Andreas Varkaris

The role of HGF/c-Met signaling in prostate cancer progression and c-Met inhibitors in clinical trials

Introduction: An increasing number of basic, translational and clinical studies demonstrate the importance of the protein tyrosine kinase receptor, c-Met, in the progression of prostate cancer. c-Met is overexpressed in primary prostate cancers, further increased in expression in bone metastases and is associated with the development of castrate-resistant disease. Because of its importance as a target, c-Met inhibitors have reached clinical trial for advanced, castrate-resistant prostate cancer. Areas covered: In this review, altered expression of c-Met and hepatocyte growth factor in prostate tumors and the microenvironment and how they contribute to growth and invasion of prostate cancer cells is described. Next, preclinical studies providing the support for use of c-Met inhibitors are discussed. Finally, early promising results from c-Met inhibitors in clinical trial, and future prospects for c-Met inhibitors in the treatment of advanced stage prostate cancer, are discussed. Expert opinion: An emerging theme in treating metastatic prostate cancer is the requirement to target both the epithelial and stromal compartments. Results from clinical trials suggest that inhibitors of c-Met that block stromal-mediated c-Met activation in prostate tumors may be important therapeutic agents in at least a subset of patients with metastatic prostate cancer. However, as many of the inhibitors have multiple targets, the efficacy of targeting c-Met alone remains to be determined.

Targeted therapies in multiple myeloma

Increasing knowledge of the biology of multiple myeloma led the way for the development of novel drugs that have changed the management of the disease. New treatments target not only to the malignant plasma cell but also target the interactions of myeloma cells with their microenvironment. Several preclinical studies have identified potential targets and drugs are developed that act on pathways crucial for myeloma cell survival, proliferation, migration and drug resistance. The identification of active agents in the laboratory is followed by rationally designed clinical studies that validate these drugs, either as single agents or in combinations with other active drugs. These novel agents may be either small molecules or monoclonal antibodies targeting receptors, kinase activity of receptors or key molecules within critical pathways, intracellular maintenance mechanisms and immune modulation.

Chitosan nanoparticle-mediated delivery of miRNA-34a decreases prostate tumor growth in the bone and its expression induces non-canonical autophagy

While several new therapies are FDA-approved for bone-metastatic prostate cancer (PCa), patient survival has only improved marginally. Here, we report that chitosan nanoparticle-mediated delivery of miR-34a, a tumor suppressive microRNA that downregulates multiple gene products involved in PCa progression and metastasis, inhibited prostate tumor growth and preserved bone integrity in a xenograft model representative of established PCa bone metastasis. Expression of miR-34a induced apoptosis in PCa cells, and, in accord with downregulation of targets associated with PCa growth, including MET and Axl and c-Myc, also induced a form of non-canonical autophagy that is independent of Beclin-1, ATG4, ATG5 and ATG7. MiR-34a-induced autophagy is anti-proliferative in prostate cancer cells, as blocking apoptosis still resulted in growth inhibition of tumor cells. Thus, combined effects of autophagy and apoptosis are responsible for miR-34a-mediated prostate tumor growth inhibition, and have translational impact, as this non-canonical form of autophagy is tumor inhibitory. Together, these results provide a new understanding of the biological effects of miR-34a and highlight the clinical potential for miR-34a delivery as a treatment for bone metastatic prostate cancer.

Src signaling pathways in prostate cancer.

Knowledge of the molecular events that contribute to prostate cancer progression has created opportunities to develop novel therapy strategies. It is now well established that c-Src, a non-receptor tyrosine kinase, regulates a complex signaling network that drives the development of castrate-resistance and bone metastases, events that signal the lethal phenotype of advanced disease. Preclinical studies have established a role for c-Src and Src Family Kinases (SFKs) in proliferation, angiogenesis, invasion and bone metabolism, thus implicating Src signaling in both epithelial and stromal mechanisms of disease progression. A number of small molecule inhibitors of SFK now exist, many of which have demonstrated efficacy in preclinical models and several that have been tested in patients with metastatic castrate-resistant prostate cancer. These agents have demonstrated provocative clinic activity, particularly in modulating the bone microenvironment in a therapeutically favorable manner. Here, we review the discovery and basic biology of c-Src and further discuss the role of SFK inhibitors in the treatment of advanced prostate cancer.

