osteoblasts into Osteosarcomas

Caveolin-1 (Cav-1) is highly expressed in normal osteoblasts. This article reports that Cav-1 down-regulation is part of osteoblast transformation and osteosarcoma progression and validates its role as oncosuppressor in human osteosarcoma. A survey of 6-year follow-up indicates a better overall survival for osteosarcoma expressing a level of Cav-1 similar to osteoblasts. However, the majority of primary osteosarcoma shows significantly lower levels of Cav-1 than normal osteoblasts. Accordingly, Met-induced osteoblast transformation is associated with Cav-1 down-regulation. In vitro, osteosarcoma cell lines forced to overexpress Cav-1 show reduced malignancy with inhibited anchorage-independent growth, migration, and invasion. In vivo, Cav-1 overexpression abrogates the metastatic ability of osteosarcoma cells. c-Src and c-Met tyrosine kinases, which are activated in osteosarcoma, colocalize with Cav-1 and are inhibited on Cav-1 overexpression. Thus, Cav-1 behaves as an oncosuppressor in osteosarcoma. Altogether, data suggest that Cav-1 down-modulation might function as a permissive mechanism, which, by unleashing c-Src and Met signaling, enables osteosarcoma cells to invade neighboring tissues. These data strengthen the rationale to target c-Src family kinases and/or Met receptor to improve the extremely poor prognosis of metastatic osteosarcoma. [Cancer Res 2007;67(16):7675–85]

The MET oncogene was causally involved in the pathogenesis of a rare tumor, i.e., the papillary renal cell carcinoma, in which activating mutations, either germline or somatic, were identified. MET activating mutations are rarely found in other human tumors, whereas at higher frequencies, MET is amplified and/or overexpressed in sporadic tumors of specific histotypes, including osteosarcoma. In this work, we provide experimental evidence that overexpression of the MET oncogene causes and sustains the full-blown transformation of osteoblasts. Overexpression of MET, obtained by lentiviral vector–mediated gene transfer, resulted in the conversion of primary human osteoblasts into osteosarcoma cells, displaying the transformed phenotype in vitro and the distinguishing features of human osteosarcomas in vivo. These included atypical nuclei, aberrant mitoses, production of alkaline phosphatase, secretion of osteoid extracellular matrix, and striking neovascularization. Although with a lower tumorigenicity, this phenotype was superimposable to that observed after transfer of the MET gene activated by mutation. Both transformation and tumorigenesis were fully abrogated when MET expression was quenched by short-hairpin RNA or when signaling was impaired by a dominant-negative MET receptor. These data show that MET overexpression is oncogenic and that it is essential for the maintenance of the cancer phenotype. (Cancer Res 2006; 66(9): 4750-7)

Supplementary Figure 1 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Table 1 from Expression Profiling in Progressive Stages of Fumarate-Hydratase Deficiency: The Contribution of Metabolic Changes to Tumorigenesis

Supplementary Figure 2 from Caveolin-1 Reduces Osteosarcoma Metastases by Inhibiting c-Src Activity and Met Signaling

Supplementary Table 2 from Spontaneous Feline Mammary Carcinoma Is a Model of HER2 Overexpressing Poor Prognosis Human Breast Cancer

Supplementary Table 1 from Caveolin-1 Reduces Osteosarcoma Metastases by Inhibiting c-Src Activity and Met Signaling

Supplementary Figure S3 from MET Overexpression Turns Human Primary Osteoblasts into Osteosarcomas

For many decades, basic and preclinical cancer research has been based on the use of established, commercially available cell lines, originally derived from patients’ samples but adapted to grow indefinitely in artificial culture conditions, and on xenograft models developed by injection of these cells in immunocompromised animals [...]

Ovarian cancer is still the most lethal gynecologic malignancy with a median five-year survival of 48%, including the less malignant and early diagnosed cases [...]

Single-cell RNA sequencing (scRNAseq) is an essential tool to investigate cellular heterogeneity. Thus, it would be of great interest being able to disclose biological information belonging to cell subpopulations, which can be defined by clustering analysis of scRNAseq data. In this manuscript, we report a tool that we developed for the functional mining of single cell clusters based on Sparsely-Connected Autoencoder (SCA). This tool allows uncovering hidden features associated with scRNAseq data. We implemented two new metrics, QCC (Quality Control of Cluster) and QCM (Quality Control of Model), which allow quantifying the ability of SCA to reconstruct valuable cell clusters and to evaluate the quality of the neural network achievements, respectively. Our data indicate that SCA encoded space, derived by different experimentally validated data (TF targets, miRNA targets, Kinase targets, and cancer-related immune signatures), can be used to grasp single cell cluster-specific functional features. In our implementation, SCA efficacy comes from its ability to reconstruct only specific clusters, thus indicating only those clusters where the SCA encoding space is a key element for cells aggregation. SCA analysis is implemented as module in rCASC framework and it is supported by a GUI to simplify it usage for biologists and medical personnel.

