Estelle Li

Targeting Amino Acid Transport in Metastatic Castration-Resistant Prostate Cancer: Effects on Cell Cycle, Cell Growth, and Tumor Development

BACKGROUND L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. METHODS We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. RESULTS LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4-7 months of neoadjuvant hormone therapy (4-7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4-mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. CONCLUSION Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Integrated genome and transcriptome sequencing identifies a novel form of hybrid and aggressive prostate cancer

Next‐generation sequencing is making sequence‐based molecular pathology and personalized oncology viable. We selected an individual initially diagnosed with conventional but aggressive prostate adenocarcinoma and sequenced the genome and transcriptome from primary and metastatic tissues collected prior to hormone therapy. The histology‐pathology and copy number profiles were remarkably homogeneous, yet it was possible to propose the quadrant of the prostate tumour that likely seeded the metastatic diaspora. Despite a homogeneous cell type, our transcriptome analysis revealed signatures of both luminal and neuroendocrine cell types. Remarkably, the repertoire of expressed but apparently private gene fusions, including C15orf21:MYC, recapitulated this biology. We hypothesize that the amplification and over‐expression of the stem cell gene MSI2 may have contributed to the stable hybrid cellular identity. This hybrid luminal‐neuroendocrine tumour appears to represent a novel and highly aggressive case of prostate cancer with unique biological features and, conceivably, a propensity for rapid progression to castrate‐resistance. Overall, this work highlights the importance of integrated analyses of genome, exome and transcriptome sequences for basic tumour biology, sequence‐based molecular pathology and personalized oncology. Copyright

Heterogeneity in the inter-tumor transcriptome of high risk prostate cancer

BackgroundGenomic analyses of hundreds of prostate tumors have defined a diverse landscape of mutations and genome rearrangements, but the transcriptomic effect of this complexity is less well understood, particularly at the individual tumor level. We selected a cohort of 25 high-risk prostate tumors, representing the lethal phenotype, and applied deep RNA-sequencing and matched whole genome sequencing, followed by detailed molecular characterization.ResultsTen tumors were exposed to neo-adjuvant hormone therapy and expressed marked evidence of therapy response in all except one extreme case, which demonstrated early resistance via apparent neuroendocrine transdifferentiation. We observe high inter-tumor heterogeneity, including unique sets of outlier transcripts in each tumor. Interestingly, outlier expression converged on druggable cellular pathways associated with cell cycle progression, translational control or immune regulation, suggesting distinct contemporary pathway affinity and a mechanism of tumor stratification. We characterize hundreds of novel fusion transcripts, including a high frequency of ETS fusions associated with complex genome rearrangements and the disruption of tumor suppressors. Remarkably, several tumors express unique but potentially-oncogenic non-ETS fusions, which may contribute to the phenotype of individual tumors, and have significance for disease progression. Finally, one ETS-negative tumor has a striking tandem duplication genotype which appears to be highly aggressive and present at low recurrence in ETS-negative prostate cancer, suggestive of a novel molecular subtype.ConclusionsThe multitude of rare genomic and transcriptomic events detected in a high-risk tumor cohort offer novel opportunities for personalized oncology and their convergence on key pathways and functions has broad implications for precision medicine.

Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

Background Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR) was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood. Methods Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels. Results Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1) and interlukin-6 (IL-6) by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion. Conclusions Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

Ultrasound-guided intramural inoculation of orthotopic bladder cancer xenografts: a novel high-precision approach.

Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23–37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice.