Ligand-independent activation of MET through IGF-1/IGF-1R signaling

The receptor tyrosine kinase, MET, has been implicated in tumorigenesis and metastasis of many solid tumors, by multiple mechanisms, including cross talk with epidermal growth factor receptor. In this study, we examined the role of insulin‐like growth factor receptor‐1 (IGF‐1R) signaling in MET activation, focusing on prostate cancer cells. Stimulation of the prostate cancer cell line PC3 with IGF‐1 induces a delayed phosphorylation of MET at multiple sites (indicative of full activation), reaching a maximum 18 hr after IGF‐1 addition. MET activation does not require the sole MET ligand hepatocyte growth factor (HGF), but does require transcription to occur. Furthermore, direct injection of IGF‐1 is sufficient to induce MET activation in vivo, in a PC3 xenograft model. Pharmacologic or genetic inhibition of the tyrosine kinase, Src, abolishes MET phosphorylation, and expression of activated Src is sufficient to induce Met phosphorylation in the absence of IGF‐1 stimulation. Activated MET is essential for IGF‐1‐mediated increased migration of PC3 cells, demonstrating an important biologic effect of IGF‐1‐mediated MET activation. Finally, we demonstrate that IGF‐1‐induced delayed MET activation occurs in multiple cell lines which express both the receptors, suggesting that IGF‐1R‐mediated MET activation may contribute to tumorigenic properties of multiple cancer types when both growth factor receptors are expressed. The results further suggest that MET may be activated by multiple receptor tyrosine kinase receptors, and dual targeting of these receptors may be important therapeutically.

Combined Inhibition of IGF-1R/IR and Src Family Kinases Enhances Antitumor Effects in Prostate Cancer by Decreasing Activated Survival Pathways

Background Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways. Materials and Methods Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively). Results In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1-induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS-754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers. Conclusions Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth.

Integrating Murine and Clinical Trials with Cabozantinib to Understand Roles of MET and VEGFR2 as Targets for Growth Inhibition of Prostate Cancer

Purpose: We performed parallel investigations in cabozantinib-treated patients in a phase II trial and simultaneously in patient-derived xenograft (PDX) models to better understand the roles of MET and VEGFR2 as targets for prostate cancer therapy. Experimental Design: In the clinical trial, radiographic imaging and serum markers were examined, as well as molecular markers in tumors from bone biopsies. In mice harboring PDX intrafemurally or subcutaneously, cabozantinib effects on tumor growth, MET, PDX in which MET was silenced, VEGFR2, bone turnover, angiogenesis, and resistance were examined. Results: In responsive patients and PDX, islets of viable pMET-positive tumor cells persisted, which rapidly regrew after drug withdrawal. Knockdown of MET in PDX did not affect tumor growth in mice nor did it affect cabozantinib-induced growth inhibition but did lead to induction of FGFR1. Inhibition of VEGFR2 and MET in endothelial cells reduced the vasculature, leading to necrosis. However, each islet of viable cells surrounded a VEGFR2-negative vessel. Reduction of bone turnover was observed in both cohorts. Conclusions: Our studies demonstrate that MET in tumor cells is not a persistent therapeutic target for metastatic castrate-resistant prostate cancer (CRPC), but inhibition of VEGFR2 and MET in endothelial cells and direct effects on osteoblasts are responsible for cabozantinib-induced tumor inhibition. However, vascular heterogeneity represents one source of primary therapy resistance, whereas induction of FGFR1 in tumor cells suggests a potential mechanism of acquired resistance. Thus, integrated cross-species investigations demonstrate the power of combining preclinical models with clinical trials to understand mechanisms of activity and resistance of investigational agents. Clin Cancer Res; 22(1); 107–21. ©2015 AACR.

