Simon J. Cooper

Pathway Signature and Cellular Differentiation in Clear Cell Renal Cell Carcinoma

Background Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer. The purpose of this study is to define a biological pathway signature and a cellular differentiation program in ccRCC. Methodology We performed gene expression profiling of early-stage ccRCC and patient-matched normal renal tissue using Affymetrix HG-U133a and HG-U133b GeneChips combined with a comprehensive bioinformatic analyses, including pathway analysis. The results were validated by real time PCR and IHC on two independent sample sets. Cellular differentiation experiments were performed on ccRCC cell lines and their matched normal renal epithelial cells, in differentiation media, to determine their mesenchymal differentiation potential. Principal Findings We identified a unique pathway signature with three major biological alterations—loss of normal renal function, down-regulated metabolism, and immune activation–which revealed an adipogenic gene expression signature linked to the hallmark lipid-laden clear cell morphology of ccRCC. Culturing normal renal and ccRCC cells in differentiation media showed that only ccRCC cells were induced to undergo adipogenic and, surprisingly, osteogenic differentiation. A gene expression signature consistent with epithelial mesenchymal transition (EMT) was identified for ccRCC. We revealed significant down-regulation of four developmental transcription factors (GATA3, TFCP2L1, TFAP2B, DMRT2) that are important for normal renal development. Conclusions ccRCC is characterized by a lack of epithelial differentiation, mesenchymal/adipogenic transdifferentiation, and pluripotent mesenchymal stem cell-like differentiation capacity in vitro. We suggest that down-regulation of developmental transcription factors may mediate the aberrant differentiation in ccRCC. We propose a model in which normal renal epithelial cells undergo dedifferentiation, EMT, and adipogenic transdifferentiation, resulting in ccRCC. Because ccRCC cells grown in adipogenic media regain the characteristic ccRCC phenotype, we have indentified a new in vitro ccRCC cell model more resembling ccRCC tumor morphology.

Stearoyl-CoA Desaturase 1 Is a Novel Molecular Therapeutic Target for Clear Cell Renal Cell Carcinoma

Purpose: We set out to identify Stearoyl-CoA desaturase 1 (SCD1) as a novel molecular target in clear cell renal cell carcinoma (ccRCC) and examine its role in tumor cell growth and viability in vitro and in vivo independently as well as in combination with current U.S. Food and Drug Administration (FDA)-approved regimens. Experimental Design: Patient normal and ccRCC tissue samples and cell lines were examined for SCD1 expression. Genetic knockdown models and targeted inhibition of SCD1 through use of a small molecule inhibitor, A939572, were analyzed for growth, apoptosis, and alterations in gene expression using gene array analysis. Therapeutic models of synergy were evaluated utilizing pharmacologic inhibition of SCD1 with the tyrosine kinase inhibitors (TKI) sunitinib and pazopanib, and the mTOR inhibitor temsirolimus. Results: Our studies identify increased SCD1 expression in all stages of ccRCC. Both genetic knockdown and pharmacologic inhibition of SCD1 decreased tumor cell proliferation and induced apoptosis in vitro and in vivo. Upon gene array, quantitative real-time PCR, and protein analysis of A939572-treated or SCD1 lentiviral knockdown samples, induction of endoplasmic reticulum stress response signaling was observed, providing mechanistic insight for SCD1 activity in ccRCC. Furthermore, combinatorial application of A939572 with temsirolimus synergistically inhibited tumor growth in vitro and in vivo. Conclusions: Increased SCD1 expression supports ccRCC viability and therefore we propose it as a novel molecular target for therapy either independently or in combination with an mTOR inhibitor for patients whose disease cannot be remedied with surgical intervention, such as in cases of advanced or metastatic disease. Clin Cancer Res; 19(9); 2368–80. ©2013 AACR.