The small heat-shock protein of 27 kDa (HSP27) is highly expressed in many cancers and is associated with aggressive tumour behaviour, metastasis, poor prognosis and resistance to chemotherapy. We aimed at assessing the role of HSP27 in modulating responses to target therapies. We selected several oncogene-addicted cancer cell lines, which undergo either cell cycle blockade or cell death in response to agents that target the specific oncogene. Surprisingly, HSP27 suppression alone resulted in the apoptotic death of MET-addicted EBC-1 lung cancer cells, epidermal growth factor receptor (EGFR)-addicted colorectal carcinoma (CRC) DiFi cells and BRAF-addicted CRC COLO205 and OXCO-1 and melanoma COLO741 cells, all of which also undergo death when treated with the specific targeted agent. In other cell lines, such as MET-addicted gastric carcinoma MKN45 and EGFR-addicted CRC SW48 lines, where oncogene inhibition only blocked proliferation, HSP27 knockdown made targeted agents switch from cytostatic to cytotoxic activity. Mechanistically, the more the cells were susceptible to HSP27 suppression, the more they were primed for death, as demonstrated by increased levels of mitochondrial outer membrane permeabilization. Priming for death was accompanied by the increase in pro-apoptotic proteins of the BCL2 family and of active caspase-3 and lamin B. Together, these data suggest that oncogene-addicted cells require HSP27 for survival and that HSP27 might interfere with the effectiveness of targeted agents.

Objective Relapsed epithelial ovarian cancer (EOC) is frequently treated with pegylated liposomal doxorubicin (PLD). Unfortunately, most patients do not benefit from treatment. Prediction of response is crucial to optimize PLD use and avoid unnecessary toxicities. We aimed at assessing the value of topoisomerase II alpha (TOP2A) expression as predictive marker of response to PLD-based therapy in patients with relapsed EOCs. Methods We retrospectively analyzed Formalin Fixed Paraffin Embedded (FFPE) tissues from 101 patients with platinum resistant (PR) or partially platinum-sensitive (PPS) EOCs treated with PLD-based chemotherapy beyond second line in three referral cancer centers between January 2010 and June 2018. TOP2A expression was measured by immunohistochemistry (IHC): images of each sample were acquired by optical microscope and analyzed by using automatic counter software. Correlation between TOP2A expression and response to PLD was assessed. Since no cut-off for positivity has been validated yet, we dichotomized TOP2A expression based on a cut-off of 18% (mean value in this study). Results TOP2A expression beyond cut-off was not prognostic for primary platinum-free interval in our series (p = 0.77) neither for optimal cytoreduction (p = 0.9). TOP2A > 18% was associated with a longer time to progression (TTP) following PLD-treatment, although not statistically significant (p = 0.394). No difference was observed between PR and PPS patients’ groups (p = 0.445 and p = 0.185, respectively). Not unexpectedly, patients with TOP2A expression > 18% treated with PLD monotherapy achieved a longer TTP compared with PLD-doublet therapy (p = 0.05). Conclusions Our data suggest that TOP2A status might predict activity of PLD in patients with PR/PPS EOCs.

Background Platinum drugs are the most powerful chemotherapeutic agents in the treatment of ovarian cancer. We demonstrated previously that unexpectedly ovarian cancer cells are sensitised to cisplatin (CDDP) by the hepatocyte growth factor (HGF), usually considered an anti-apoptotic factor. Methods We used quantitative polymerase chain reaction and Western blot analysis to evaluate gene and protein expression, immunofluorescence to evaluate protein localisation and functional assays to measure cell viability and apoptosis. Results In ovarian cancer cells, CDDP induced the phosphorylation, i.e. the activation, of the p90RSK. Surprisingly, a 48-h-long cell pre-treatment with HGF reverted this activation. HGF pre-treatment also resulted in the increased expression of the integrin-linked kinase (ILK)-associated phosphatase (ILKAP) that dephosphorylated the p90RSK. Conversely, CDDP down-modulated ILKAP expression. This impaired CDDP efficacy, as ILKAP silencing protected cells from CDDP-induced death. In line, the biochemical inhibition of the p90RSK or the combined silencing of the most expressed RSK isoforms, namely RSK1 and RSK2, increased the efficacy of CDDP. However, p90RSK inhibition was not sufficient to revert cell protection from death after ILKAP suppression, because of the simultaneous increased activity of the anti-apoptotic kinases ILK and ILK substrate AKT, which were both dephosphorylated, i.e. negatively regulated, by ILKAP. Only the combined inhibition of p90RSK and ILK reverted the effect of ILKAP suppression. Conclusions As RSKs, ILK and AKT are vital kinases for ovarian cancer onset and progression, data suggest that ILKAP is a regulatory hub of ovarian cancer cell survival by controlling the activation of these kinases.

