Cabozantinib and dasatinib synergize to induce tumor regression in non-clear cell renal cell carcinoma.

The lack of effective treatment options for advanced non-clear cell renal cell carcinoma (NCCRCC) is a critical unmet clinical need. Applying a high-throughput drug screen to multiple human kidney cancer cells, we identify the combination of the VEGFR-MET inhibitor cabozantinib and the SRC inhibitor dasatinib acts synergistically in cells to markedly reduce cell viability. Importantly, the combination is well tolerated and causes tumor regression in vivo. Transcriptional and phosphoproteomic profiling reveals that the combination converges to downregulate the MAPK-ERK signaling pathway, a result not predicted by single-agent analysis alone. Correspondingly, the addition of a MEK inhibitor synergizes with either dasatinib or cabozantinib to increase its efficacy. This study, by using approved, clinically relevant drugs, provides the rationale for the design of effective combination treatments in NCCRCC that can be rapidly translated to the clinic.

Multi-Omics Analyses Detail Metabolic Reprogramming in Lipids, Carnitines, and Use of Glycolytic Intermediates between Prostate Small Cell Neuroendocrine Carcinoma and Prostate Adenocarcinoma.

As the most common cancer in men, prostate cancer is molecularly heterogeneous. Contributing to this heterogeneity are the poorly understood metabolic adaptations of the two main types of prostate cancer, i.e., adenocarcinoma and small cell neuroendocrine carcinoma (SCNC), the latter being more aggressive and lethal. Using transcriptomics, untargeted metabolomics and lipidomics profiling on LASCPC-01 (prostate SCNC) and LNCAP (prostate adenocarcinoma) cell lines, we found significant differences in the cellular phenotypes of the two cell lines. Gene set enrichment analysis on the transcriptomics data showed 62 gene sets were upregulated in LASCPC-01, while 112 gene sets were upregulated in LNCAP. ChemRICH analysis on metabolomics and lipidomics data revealed a total of 25 metabolite clusters were significantly different. LASCPC-01 exhibited a higher glycolytic activity and lower levels of triglycerides, while the LNCAP cell line showed increases in one-carbon metabolism as an exit route of glycolytic intermediates and a decrease in carnitine, a mitochondrial lipid transporter. Our findings pinpoint differences in prostate neuroendocrine carcinoma versus prostate adenocarcinoma that could lead to new therapeutic targets in each type.

Metabolic reprogramming ensures cancer cell survival despite oncogenic signaling blockade.

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5 Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.

Androgen receptor amplification is concordant between circulating tumor cells and biopsies from men undergoing treatment for metastatic castration resistant prostate cancer.

Increased AR activity has been shown to be preserved in spatially distinct metastatic tumors from the same patient suggesting the requirement for lineage-specific dependencies for metastatic castration resistant prostate cancer (mCRPC). Amplification of the AR gene is a common mechanism by which mCRPC increase AR activity. To determine whether AR amplification in circulating tumor cells (CTC) could complement metastatic tissue biopsies in men undergoing treatment for mCRPC, we developed a novel two-step assay to isolate CTCs and subsequently analyzed AR amplification status in CTCs and matched biopsy tissue from the same patient by fluorescence in situ hybridization (FISH). AR gene status in CTCs showed strong concordance with AR gene status in matched tissue samples in 24 of 25 patients (Correlation: 96%; Kappa: 0.83; Sensitivity: 100%, Specificity: 83%). Our work demonstrates that AR amplification is conserved between CTCs and biopsies and that CTCs can serve as non-invasive surrogate to document AR amplification in mCRPC.

Src and STAT3 inhibitors synergize to promote tumor inhibition in renal cell carcinoma.

