Mukherjee, Priyabrata

Abstract Purpose: Although the B-cell receptor (BCR) signal plays a critical role in chronic lymphocytic leukemia (CLL) cell survival and a target of current therapies (ibrutinib targets Bruton’s tyrosine kinase; idelalisib targets PI3Kδ), contribution of the cytokine-driven JAK2 pathway to the “CLL cell-survival signaling network” is largely undefined. Experimental Design: Patients with CLL were enrolled to investigate expression/activation of JAK2 and acylglycerol kinase (AGK), and their functional implication in primary CLL cell survival. A series of biochemical and molecular biology assays were employed to uncover the underlying mechanism. Results: We detected that compared with normal B cells, CLL cells aberrantly express constitutively active JAK2. Mechanistically, HSP90 forms a chaperoning complex with JAK2, resulting in its aberrant accumulation in CLL cells. We also discovered aberrant upregulation of a novel mitochondrial lipid kinase, AGK, which remains complexed with HSP90 in CLL cells activating JAK2. Although AGK is typically mitochondrial, we detected its nuclear localization in association with JAK2 in some CLL cells. Functionally, JAK2 phosphorylates its noncanonical substrate, histone H3(Y41), but not STAT3, activating transcription of diverse sets of genes in a patient-specific manner. Additionally, JAK2 activates the BCR signal in CLL cells via LYN/Bruton’s tyrosine kinase axis. Targeted inhibition of JAK2 as monotherapy, or in combination with the BCR inhibitors or venetoclax (a BCL2 inhibitor), induced apoptosis synergistically in CLL cells. Conclusions: These findings suggest that aberrantly expressed AGK activates JAK2, independent of cytokine, leading to activation of diverse sets of gene transcription in CLL cells. Combined targeting of JAK2 and BCR signals or BCL2 may be effective in some patients with CLL.

Ovarian cancer is the eighth most common cancer and the fifth leading cause of cancer death among US women. Hepatoma‐derived growth factor (HDGF) is a heparin binding, secreted mitogen with prognostic value in multiple cancers. We previously reported that HDGF is overexpressed in ovarian cancer. Surprisingly, the mechanisms by which HDGF acts in any cell type remains to be fully elucidated. We hypothesized that overexpression of HDGF in ovarian cancer cells is functionally relevant and that HDGF confers pro‐survival and proliferative properties, promoting cancer growth. To elucidate intracellular and extracellular signaling mechanisms, we examined the effect of HDGF knockdown via siRNA, overexpression of HDGF and stimulation by full‐length recombinant protein. Immunofluorescence image analysis showed nuclear localization of HDGF in OV202 cell line. Additionally, HDGF was minimally secreted but passively released in greater amounts by necrotic cells. Extracellular HDGF stimulated phosphorylation of ERK and p38. In contrast, modulating intracellular HDGF did not affect MAPK pathways. In fact, intracellular HDGF functioned in a distinct manner by regulating cellular metabolism. This suggests that HDGF acts in ovarian cancer cells via two distinct mechanisms, both of which could be exploited for ovarian cancer therapy. Supported by the Mayo Graduate School (Giri) and the Department of Anesthesiology, Mayo Clinic (Prakash).

Abstract Macropinocytosis supports the metabolic requirement of RAS‐transformed pancreatic ductal adenocarcinoma cells (PDACs). However, regulators of RAS‐transformation (activation) that lead to macropinocytosis have not been identified. Herein, we report that UBAP2 (ubiquitin‐binding associated protein 2), regulates the activation of KRAS and macropinocytosis in pancreatic cancer. We demonstrate that UBAP2 is highly expressed in both pancreatic cancer cell lines and tumor tissues of PDAC patients. The expression of UBAP2 is associated with poor overall survival in several cancers, including PDAC. Silencing UBAP2 decreases the levels of activated KRAS, and inhibits macropinocytosis, and tumor growth in vivo. Using a UBAP2‐deletion construct, we demonstrate that the UBA‐domain of UBAP2 is critical for the regulation of macropinocytosis and maintaining the levels of activated KRAS. In addition, UBAP2 regulates RAS downstream signaling and helps maintain RAS in the GTP‐bound form. However, the exact mechanism by which UBAP2 regulates KRAS activation is unknown and needs further investigation. Thus, UBAP2 may be exploited as a potential therapeutic target to inhibit macropinocytosis and tumor growth in activated KRAS‐driven cancers.