Platinum-based chemotherapy is widely used to treat various cancers, but many patients ultimately relapse due to drug resistance. We employed phosphoproteomic analysis and functional assays of the response of SK-OV-3 ovarian cancer cells to cisplatin as a strategy to identify kinases as candidate druggable targets to sensitize cells to platinum. A SILAC-based approach combined with TiO2-based phosphopeptide enrichment allowed the direct identification of ERK1/2, p90RSK, and ERBB2 as kinases whose phosphorylation is regulated by cisplatin. Bioinformatic analysis revealed enrichment in linear phosphorylation motifs predicted to be targets of p38MAPK, CDK2, and PIM2. All three PIM kinases were found expressed in a panel of 10 ovarian cancer cell lines, with the oncogenic PIM2 being the most commonly induced by cisplatin. Targeting PIM2 kinase by either biochemical inhibitors or RNA interference impaired cell growth, decreased cisplatin-triggered BAD phosphorylation, and sensitized ovarian cancer cells to drug-induced apoptosis. Overexpression of PIM2 triggered anchorage-independent growth and resulted in increased BAD phosphorylation and cell resistance to DNA damaging agents. The data show that the PIM2 kinase plays a role in the response of ovarian cancer cells to platinum drugs and suggest that PIM inhibitors may find clinical application as an adjunct to platinum-based therapies.

The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.—Musiani, D., Konda, J. D., Pavan, S., Torchiaro, E., Sassi, F., Noghero, A., Erriquez, J., Perera, T., Olivero, M., Di Renzo, M. F. Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy.

Differentiation therapy is an attractive treatment for osteosarcoma (OS). CD99 is a cell surface molecule expressed in mesenchymal stem cells and osteoblasts that is maintained during osteoblast differentiation while lost in OS. Herein, we show that whenever OS cells regain CD99, they become prone to reactivate the terminal differentiation program. In differentiating conditions, CD99-transfected OS cells express osteocyte markers, halt proliferation, and largely die by apoptosis, resembling the fate of mature osteoblasts. CD99 induces ERK activation, increasing its membrane-bound/cytoplasmic form rather than affecting its nuclear localization. Through cytoplasmic ERK, CD99 promotes activity of the main osteogenic transcriptional factors AP1 and RUNX2, which in turn enhance osteocalcin and p21WAF1/CIP1, leading to G0/G1 arrest. These data underscore the alternative positions of active ERK into distinct subcellular compartments as key events for determining OS fate. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Patients with advanced ovarian cancers have experienced little improvement in overall survival with standard treatments even after the incorporation of anti-angiogenic therapies. Besides anti-PARP inhibitors, matching individual critical genomic alterations with the best available drugs has not advanced as in other cancers, likely because a handful of cancer-related genes are mutated at high frequency, while many more are found mutated at much lower frequencies. This so called “mutation tail” is not only long but also mostly unexplored. We used Patient Derived Xenografts (PDXs) to identify actionable cancer genes and PDX Derived Tumor Cells (PDTCs) to accelerate the discovery of treatment options. We envisioned that the alleged weakness of PDX models, i.e. lack of human stromal and immune cells, might be instrumental to identify mutations in cancer and to test approved or experimental targeted drugs as monotherapy or in different combinations to link biomarkers to treatments. Forty-nine PDX lines from metastatic epithelial ovarian carcinomas have been propagated and fully characterized as far as histology, immunohistochemistry of epithelial and high-grade serous-specific markers and presence of TP53 and BRCA1/2 mutations. Copy number variations (CNV) analysis and Whole Exome Sequencing (WES) were carried out PDX lines derived from naïve metastatic high-grade epithelial ovarian carcinomas, which came out to be refractory/resistant to platinum drugs. We studied non-synonymous mutations with allele frequencies ≥0.1. Only mutations in cancer genes listed in databases were further analyzed. SNPdb allowed ruling out polymorphisms. SIFT and PROVEAN softwares predicted deleterious or damaging effects onto the protein sequences. DGIdb helped selecting actionable genes. We identified in one PDX line, a possibly loss-of-function mutation of the PIK3R1 gene (encoding the p85alpha regulatory subunit of PI3K) had an allele frequency=0.9 in early and late passages. Moreover, in two micro-dissected FFPE samples of the source tumor this mutation had an allele frequency nearly identical to that of the mutated TP53. Hence, PIK3R1W624R could be a trunk mutation in the PDX line and possibly in the human counterpart. Treatment options were assayed ex-vivo, on short-term cultures of PDTCs of the PIK3R1W624R PDX line. Buparlisib, a pan-class I PI3K inhibitor, showed the ability to block proliferation of PDTCs and the growth in vivo of PDXs in regression preclinical trial. These data proofed-the-concept that a PDX-based pipeline is able to unveil actionable pathways for the treatment of advanced/metastatic ovarian cancer. Citation Format: Martina Olivero, Jessica Erriquez, Maddalena Arigoni, Sonia Capellero, Concetta D'Ambrosio, Gloria Mittica, Fulvio Borella, Dionyssios Katsaros, Silvana Privitera, Enrico Berrino, Tiziana Venesio, Giorgio Valabrega, Raffaele Calogero and Maria Flavia Di Renzo. PATIENT DERIVED XENOGRAFTS (PDXS) AND PDX DERIVED TUMOR CELLS (PDTC) ALLOW THE IDENTIFICATION OF ACTIONABLE CANCER GENES AND TREATMENT OPTIONS FOR PLATINUM REFRACTORY/RESISTANT OVARIAN CARCINOMAS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-023.