The intracytoplasmic tyrosine kinase Src serves both as a conduit and a regulator for multiple processes required for the proliferation and survival cancer cells. In some cancers, Src engages with receptor tyrosine kinases to mediate downstream signaling and in other cancers, it regulates gene expression. Src therefore represents a viable oncologic target. However, clinical responses to Src inhibitors, such as dasatinib have been disappointing to date. We identified Stat3 signaling as a potential bypass mechanism that enables renal cell carcinoma (RCC) cells to escape dasatinib treatment. Combined Src-Stat3 inhibition using dasatinib and CYT387 (a JAK/STAT inhibitor) synergistically reduced cell proliferation and increased apoptosis in RCC cells. Moreover, dasatinib and CYT387 combine to suppress YAP1, a transcriptional co-activator that promotes cell proliferation, survival and organ size. Importantly, this combination was well tolerated, and caused marked tumor inhibition in RCC xenografts. These results suggest that combination therapy with inhibitors of Stat3 signaling may be a useful therapeutic approach to increase the efficacy of Src inhibitors.

LIV-1 promotes prostate cancer epithelial-to-mesenchymal transition and metastasis through HB-EGF shedding and EGFR-mediated ERK signaling.

LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there was no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In the present communication, we found increased LIV-1 expression in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives human prostate cancer EMT in an androgen-refractory prostate cancer cells (ARCaP) prostate cancer bone metastasis model. LIV-1, when overexpressed in ARCaP(E) (derivative cells of ARCaP with epithelial phenotype) cells, promoted EMT irreversibly. LIV-1 overexpressed ARCaP(E) cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaP(E) cells that elicited constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promoted EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.

Beta2-microglobulin signaling blockade inhibited androgen receptor axis and caused apoptosis in human prostate cancer cells.

PURPOSE: beta2-Microglobulin (beta2M) has been shown to promote osteomimicry and the proliferation of human prostate cancer cells. The objective of this study is to determine the mechanism by which targeting beta2M using anti-beta2M antibody inhibited growth and induced apoptosis in prostate cancer cells. EXPERIMENTAL DESIGN: Polyclonal and monoclonal beta2M antibodies were used to interrupt beta2M signaling in human prostate cancer cell lines and the growth of prostate tumors in mice. The effects of the beta2M antibody on a survival factor, androgen receptor (AR), and its target gene, prostate-specific antigen (PSA) expression, were investigated in cultured cells and in tumor xenografts. RESULTS: The beta2M antibody inhibited growth and promoted apoptosis in both AR-positive and PSA-positive, and AR-negative and PSA-negative, prostate cancer cells via the down-regulation of the AR in AR-positive prostate cancer cells and directly caused apoptosis in AR-negative prostate cancer cells in vitro and in tumor xenografts. The beta2M antibody had no effect on AR expression or the growth of normal prostate cells. CONCLUSIONS: beta2M downstream signaling regulates AR and PSA expression directly in AR-positive prostate cancer cells. In both AR-positive and AR-negative prostate cancer cells, interrupting beta2M signaling with the beta2M antibody inhibited cancer cell growth and induced its apoptosis. The beta2M antibody is a novel and promising therapeutic agent for the treatment of human prostate cancers.

Epithelial to mesenchymal transition (EMT) in human prostate cancer: lessons learned from ARCaP model.

Androgen refractory cancer of the prostate (ARCaP) cells contain androgen receptor (AR) and synthesize and secrete prostate specific antigen (PSA). We isolated epithelia-like ARCaP(E) from parental ARCaP cells and induced them to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors including TGFbeta1 plus EGF, IGF-1, beta2-microglobulin (beta2-m), or a bone microenvironment. The molecular and behavioral characteristics of the resultant ARCaP(M) were characterized extensively in comparison to the parental ARCaP(E) cells. In addition to expressing mesenchymal biomarkers, ARCaP(M) gained 100% incidence of bone metastasis. ARCaP(M) cells express receptor activator of NF-kappaB ligand (RANKL), which was shown to increase tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in culture, and when metastatic to bone in vivo. We provide evidence that RANKL expression was promoted by increased cell signaling mediated by the activation of Stat3-Snail-LIV-1. RANKL expressed by ARCaP(M) cells is functional both in vitro and in vivo. The lesson we learned from the ARCaP model of EMT is that activation of a specific cell signaling pathway by soluble factors can lead to increased bone turnover, mediated by enhanced RANKL expression by tumor cells, which is implicated in the high incidence of prostate cancer bone colonization. The ARCaP EMT model is highly attractive for developing new therapeutic agents to treat prostate cancer bone metastasis.