Crosstalk Between Nuclear MET and SOX9/β-Catenin Correlates with Castration-Resistant Prostate Cancer

Castration-resistant prostate cancer (PCa) (CRPC) is relapse after various forms of androgen ablation therapy and causes a major mortality in PCa patients, yet the mechanism remains poorly understood. Here, we report the nuclear form of mesenchymal epithelial transition factor (nMET) is essential for CRPC. Specifically, nMET is remarkably increased in human CRPC samples compared with naïve samples. Androgen deprivation induces endogenous nMET and promotes cell proliferation and stem-like cell self-renewal in androgen-nonresponsive PCa cells. Mechanistically, nMET activates SRY (sex determining region Y)-box9, β-catenin, and Nanog homeobox and promotes sphere formation in the absence of androgen stimulus. Combined treatment of MET and β-catenin enhances the inhibition of PCa cell growth. Importantly, MET accumulation is detected in nucleus of recurrent prostate tumors of castrated Pten/Trp53 null mice, whereas MET elevation is predominantly found in membrane of naïve tumors. Our findings reveal for the first time an essential role of nMET association with SOX9/β-catenin in CRPC in vitro and in vivo, highlighting that nuclear RTK activate cell reprogramming to drive recurrence, and targeting nMET would be a new avenue to treat recurrent cancers.

ER stress protein GRP78 as a therapeutic target combined with antiangiogenic therapy in renal cell carcinoma.

428 Background: Antiangiogenic therapy deprives oxygen and nutrition from the tumor. These stresses cause unfolded proteins in tumor cells. Glucose regulated protein 78 (GRP78) binds to unfolded proteins and its subsequent activation suppresses global mRNA translation to protect cells from excessive unfolded proteins. We investigated a potential role of GRP78 as a combined therapeutic target in renal cell carcinoma treated with antiangiogenic therapy. METHODS Renal cell carcinoma cells (Caki-1, Caki-2, UMRC-3, and UMRC-6) were used to investigate the effect of GRP78 knockdown, which was performed by small interfering RNA. Caki-1 xenografts were developed and treated with sunitinib 40mg/kg/day to evaluate in vivo expression of GRP78 during antiangiogenic therapy. Caki-1 cells stably overexpressing GRP78 were developed to investigate the role of GRP78 in cancer cell. Hypoxic stress was induced by 1% hypoxia chamber and hypoglycaemic stress was induced by glucose-free media. Downstream signalling pathways of GRP78 were evaluated by Western blots. RESULTS In vitro hypoxia and/or glucose deprivation induced GRP78 upregulation in Caki-1 cells. GRP78 was also induced in Caki-1 xenografts treated by sunitinib. Overexpression of GRP78 increased tumor proliferation in hypoxic and/or hypoglycemic stresses by activating PERK/eIF2α pathway and protected tumor cells from stress-induced apoptosis. Knockdown of GRP78 using small interference RNA inhibited cancer cell survival and induced apoptosis in renal cell carcinoma cells in vitro. GRP78 knockdown also sensitized renal cell carcinoma cells to ER stress-induced apoptosis and hypoxic and hypoplycemic stress-induces apoptosis. CONCLUSIONS Antiangiogenic therapy induces ER stress by depriving oxygen and glucose from renal cell carcinoma. ER protein GRP78 has a critical role in protecting renal cell carcinoma cells from hypoxic and hypoglycemic stress induced by antiangiogenic therapy. Knockdown of GRP78 sensitizes RCC cells to apoptotic cell death from anti-angiogenic stresses. Our results suggest that GRP78 is a novel therapeutic target in renal cell carcinoma management.