The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.

// Jessica Erriquez 2, * , Martina Olivero 1, 2, * , Gloria Mittica 1, 2 , Maria Stella Scalzo 3 , Marco Vaira 2 , Michele De Simone 2 , Riccardo Ponzone 2 , Dionyssios Katsaros 4 , Massimo Aglietta 1, 2 , Raffaele Calogero 5 , Maria Flavia Di Renzo 1, 2 , Giorgio Valabrega 1, 2 1 Department of Oncology, University of Torino, Candiolo, Torino, Italy 2 Candiolo Cancer Institute, FPO-IRCCS Candiolo, Torino, Italy 3 Department of Medical Sciences, University of Torino, Torino, Italy 4 Department of Surgical Sciences, Gynecologic Oncology, AO-Universitaria Citta della Salute, Torino, Italy 5 Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Italy * These authors have contributed equally to this work Correspondence to: Maria Flavia Di Renzo, e-mail: mariaflavia.direnzo@unito.it Keywords: ovarian cancer, chemotherapy, patient derived xenograft Received: December 14, 2015 Accepted: March 14, 2016 Published: March 24, 2016 ABSTRACT Platinum-based chemotherapy is the recommended first-line treatment for high-grade serous (HGS) epithelial ovarian cancer (EOC). However, most patients relapse because of platinum refractory/resistant disease. We aimed at assessing whether other drugs, commonly used to treat relapsed HGS-EOC and poorly active in this clinical setting, might be more effective against chemotherapy-naive cancers. We collected couples of HGS-EOC samples from the same patients before and after neo-adjuvant platinum-based chemotherapy. Samples were propagated as Patient Derived Xenografts (PDXs) in immunocompromised mice (“xenopatients”). Xenopatients were treated in parallel with carboplatin, gemcitabine, pegylated liposomal doxorubicin (PLD) and trabectedin. PDXs derived from a naive HSG-EOC showed responsiveness to carboplatin, trabectedin and gemcitabine. The PDXs propagated from a tumor mass of the same patient, grown after carboplatin therapy, did no longer respond to trabectedin and gemcitabine and showed heterogeneous response to carboplatin. In line, the patient experienced clinically platinum-sensitivity first and then discordant responses of different tumor sites to platinum re-challenge. Loss of PDX responsiveness to drugs was associated with 4-fold increase of NR2F2 gene expression. PDXs from another naive tumor showed complete response to PLD, which was lost in the PDXs derived from a mass grown in the same patient after platinum-based chemotherapy. This patient showed platinum refractoriness and responded poorly to PLD as second-line treatment. PDX response to PLD was associated with high expression of TOP2A protein. PDXs demonstrated that chemotherapy-naive HGS-EOC might display susceptibility to agents not used commonly as first line treatment. Data suggest the importance of personalizing also chemotherapy.