Reactivation of Suppressed RhoB is a Critical Step for the Inhibition of Anaplastic Thyroid Cancer Growth

Anaplastic thyroid carcinoma (ATC) is a highly aggressive form of the disease for which new therapeutic options are desperately needed. Previously, we showed that the high-affinity peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, RS5444, inhibits cell proliferation of ATC cells via induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21). We show here that up-regulation of RhoB is a critical step in PPARgamma-mediated activation of p21-induced cell stasis. Using multiple independently derived ATC cell lines, we found that treatment with RS5444 leads to the up-regulation of RhoB and subsequent activation of p21, and that silencing of RhoB by RNAi blocks the ability of RS5444 to induce p21 and to inhibit cell proliferation. Our results show that transcriptional regulation of RhoB by the nuclear transcription factor PPARgamma is responsible for the induction of p21 mRNA and protein. We further implicate RhoB as a key signaling effector for the growth inhibition of ATC, as treatment with a histone deacetylase inhibitor shown to increase RhoB expression in lung cancer cells caused the up-regulation of RhoB in ATC cells accompanied by increased expression of p21 and inhibition of cell proliferation; this effect occurred even in ATC cells that were unresponsive to RS5444 due to a lack of expression of PPARgamma. Our results implicate RhoB as a novel intermediate in critical signaling pathways and as an additional target for therapeutic intervention in ATC.

p38 MAP kinase plays a functional role in UVB-induced mouse skin carcinogenesis.

UVB irradiation of epidermal keratinocytes results in the activation of the p38 mitogen‐activated protein kinase (MAPK) pathway and subsequently activator protein‐1 (AP‐1) transcription factor activation and cyclooxygenase‐2 (COX‐2) expression. AP‐1 and COX‐2 have been shown to play functional roles in UVB‐induced mouse skin carcinogenesis. In this study, the experimental approach was to express a dominant negative p38α MAPK (p38DN) in the epidermis of SKH‐1 hairless mice and assess UVB‐induced AP‐1 activation, COX‐2 expression, and the skin carcinogenesis response in these mice compared to wild‐type littermates. We observed a significant inhibition of UVB‐induced AP‐1 activation and COX‐2 expression in p38DN transgenic mice, leading to a significant reduction of UVB‐induced tumor number and growth compared to wild‐type littermates in a chronic UVB skin carcinogenesis model. A potential mechanism for this reduction in tumor number and growth rate is an inhibition of chronic epidermal proliferation, observed as reduced Ki‐67 staining in p38DN mice compared to wild‐type. Although we detected no difference in chronic apoptotic rates between transgenic and nontransgenic mice, analysis of acutely irradiated mice demonstrated that expression of the p38DN transgene significantly inhibited UVB‐induced apoptosis of keratinocytes. These results counter the concerns that inhibition of p38 MAPK in a chronic situation could compromise the ability of the skin to eliminate potentially tumorigenic cells. Our data indicate that p38 MAPK is a good target for pharmacological intervention for UV‐induced skin cancer in patients with sun damaged skin, and suggest that inhibition of p38 signaling reduces skin carcinogenesis by inhibiting COX‐2 expression and proliferation of UVB‐irradiated cells.

Detailed Molecular Fingerprinting of Four New Anaplastic Thyroid Carcinoma Cell Lines and Their Use for Verification of RhoB as a Molecular Therapeutic Target

CONTEXT Anaplastic thyroid carcinoma (ATC) is a highly aggressive carcinoma in need of therapeutic options. One critical component of drug discovery is the availability of well-characterized cell lines for identification of molecular mechanisms related to tumor biology and drug responsiveness. Up to 42% of human thyroid cancer cell lines are redundant or not of correct tissue origin, and a comprehensive analysis is currently nonexistent. Mechanistically, RhoB has been identified as a novel molecular target for ATC therapy. OBJECTIVE The aim was to develop four ATC cell lines detailing genetic, molecular, and phenotypic characteristics and to test five classes of drugs on the cell lines to determine whether they inhibited cell proliferation in a RhoB-dependent fashion. DESIGN Four cell lines were derived from ATC tumors. Short tandem DNA repeat and mutational status of the originating tumors and cell lines were performed along with molecular and phenotypic characterizations. Compounds were tested for growth inhibition and ability to up-regulate RhoB. RESULTS Cell line authenticity was confirmed by DNA short tandem repeat analysis. Each proved unique regarding expression of thyroid markers, oncogene status, amplified and deleted genes, and proliferative growth rates. FTI-277, GGTI-286, lovastatin, romidepsin, and UCN-01 up-regulated RhoB and inhibited cell proliferation in a dose-responsive fashion with only romidepsin and FTI-277 being RhoB dependent. CONCLUSIONS Molecular descriptions of thyroid lines were matched to the originating tumors, setting a new standard for cell line characterization. Furthermore, suppressed RhoB is implicated as a molecular target for therapy against ATC because five classes of drugs up-regulate RhoB and inhibit growth dose-responsively.