Increased serum insulin-like growth factor-1 levels are associated with prolonged response to dasatinib-based regimens in metastatic prostate cancer.

Dasatinib, an inhibitor of Src‐family kinases, combined with docetaxel in men with castrate‐resistant prostate cancer (CRPC), affects bone turnover markers in a phase I/II clinical trial in metastatic CRPC. Only a subset of men benefit from this therapy, and predictive markers are lacking. We hypothesized a role for insulin‐like growth factor‐1 (IGF‐1) as a predictive marker, since IGF‐1 is important in both prostate cancer progression and bone development. Hence, we determined the association of IGF‐1 expression to treatment response, and whether this expression resulted from tumor cells, the microenvironment, or their interactions.

Modeling of Patient Derived Xenografts in Colorectal Cancer

Developing realistic preclinical models using clinical samples that mirror complex tumor biology and behavior are vital to advancing cancer research. While cell line cultures have been helpful in generating preclinical data, the genetic divergence between these and corresponding primary tumors has limited clinical translation. Conversely, patient-derived xenografts (PDX) in colorectal cancer are highly representative of the genetic and phenotypic heterogeneity in the original tumor. Coupled with high-throughput analyses and bioinformatics, these PDXs represent robust preclinical tools for biomarkers, therapeutic target, and drug discovery. Successful PDX engraftment is hypothesized to be related to a series of anecdotal variables namely, tissue source, cancer stage, tumor grade, acquisition strategy, time to implantation, exposure to prior systemic therapy, and genomic heterogeneity of tumors. Although these factors at large can influence practices and patterns related to xenotransplantation, their relative significance in determining the success of establishing PDXs is uncertain. Accordingly, we systematically examined the predictive ability of these factors in establishing PDXs using 90 colorectal cancer patient specimens that were subcutaneously implanted into immunodeficient mice. Fifty (56%) PDXs were successfully established. Multivariate analyses showed tissue acquisition strategy [surgery 72.0% (95% confidence interval (CI): 58.2–82.6) vs. biopsy 35% (95% CI: 22.1%–50.6%)] to be the key determinant for successful PDX engraftment. These findings contrast with current empiricism in generating PDXs and can serve to simplify or liberalize PDX modeling protocols. Better understanding the relative impact of these factors on efficiency of PDX formation will allow for pervasive integration of these models in care of colorectal cancer patients. Mol Cancer Ther; 16(7); 1435–42. ©2017 AACR.

Serum insulin-like growth factor-1 levels in response to dasatinib-based regimens in bone-metastatic prostate cancer.

166 Background: Dasatinib, an inhibitor of Src-family kinases, combined with docetaxel in men with castrate-resistant prostate cancer (CRPC), affects bone turnover markers. Only a subset of men benefit from this therapy, and predictive markers are lacking. We hypothesized a role for insulin-like growth factor-1 (IGF-1) as a predictive marker, since IGF-1 is important in both prostate cancer (PCa) progression and bone development. Hence, we determined the association of IGF-1 expression to treatment response, and whether this expression resulted from tumor cells, the microenvironment, or their interactions. METHODS We first measured serum IGF-1 levels in men with CRPC treated with dasatinib plus docetaxel. To investigate the source of IGF-1, we utilized different mouse models harboring human PCa cells, and used species-specific IGF-1 ELISA kits (mouse vs. human). RESULTS In men with CRPC, an increase in IGF-1 levels after one cycle of treatment is associated with a higher response rate and longer duration of treatment with docetaxel and dasatinib. Xenograft experiments with subcutaneous, and intratibial injection of PCa cells and treatment of mice with dasatinib-based combinations suggest that direct interaction of PCa cells with bone microenvironment is necessary for IGF-1 induction, is entirely host-derived, and occurs only in mice that respond to dasatinib-based therapy. CONCLUSIONS Our results support a role for serum IGF-1 as a potential biomarker for dasatinib-based combination treatments in CRPC. Inoculation of human PCa cells into murine hosts was essential in determining that IGF-1 results from the bone microenvironment. Further studies are warranted to validate these findings in a larger cohort of patients in a prospective manner.