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer.

The MET oncogene is aberrantly overexpressed in human osteosarcomas. We have previously converted primary cultures of human bone-derived cells into osteosarcoma cells by overexpressing MET. To determine whether MET transforms mesenchymal stem cells or committed progenitor cells, here we characterize distinct MET overexpressing osteosarcoma (MET-OS) clones using genome-wide expression profiling, cytometric analysis, and functional assays. All the MET-OS clones consistently display mesenchymal and stemness markers, but not most of the mesenchymal–stem cell-specific markers. Conversely, the MET-OS clones express genes characteristic of early osteoblastic differentiation phases, but not those of late phases. Profiling of mesenchymal stem cells induced to differentiate along osteoblast, adipocyte, and chondrocyte lineages confirms that MET-OS cells are similar to cells at an initial phase of osteoblastic differentiation. Accordingly, MET-OS cells cannot differentiate into adipocytes or chondrocytes, but can partially differentiate into osteogenic-matrix-producing cells. Moreover, in vitro MET-OS cells form self-renewing spheres enriched in cells that can initiate tumors in vivo. MET kinase inhibition abrogates the self-renewal capacity of MET-OS cells and allows them to progress toward osteoblastic differentiation. These data show that MET initiates the transformation of a cell population that has features of osteo-progenitors and suggest that MET regulates self-renewal and lineage differentiation of osteosarcoma cells. © 2012 American Society for Bone and Mineral Research.

Purpose: We determined the gene copy numbers for MET, for its transcriptional activator MACC1 and for its ligand hepatocyte growth factor (HGF) in liver metastases from colorectal carcinoma (mCRC). We correlated copy numbers with mRNA levels and explored whether gain and/or overexpression of MET and MACC1 predict response to anti-Met therapies. Finally, we assessed whether their genomic or transcriptional deregulation correlates with pathologic and molecular parameters of aggressive disease. Experimental Design: One hundred three mCRCs were analyzed. Copy numbers and mRNA were determined by quantitative PCR (qPCR). Thirty nine samples were implanted and expanded in NOD (nonobese diabetic)/SCID (severe combined immunodeficient) mice to generate cohorts that were treated with the Met inhibitor JNJ-38877605. In silico analysis of MACC1 targets relied on genome-wide mapping of promoter regions and on expression data from two CRC datasets. Results: No focal, high-grade amplifications of MET, MACC1, or HGF were detected. Chromosome 7 polysomy and gain of the p-arm were observed in 21% and 8% of cases, respectively, and significantly correlated with higher expression of both Met and MACC1. Met inhibition in patient-derived xenografts did not modify tumor growth. Copy number gain and overexpression of MACC1 correlated with unfavorable pathologic features better than overexpression of Met. Bioinformatic analysis of putative MACC1 targets identified elements besides Met, whose overexpression cosegregated with aggressive forms of colorectal cancer. Conclusions: Experiments in patient-derived xenografts suggest that mCRCs do not rely on Met genomic gain and/or overexpression for growth. On the basis of pathologic correlations and bioinformatic analysis, MACC1 could contribute to CRC progression through mechanisms other than or additional to Met transcriptional upregulation. Clin Cancer Res; 17(10); 3146–56. ©2011 AACR.

Companion animal spontaneous tumors are suitable models for human cancer, primarily because both animal population and the tumors are genetically heterogeneous. Feline mammary carcinoma (FMC) is a highly aggressive, mainly hormone receptor–negative cancer, which has been proposed as a model for poor prognosis human breast cancer. We have identified and studied the feline orthologue of the HER2 gene, which is both an important prognostic marker and therapeutic target in human cancer. Feline HER2 (f-HER2) gene kinase domain is 92% similar to the human HER2 kinase. F-HER2–specific mRNA was found 3- to 18-fold increased in 3 of 3 FMC cell lines, in 1 of 4 mammary adenomas and 6 of 11 FMC samples using quantitative reverse transcription-PCR. Western blot showed that an anti-human HER2 antibody recognized a protein comigrating with the human p185HER2 in FMC cell lines. The same antibodies strongly stained 13 of 36 FMC archival samples. These data show that feline HER2 overexpression qualifies FMC as homologous to the subset of HER2 overexpressing, poor prognosis human breast carcinomas and as a suitable model to test innovative approaches to therapy of aggressive tumors.