Expression of the type III TGF-β receptor is negatively regulated by TGF-β

The type III transforming growth factor-beta receptor (TbetaRIII or betaglycan) is a ubiquitously expressed transforming growth factor-beta (TGF-beta) superfamily coreceptor with essential roles in embryonic development. Recent studies have defined a role for TbetaRIII in the pathogenesis of human cancers, with frequent loss of TbetaRIII expression at the message and protein level. Mechanisms for the loss of TbetaRIII expression remain to be fully defined. Advanced human cancers often have elevated circulating levels of TGF-beta1. Here, we define a specific role for TGF-beta1 in negatively regulating TbetaRIII at the message level in breast and ovarian cancer models. TGF-beta1 decreased TbetaRIII message and protein levels in ovarian (Ovca420) and breast cancer (MDA-MB-231) cell lines in both a dose- and time-dependent manner. TGF-beta1-mediated TbetaRIII repression is mediated by the type I TGF-beta receptor/Smad2/3 pathway as the activin receptor-like kinase 5 (ALK5) inhibitor, SB431542, abrogated this effect, while the expression of constitutively active ALK5 was sufficient to repress TbetaRIII expression. Mechanistically, TGF-beta1 does not affect TbetaRIII messenger RNA (mRNA) stability, but instead directly regulates the TbetaRIII promoter. We define alternative promoters for the TGFBR3 gene, a distal and proximal promoter. Although both promoters are active, only the proximal promoter was responsive and negatively regulated by TGF-beta1 and constitutively active ALK5. Taken together, these studies define TGF-beta1-mediated downregulation of TbetaRIII mRNA expression through effects on the ALK5/Smad2/3 pathway on the TGFBR3 gene proximal promoter as a potential mechanism for decreased TbetaRIII expression in human cancers.

Reexpression of Tumor Suppressor, sFRP1, Leads to Antitumor Synergy of Combined HDAC and Methyltransferase Inhibitors in Chemoresistant Cancers

Metastatic solid tumors are aggressive and mostly drug resistant, leading to few treatment options and poor prognosis as seen with clear cell renal cell carcinoma (ccRCC) and triple-negative breast cancer (TNBC). Therefore, the identification of new therapeutic regimes for the treatment of metastatic disease is desirable. ccRCC and TNBC cell lines were treated with the HDAC inhibitor romidepsin and the methyltransferase inhibitor decitabine, two epigenetic modifying drugs approved by the U.S. Food and Drug Administration for the treatment of various hematologic malignancies. Cell proliferation analysis, flow cytometry, quantitative PCR, and immunoblotting techniques were used to evaluate the antitumor synergy of this drug combination and identify the reexpression of epigenetically silenced tumor suppressor genes. Combinatorial treatment of metastatic TNBC and stage IV ccRCC cell lines with romidepsin/decitabine leads to synergistic inhibition of cell growth and induction of apoptosis above levels of individual drug treatments alone. Synergistic reexpression of the tumor suppressor gene secreted frizzled-related protein one (sFRP1) was observed in combinatorial drug-treated groups. Silencing sFRP1 (short hairpin RNA) before combinatorial drug treatment showed that sFRP1 mediates the growth inhibitory and apoptotic activity of combined romidepsin/decitabine. Furthermore, addition of recombinant sFRP1 to ccRCC or TNBC cells inhibits cell growth in a dose-dependent manner through the induction of apoptosis, identifying that epigenetic silencing of sFRP1 contributes to renal and breast cancer cell survival. Combinatorial treatment with romidepsin and decitabine in drug resistant tumors is a promising treatment strategy. Moreover, recombinant sFRP1 may be a novel therapeutic strategy for cancers with suppressed sFRP1 expression. Mol Cancer Ther; 11(10); 2105–15. ©2012 AACR.