Abstract 400: Coordinated loss of CD9 and IGSF8 expression on the prostate cancer cell surface contributes to metastasis and hormone responsiveness.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The tetraspanin web is a complex multi-molecular cell structure that tethers cell surface signalling proteins (i.e. integrins) to their intermediate intracellular signalling partners (i.e. small GTPases) and anchors actin filaments. Loss of certain components of the web (CD9, CD82 or CD316) are associated with increased invasive and metastatic behaviors of prostate cancer (PCa) cells and here we demonstrate that two of these tetraspanin web components, CD9 and CD316, also play a role in regulating androgen signaling in PCa cells. Additionally, we utilized human prostate cancer tissue microarrays (TMAs) containing 261 prostate/PCa specimens of differing stages, grades and states of progression (including 60 castration resistant tumors [CRPCs]) to study the relationship between CD9 and CD316 expressions and progression to CRPC. Using androgen-dependent LNCaP cells as a model, we found that androgen deprivation (AD) suppressed expression of CD9 protein and mRNA without affecting CD316 expression. FACS analysis showed that the presence of CD9 on the cell surface was coordinately reduced by AD as was cell surface CD316 which was relocalized to the cytoplasm. This effect was reversed by addition of R1881. Knockdown of CD9 or CD316 in LNCaP cells using siRNA reduced expression of androgen receptor (AR) mRNA but increased expression of PSA, an AR target gene. CD9 or CD316 knockdown also increased LNCaP growth in fetal bovine serum (FBS) containing medium but not in charcoal-stripped FBS (CS-FBS) whereas dual-knockdown further increased growth in FBS as well as in CS-FBS. TMAs immunostained for CD9 or CD316 were assessed for correlation to clinicopathological parameters. For untreated, primary tumors, CD9 and CD316 expression were inversely correlated with Gleason scores (R2=0.9612). CD9 expression was diminished during progression to metastatic (p<0.0038, compared to non-metastatic) and further in CRPC (p<0.040, compared to metastatic non-treated) disease. Conversely, CD316 expression was increased in primary (non-metastatic) as well as in metastatic untreated disease but then reduced (p<0.046) during progression to metastatic CRPC. We propose that androgen deprivation (first line therapy for advanced PCa) may potentially trigger loss of both CD9 and IGSF8 expression on the cell surface that contributes to metastatic behaviors as well as to responsiveness to androgen ablation therapies. This work is supported by the Cancer Research Society (Canada). Citation Format: Na Li, Ladan Fazli, Estelle Li, Ralph Buttyan. Coordinated loss of CD9 and IGSF8 expression on the prostate cancer cell surface contributes to metastasis and hormone responsiveness. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2013-400

The effect of activation of angiogenesis pathways independent of VEGF in renal cell carcinoma on resistance to VEGF-targeted antiangiogenic therapy.

409 Background: VEGF-targeted anti-angiogenic therapy provides significant growth inhibition in clear cell type renal cell carcinoma (CCRCC). However, evasive resistance develops in most responding cases due to unclear mechanisms. We investigated the mechanism of resistance to VEGF-targeted therapy in CCRCC both in vitro and in vivo. METHODS Two different conditioned cell lines were developed from wild type Caki-1. Sunitinib-conditioned Caki-1 was developed by chronic exposures to sunitinib (up to 15uM) and hypoxia-conditioned Caki-1 was developed by chronic exposures to hypoxia (1% oxygen). We characterized these conditioned cells in vitro and response patterns to sunitinib were evaluated using subcutaneous xenograft models with parental and conditioned cells. In vivo angiogenesis assays were performed to characterize angiogenesis potentials in these cells. Finally, mRNA microarray was performed to find pathways that induce resistance to anti-angiogenic therapy. RESULTS Sunitinib inhibited proliferation of HUVEC cells, but did not inhibit tumor proliferation in CCRCC cells at pharmacologically relevant doses. In vitro sunitinib-conditioned Caki-1 cells did not show obvious resistance to sunitinib compared to parental cells, but when tested in vivo these cells appeared to be highly resistant to sunitinib therapy. In contrast, hypoxia-conditioned Caki-1, although more resistant to hypoxia and showing increased vascularity by upregulating VEGF production, did not develop sunitinib resistance either in vitro or in vivo compared to parental cells. In vivo matrigel plug assay with sunitinib treatment confirmed that tumor angiogenesis was relatively intact and less affected by sunitinib treatment in xenografts of sunitinib-conditioned cells compared to parental cells. CONCLUSIONS Resistance to VEGF-targeted therapy is acquired by activation of VEGF-independent angiogenesis pathways induced by interactions with VEGF-targeted drug but not by hypoxia. Our results suggest that more broad inhibitions of tumor angiogenesis are required to prevent development of resistance to anti-angiogenic therapy in CCRCC.