CDT2/L2DTL/RAMP is one of the substrate receptors of the Cullin Ring Ubiquitin Ligase 4 that targets for ubiquitin mediated degradation a number of substrates, such as CDT1, p21 and CHK1, involved in the regulation of cell cycle and survival. Here we show that CDT2 depletion was alone able to induce the apoptotic death in 12/12 human cancer cell lines from different tissues, regardless of the mutation profile and CDT2 expression level. Cell death was associated to rereplication and to loss of CDT1 degradation. Conversely, CDT2 depletion did not affect non-transformed human cells, such as immortalized kidney, lung and breast cell lines, and primary cultures of endothelial cells and osteoblasts. The ectopic over-expression of an activated oncogene, such as the mutation-activated RAS or the amplified MET in non-transformed immortalized breast cell lines and primary human osteoblasts, respectively, made cells transformed in vitro, tumorigenic in vivo, and susceptible to CDT2 loss. The widespread effect of CDT2 depletion in different cancer cells suggests that CDT2 is not in a synthetic lethal interaction to a single specific pathway. CDT2 likely is a non-oncogene to which transformed cells become addicted because of their enhanced cellular stress, such as replicative stress and DNA damage.

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals.

Background The Interferon Regulatory Factors (IRFs) are transcription factors involved in immune responses and oncogenesis and most of them are classified as tumour suppressors. The expression and activation of IRF(s) are stimulated by several cytokines and by DNA damage. Here we examine the role of the IRF-1 in the response of ovarian cancer cells to the front-line chemotherapeutic drug cisplatin (CDDP). Methods We evaluated the transcriptional response of three ovarian cancer cell lines to CDDP both under control conditions and after IRF-1 silencing using expression microarrays. The role played by IRF-1 in the response of these cells to CDDP was evaluated after silencing and overexpressing IRF-1. We studied cell cycle progression, colony forming ability in monolayer culture and semisolid medium, and apoptosis in the response to the drug. Results The treatment of ovarian cancer cells with CDDP boosted the expression and the nuclear translocation of IRF-1, which in turn modulated the expression of putative IRF-1 target genes. Accordingly, IRF-1 silencing re-orchestrated the expression profiles of CDDP-treated cells. In agreement with its role as a tumour suppressor, overexpressing IRF-1 suppressed the transformed phenotype of ovarian cancer cells. Nevertheless, IRF-1 silencing sensitized cells to the apoptotic death induced by CDDP. Over-expression was associated with cell G1 arrest and p21 induction irrespective of p53 proficiency, while IRF-1 silencing reduced the induction of p21 by CDDP. Conclusions These data demonstrate that IRF-1 is up-regulated by CDDP in ovarian cancer cells and might limit the cell response to CDDP, likely by inhibiting cell proliferation. Data suggest that IRF-1 induction might interfere with the effectiveness of combination therapy with platinum drugs and cytokines.

The c-met oncogene encodes the receptor for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional cytokine able to mediate morphogenesis as well as mitogenesis, motogenesis and invasiveness of epithelial cells. HGF/SF has been implicated in branching tubulogenesis of the developing kidney and in regeneration after renal injury and nephrectomy. We have examined the expression of the met/HGF receptor in normal human kidney and tissues of the genito-urinary tract, and in 50 kidney neoplasms of different histotypes, using monoclonal antibodies (MAbs) against the met/HGF receptor and immunohistochemistry. In normal kidneys, weak staining restricted to the distal tubules was observed. Transitional cell carcinomas were consistently negative, whereas increased expression at various levels was found in 87% of renal cell carcinomas with different cytological features and histological patterns. Western blot analysis of samples showed that the met/HGF receptor found in the malignant cells exhibits features of the normal receptor. The met/HGF receptor is also overexpressed in a renal cell carcinoma cell line, whose motility is triggered by HGF/SF. Our data suggest that expression of the met/HGF receptor may be involved in the onset and progression of renal cell carcinomas.