Abstract Like VEGF, morphine stimulates MAPK/ERK and Akt, leading to the promotion of angiogenesis via NO dependent signaling (Cancer Res62: 4491, 2002). Morphine acts via pertussis toxin (PT)-dependent G-protein coupled receptors (GPCRS), while VEGF acts via receptor tyrosine kinases (RTKs). We showed that PT-dependent GPCRs transactivate VEGF receptor-2/Flk1 via small GTPase RhoA (JBC277: 4679, 2002; JBC278:20738, 2003). Therefore, we hypothesized that morphine via the mu opioid receptor (MOR) transactivates Flk1 and promotes a pro-angiogenic microenvironment. Morphine-induced proliferation of human umbilical vein endothelial cells (HUVEC) was completely abrogated by Y-27632 (100 μM), a highly selective and potent inhibitor of Rho-associated protein kinases, suggesting the activation of Rho signaling by morphine. Addition of 1 μM morphine potentiated VEGF-induced (10 ng/ml) proliferation of HUVEC by 25%. We observed a 30% increase in intracellular calcium release after VEGF stimulation of HUVEC pre-incubated with morphine as compared to HUVEC pre-incubated with PBS, detected by a change in the fluorescence ratio of the Fura-2 AM dye. These findings show that morphine, via MOR and Rho signaling, transactivates Flk1 leading to the stimulation of calcium signaling and endothelial cell proliferation. To functionally corroborate our hypothesis, we used MOR knockout (MOR-KO) mice and injected them with MOR-replete T241 fibrosarcoma cells. T241 fibrosarcoma tumor growth in vivo showed appearance of palpable and measurable tumors 2 days earlier in wild type (wt) as compared to MOR-KO mice. Tumor growth and angiogenesis were decreased by 20–35% in MOR-KO mice as compared to wt littermates during 3 weeks of tumor growth. None of the MOR-KO showed signs of lung metastasis versus 40% wt mice with metastasis. Morphine (1.42 for the first 2 wks and 2.14 mg/Kg/day later, respectively) stimulated 20–35% tumor growth in wt, but not in MOR-KO mice. Western immunoblotting showed a 10-fold increase in the expression of phospho-Flk1 in morphine treated wt tumors as compared to PBS-treated wt mice. Morphine did not stimulate phospho-Flk1 expression in MOR-KO mice. Western analysis of immunoprecipitates obtained with α-MOR antibody showed the expression of Flk1 and phospho-Flk1 in wt, but were not expressed in MOR-KO tumors. Thus, MOR stimulates the transactivation of Flk1 in wt mice but not in MOR-KO. These in vitro and in vivo data using MOR-KO mice and the MOR agonist, morphine, show that MOR stimulates endothelial proliferation, angiogenesis and promotes tumor growth and metastasis directly as well as by transactivating Flk1 phosphorylation. We speculate that MOR is a critical component of the ‘angiogenic switch’, which regulates the pro-angiogenic and growth promoting tumor microenvironment. Thus, MOR provides a novel target for developing anti-angiogenic therapies.

Abstract Hydrogen sulfide (H2S), the third gasotransmitter, has recently attracted a lot of attention for its cytoprotective action and role in angiogenesis. Dysregulation of the major enzymes which produces H2S namely, cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) have also been implicated in a large number of pathological conditions that range from neurological disorders to cardiovascular diseases. Surprisingly, studies on the role of these enzymes in cancer is scarce, especially, that of CBS. Relapse with a drug resistant and aggressive phenotype is a common occurrence in ovarian cancer with an overall 5-year survival of approximately 30%. Here we report that, CBS, a key enzyme required for sulphur amino acid metabolism, plays a critical role in promoting ovarian cancer growth, metastasis and drug resistance. Overexpression of CBS was observed in a majority of ovarian cancer cell lines utilized in our study compared to normal ovarian surface epithelial cells. Silencing CBS in A2780 cells using siRNA or a CBS-specific inhibitor, aminooxyacetic acid reduced proliferation of ovarian cancer cells by dramatically reducing total glutathione levels, H2S levels and thereby enhancing oxidative stress. Downregulation of CBS reduced activation of NF-kB and upregulated p53 protein levels thereby inactivating the antioxidant network of the cells. A deeper insight into the intracellular localization of CBS by confocal fluorescence microscopy demonstrated a mitochondria specific distribution and silencing CBS caused mitochondrial superoxide accumulation that resulted in decreased mitochondrial respiration, reduced ATP production and increased ADP-to-ATP ratio. Furthermore, silencing CBS, augmented the activity of cisplatin in vitro in A2780 cells and in vivo in an orthotopic model of advanced ovarian cancer. Nanoliposomal delivery of CBS siRNA significantly reduced the number of tumor nodules in a chemoresistant orthotopic mouse model of ovarian cancer. To our knowledge, this is the first study demonstrating the importance of CBS in ovarian cancer progression and drug resistance and therefore is a viable target for future anti-cancer therapies. Citation Format: Sounik Saha, Sanjib Bhattacharyya, Karuna Giri, Ian R. Lanza, K. Sreekumar Nair, Nicholas B. Jennings, Rakesh Kumar, Anil K. Sood, Resham Bhattacharya, Priyabrata Mukherjee. Role of cystathionine beta-synthase in ovarian cancer growth, metastasis and drug sensitivity. [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 1882. doi:10.1158/1538-7445.AM2013-1882