Loss of type III transforming growth factor-β receptor expression is due to methylation silencing of the transcription factor GATA3 in renal cell carcinoma

Loss of transforming growth factor-β receptor III (TβRIII) correlates with loss of transforming growth factor-β (TGF-β) responsiveness and suggests a role for dysregulated TGF-β signaling in clear cell renal cell carcinoma (ccRCC) progression and metastasis. Here we identify that for all stages of ccRCC TβRIII expression is downregulated in patient-matched tissue samples and cell lines. We find that this loss of expression is not due to methylation of the gene and we define GATA3 as the first transcriptional factor to positively regulate TβRIII expression in human cells. We localize GATA3′s binding to a 10-bp region of the TβRIII proximal promoter. We demonstrate that GATA3 mRNA is downregulated in all stages, of ccRCC, mechanistically show that GATA3 is methylated in ccRCC patient tumor tissues as well as cell lines, and that inhibiting GATA3 expression in normal renal epithelial cells downregulates TβRIII mRNA and protein expression. These data support a sequential model whereby loss of GATA3 expression through epigenetic silencing decreases TβRIII expression during ccRCC progression.

Type III Transforming Growth Factor-β (TGF-β) Receptor Mediates Apoptosis in Renal Cell Carcinoma Independent of the Canonical TGF-β Signaling Pathway

Purpose: Alterations in transforming growth factor-β (TGF-β) signaling occur early during malignant transformation of renal epithelial cells and are associated with loss of type III TGF-β receptor (TβRIII) expression. We evaluated the role of TβRIII in mediation of apoptosis using in vitro cell culture and in vivo animal models of clear cell renal cell carcinoma. Experimental Design: TβR3 expression was manipulated with adenoviral gene vector delivery system in vitro and in vivo. Induction of apoptosis and signaling through the Smad and mitogen-activated protein kinase (MAPK) pathways were examined at various time points after infection. To study viral oncolysis in vivo, human renal cell carcinoma cells were implanted s.c. in the flanks of nude mice and treated with intratumoral injections of adenovirus. Results: Restoring TβRIII expression in clear cell renal cell carcinoma resulted in a marked induction of apoptosis using in vitro cell culture and in vivo animal models. The expression of the cytoplasmic domain, but not the extracellular domain, of TβRIII mimicked the induction of apoptosis by full-length TβRIII in cell culture and the growth inhibition of tumors in athymic nude mice. TβRIII-associated apoptosis was not dependent on signaling through the canonical TGF-β/Smad pathway but was mediated through p38 MAPK. Conclusion: These findings indicate a novel mechanistic antitumor function for TβRIII and further support its role as an important tumor suppressor in clear cell renal cell carcinoma.

Neuronal Pentraxin 2 Supports Clear Cell Renal Cell Carcinoma by Activating the AMPA-Selective Glutamate Receptor-4

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and has the highest propensity to manifest as metastatic disease. Recent characterizations of the genetic signature of ccRCC have revealed several factors correlated with tumor cell migration and invasion; however, the specific events driving malignancy are not well defined. Furthermore, there remains a lack of targeted therapies that result in long-term, sustainable response in patients with metastatic disease. We show here that neuronal pentraxin 2 (NPTX2) is overexpressed specifically in ccRCC primary tumors and metastases, and that it contributes to tumor cell viability and promotes cell migration through its interaction with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR4. We propose NPTX2 as a novel molecular target for therapy for patients with ccRCC diagnosed with or at risk of developing metastatic disease.