Platinum-based chemotherapy is the recommended first-line treatment for high-grade serous (HGS) epithelial ovarian cancer (EOC). However, most patients relapse because of platinum refractory/resistant disease. We aimed at assessing whether other drugs, commonly used to treat relapsed HGS-EOC and poorly active in this clinical setting, might be more effective against chemotherapy-naïve cancers. We collected samples of advanced HGS-EOC from the same patients before and after neo-adjuvant platinum-based chemotherapy. Samples were propagated as Patient Derived Xenografts (PDXs) in immunocompromised mice (“xenopatients”). Xenopatients were treated with carboplatin, gemcitabine, pegylated liposomal doxorubicin (PLD) and trabectedin. One patient was studied who experienced clinically platinum-sensitivity first and then discordant responses of different tumor sites to platinum re-challenge. PDXs derived from this patient before chemotherapy showed responsiveness to carboplatin, trabectedin and gemcitabine. The PDXs from the same patient after chemotherapy did no longer respond to trabectedin and gemcitabine and showed a heterogeneous response to carboplatin. Expression profiling showed that loss of responsiveness to drugs of the post-chemotherapy PDXs was associated with the up-regulation of NR2F2 gene expression. A second patient with platinum refractory HGS-EOC responded poorly to PLD as second-line treatment. PDXs obtained from this patient's tumor before chemotherapy showed a complete response to PLD, which was lost in the post-chemotherapy PDXs. Response to PLD was associated with the over-expression of the TOP2A protein, which was lost in the post-chemotherapy PDXs. Thus, PDXs demonstrated that chemotherapy-naïve HGS-EOC might display susceptibility to agents not used commonly as first line treatment. These data suggest the importance of tailoring chemotherapy. Citation Format: Jessica Erriquez, Martina Olivero, Gloria Mittica, Maria Stella Scalzo, Marco Vaira, Michele De Simone, Riccardo Ponzone, Dionyssios Katsaros, Massimo Aglietta, Raffaele Calogero, Maria Flavia Di Renzo, Giorgio Valabrega. Xenopatients help in redefining medical therapeutic algorithms in high risk ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-042.

Background Hepatocyte Growth Factor (HGF) enhances cytotoxicity of paclitaxel (PTX) and cisplatin (CDDP) in human ovarian cancer cells. Because of potential pitfalls of HGF exogenous administration, we investigated whether HGF serum concentration might be alternatively raised in vivo by administering low molecular weight heparin (LMWH). Methods The main HGF pharmacokinetic parameters were evaluated following acute and chronic LMWH treatment. First, women, operated on for gynaecological tumors, were treated with a single dose of calcium nadroparin and studied for 12 hours. Next, women operated on for benign or malignant gynaecological tumors were treated daily with calcic nadroparin for one month. Subsequently, the biological activity of the measured HGF serum levels was tested in assays of ovarian cancer cell sensitization to drugs. Results In the short-term treated group, median HGF AUCss, Cmax and Caverage were about four-fold that of the control group, whereas Cmin was three-fold. In the patients treated chronically median HGF serum levels rose about six-fold in the first week, and decreased but remained significantly higher after one month. The pharmacokinetic of nadroparin-dependent HGF increase were similar in the two groups. The HGF concentrations measured after both acute and chronic treatment were found to be effective in sensitising ovarian cancer cells to chemotherapeutics. Conclusions This study raises the possibility of using LMWH to increase HGF serum concentration and to take advantage of its biological activities. In particular, nadroparin might be used as a chemo-potentiating agent in epithelial cell ovarian carcinoma through its action on HGF serum concentration. Trial registration ClinicalTrials.gov ID: NCT01523652