Although biomedical applications of nanotechnology, which typically involve functionalized nanoparticles, have taken significant strides, biological characterization of unmodified nanoparticles remains underinvestigated. Herein we demonstrate that unmodified gold nanoparticles (AuNPs) inhibit the proliferation of cancer cells in a size- and concentration-dependent manner by abrogating MAPK-signaling. In addition, these AuNPs reverse epithelial-mesenchymal transition (EMT) in cancer cells by reducing secretion of a number of proteins involved in EMT, up-regulating E-Cadherin, and down-regulating Snail, N-Cadherin, and Vimentin. Inhibition of MAPK signaling and reversal of EMT upon AuNP treatment inhibits tumor growth and metastasis in two separate orthotopic models of ovarian cancer. Western blot analyses of tumor tissues reveal up-regulation of E-Cadherin and down-regulation of Snail and phospho-MAPK, confirming the reversal of EMT and inhibition of MAPK signaling upon AuNP treatment. The ability of a single self-therapeutic nanoparticle to abrogate signaling cascades of multiple growth factors is distinctive and purports possible medical applications as potential antitumor and antimetastatic agent.

Treatment with monoclonal antibody (mAbs) is a viable therapeutic option in cancer. Recently, these mAbs such as cetuximab, herceptin, etc., have been used as targeting agents to selectively deliver chemotherapeutics to cancerous cells. However, mechanisms of nanoparticles-mAbs interactions with the target cells and its effect on intracellular trafficking and mechanism are currently unknown. In this paper, we demonstrate that the distinct patterning and dynamics of anti-EGFR (epidermal growth factor receptor) antibody cetuximab (C225)- induced EGFR internalization in pancreatic cancer cells with variable receptor expression is altered upon nanoconjugation. Nanoconjugation uniformly enhanced C225-induced EGFR endocytosis in PANC-1, AsPC-1, and MiaPaca-2 cells, influenced its compartmentalization and regulated the involvement of dynamin-2 in the endocytic processes. Receptor endocytosis and its intracellular trafficking were monitored by confocal microscopy and transmission electron microscopy. The role of dynamin-2 in EGFR endocytosis was determined after overexpressing either wild-type dynamin-2 or mutant dynamin-2 in pancreatic cancer cells followed by tracking the receptor-antibody complex internalization by confocal microscopy. Significantly, these findings demonstrate that the nanoconjugation cannot be construed as an innocuous reaction involved in attaching the targeting agent to the nanoparticle, instead it may distinctly alter the cellular processes at the molecular level, at least antibody induced receptor endocytosis. This information is critical for successful design of a nanoparticle-based targeted drug delivery system for future clinical translation.

Abstract Oncogenic activation of Bmi-1 is found in a wide variety of epithelial malignancies including ovarian cancer, yet a specific mechanism for overexpression of Bmi-1 has not been determined. Thus, realizing the immense pathologic significance of Bmi-1 in cancer, we wanted to investigate if microRNA (miRNA) aberrations played a role in the regulation of Bmi-1 in ovarian cancer. In this report, we identify two miRNAs, miR-15a and miR-16, that are underexpressed in ovarian cell lines and in primary ovarian tissues. We show that these miRNAs directly target the Bmi-1 3′ untranslated region and significantly correlate with Bmi-1 protein levels in ovarian cancer patients and cell lines. Furthermore, Bmi-1 protein levels are downregulated in response to miR-15a or miR-16 expression and lead to significant reduction in ovarian cancer cell proliferation and clonal growth. These findings suggest the development of therapeutic strategies by restoring miR-15a and miR-16 expression in ovarian cancer and in other cancers that involve upregulation of Bmi-1. [Cancer Res 2009;69(23):9090–5]