The cellular apoptosis susceptibility gene CAS/CSE1L is overexpressed in cancer, although it was originally identified as a gene that renders cells vulnerable to apoptotic stimuli. CAS/CSE1L has roles in the nucleocytoplasmic recycling of importin-α and in the regulation of gene expression, cell migration, and secretion. We identified CAS/CSE1L as a survival factor for ovarian cancer cells in vitro and in vivo. In 3/3 ovarian cancer cell lines, CAS/CSE1L was down-modulated by the unorthodox proapoptotic signaling of the MET receptor. CAS/CSE1L knockdown with RNA interference committed the ovarian cancer cells to death, but not immortalized normal cells and breast and colon cancer cells. In 70 and 95% of these latter cells, respectively, CAS/CSE1L was localized in the cytoplasm, while it accumulated in the nucleus in90% of ovarian cancer cells. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells. In the nucleus, CAS/CSE1L regulated the expression of the proapoptotic Ras-association domain family 1 gene products RASSF1C and RASSF1A, which mediated death signals evoked by depletion of CAS/CSE1L. Our data show that CAS/CSE1L protects ovarian cancer cells from death through transcriptional suppression of a proapoptotic gene and suggest that the localization of CAS/CSE1L dictates its function.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by mutations in the Krebs cycle enzyme fumarate hydratase (FH). It has been proposed that “pseudohypoxic” stabilization of hypoxia-inducible factor-α (HIF-α) by fumarate accumulation contributes to tumorigenesis in HLRCC. We hypothesized that an additional direct consequence of FH deficiency is the establishment of a biosynthetic milieu. To investigate this hypothesis, we isolated primary mouse embryonic fibroblast (MEF) lines from Fh1-deficient mice. As predicted, these MEFs upregulated Hif-1α and HIF target genes directly as a result of FH deficiency. In addition, detailed metabolic assessment of these MEFs confirmed their dependence on glycolysis, and an elevated rate of lactate efflux, associated with the upregulation of glycolytic enzymes known to be associated with tumorigenesis. Correspondingly, Fh1-deficient benign murine renal cysts and an advanced human HLRCC-related renal cell carcinoma manifested a prominent and progressive increase in the expression of HIF-α target genes and in genes known to be relevant to tumorigenesis and metastasis. In accord with our hypothesis, in a variety of different FH-deficient tissues, including a novel murine model of Fh1-deficient smooth muscle, we show a striking and progressive upregulation of a tumorigenic metabolic profile, as manifested by increased PKM2 and LDHA protein. Based on the models assessed herein, we infer that that FH deficiency compels cells to adopt an early, reversible, and progressive protumorigenic metabolic milieu that is reminiscent of that driving the Warburg effect. Targets identified in these novel and diverse FH-deficient models represent excellent potential candidates for further mechanistic investigation and therapeutic metabolic manipulation in tumors. Cancer Res; 70(22); 9153–65. ©2010 AACR.

Loss of the fumarate hydratase (FH) tumor suppressor gene results in the development of benign tumors that rarely, but regrettably, progress to very aggressive cancers. Using mouse embryo fibroblasts (MEFs) to model transformation, we found that fh knockdown results in increased expression of the met oncogene-encoded tyrosine kinase receptor through hypoxia-inducible factor (hif) stabilization. MET-increased expression was alone able to stabilize hif, thus establishing a feed forward loop that might enforce tumor progression. The fh-defective MEFs showed increased motility and protection from apoptosis. Motility, but not survival, relied on hif-1alpha and was greatly enhanced by MET ligand hepatocyte growth factor. Met cooperated with a weakly oncogenic ras in making MEFs transformed and tumorigenic, as shown by in vitro and in vivo assays. Loss of fh was not equally effective by itself but enhanced the transformed and tumorigenic phenotype induced by ras and MET. Consistently, the rescue of fumarase expression abrogated the motogenic and transformed phenotype of fh-defective MEFs. In conclusion, the data suggest that the progression of tumors where FH is lost might be boosted by activation of the MET oncogene, which is able to drive cell-autonomous tumor progression and is a strong candidate for targeted therapy.

Estrogen receptor alpha (ERalpha) and E-cadherin are primary markers of luminal epithelial breast cancer cells with E-cadherin being a main caretaker of the epithelial phenotype. E-cadherin repression is needed for cancer cells to acquire motile and invasive properties, and it is known that in ER-positive breast cancer cells, estrogen down-regulate E-cadherin gene transcription. We report here that ERalpha is bound to the E-cadherin promoter in both the presence and the complete absence of estrogen, suggesting an unexpected role for unliganded ERalpha in E-cadherin transcription. Indeed, our data reveal that activation by unliganded ERalpha and repression by estrogen-activated ERalpha require direct binding to a half-estrogen response element within the E-cadherin promoter and exchange from associated coactivators to corepressors. Therefore, these results suggest a pivotal role for unliganded ERalpha in controlling a fundamental caretaker of the epithelial phenotype in breast cancer cells. Here, we show that ERalpha-positive breast cancer T47D cells transduced with the sfRON kinase undergo a full epithelial-mesenchymal conversion and lose E-cadherin and ERalpha expression. Our data show that, although the E-cadherin gene becomes hypermethylated and heterochromatic, kinase inhibitors can restore E-cadherin expression, together with an epithelial morphology in an ERalpha-dependent fashion. Similarly, transfection of ERalpha, in the absence of ligands, was sufficient to restore E-cadherin transcription in both sfRON-T47D and other ERalpha-, E-cadherin-negative cells. Therefore, our results suggest a novel role for the ERalpha that plays the dual role of ligand-independent activator and ligand-dependent repressor of E-cadherin in breast cancer cells.