Catherine A. Gallo

VHL-Regulated MiR-204 Suppresses Tumor Growth through Inhibition of LC3B-Mediated Autophagy in Renal Clear Cell Carcinoma

The von Hippel-Lindau tumor-suppressor gene (VHL) is lost in most clear cell renal cell carcinomas (ccRCC). Here, using human ccRCC specimens, VHL-deficient cells, and xenograft models, we show that miR-204 is a VHL-regulated tumor suppressor acting by inhibiting macroautophagy, with MAP1LC3B (LC3B) as a direct and functional target. Of note, higher tumor grade of human ccRCC was correlated with a concomitant decrease in miR-204 and increase in LC3B levels, indicating that LC3B-mediated macroautophagy is necessary for RCC progression. VHL, in addition to inducing endogenous miR-204, triggered the expression of LC3C, an HIF-regulated LC3B paralog, that suppressed tumor growth. These data reveal a function of VHL as a tumor-suppressing regulator of autophagic programs.

Akt-dependent glucose metabolism promotes Mcl-1 synthesis to maintain cell survival and resistance to Bcl-2 inhibition.

Most cancer cells utilize aerobic glycolysis, and activation of the phosphoinositide 3-kinase/Akt/mTOR pathway can promote this metabolic program to render cells glucose dependent. Although manipulation of glucose metabolism may provide a means to specifically eliminate cancer cells, mechanistic links between cell metabolism and apoptosis remain poorly understood. Here, we examined the role and metabolic regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in cell death upon inhibition of Akt-induced aerobic glycolysis. In the presence of adequate glucose, activated Akt prevented the loss of Mcl-1 expression and protected cells from growth factor deprivation-induced apoptosis. Mcl-1 associated with and inhibited the proapoptotic Bcl-2 family protein Bim, contributing to cell survival. However, suppression of glucose metabolism led to induction of Bim, decreased expression of Mcl-1, and apoptosis. The proapoptotic Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, shows clinical promise, but Mcl-1 upregulation can promote resistance. Importantly, inhibition of glucose metabolism or mTORC1 overcame Mcl-1-mediated resistance in diffuse large B cell leukemic cells. Together these data show that Mcl-1 protein synthesis is tightly controlled by metabolism and that manipulation of glucose metabolism may provide a mechanism to suppress Mcl-1 expression and sensitize cancer cells to apoptosis.

Requirement for ribosomal protein S6 kinase 1 to mediate glycolysis and apoptosis resistance induced by Pten deficiency

Pten inactivation promotes cell survival in leukemia cells by activating glycolytic metabolism. We found that targeting ribosomal protein S6 kinase 1 (S6K1) in Pten-deficient cells suppressed glycolysis and induced apoptosis. S6K1 knockdown decreased expression of HIF-1α, and HIF-1α was sufficient to restore glycolysis and survival of cells lacking S6K1. In the Ptenfl/fl Mx1-Cre+ mouse model of leukemia, S6K1 deletion delayed the development of leukemia. Thus, S6K1 is a critical mediator of glycolytic metabolism, cell survival, and leukemogenesis in Pten-deficient cells.

Alternatively spliced tissue factor contributes to tumor spread and activation of coagulation in pancreatic ductal adenocarcinoma

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF‐overexpressing high‐grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell‐derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell‐derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

S6K1 determines the metabolic requirements for BCR-ABL survival

In chronic myelogenous leukemia, the constitutive activation of the BCR-ABL kinase transforms cells to an addicted state that requires glucose metabolism for survival. We investigated S6K1, a protein kinase that drives glycolysis in leukemia cells, as a target for counteracting glucose-dependent survival induced by BCR-ABL. BCR-ABL potently activated S6K1-dependent signaling and glycolysis. Although S6K1 knockdown or rapamycin treatment suppressed glycolysis in BCR-ABL-transformed cells, these treatments did not induce cell death. Instead, loss of S6K1 triggered compensatory activation of fatty-acid oxidation, a metabolic program that can support glucose-independent cell survival. Fatty-acid oxidation in response to S6K1 inactivation required the expression of the fatty-acid transporter carnitine palmitoyl transferase 1c, which was recently linked to rapamycin resistance in cancer. Finally, addition of an inhibitor of fatty-acid oxidation significantly enhanced cytotoxicity in response to S6K1 inactivation. These data indicate that S6K1 dictates the metabolic requirements mediating BCR-ABL survival and provide a rationale for combining targeted inhibitors of signal transduction, with strategies to interrupt oncogene-induced metabolism.

Interleukin-6 receptor enhances early colonization of the murine omentum by upregulation of a mannose family receptor, LY75, in ovarian tumor cells

One of the earliest metastatic events in human ovarian cancer, tumor spread to the omentum, may be influenced by expression of interleukin 6 (IL6) and its cognate receptor (IL6Rα). Previous reports have shown that IL6 and IL6Rα expression is elevated in the serum and ascites of patients with ovarian cancer and that this can influence in vitro processes such as cell survival, proliferation and migration. In this study, overexpression of IL6Rα, and to a lesser extent IL6, enhanced tumor growth on the omentum. Moreover, adherence to plastic and to peritoneal extracellular matrix components was enhanced in tumor cells overexpressing IL6 or IL6Rα. Host production of IL6 and IL6Rα was also sufficient to influence tumor adherence to the omentum. Expression of LY75/CD205/DEC205, a collagen-binding mannose family receptor, was directly influenced by IL6Rα expression. Blocking LY75 with antibody reduced the adherence of tumor cells overexpressing IL6Rα to matrices in vitro and to the omentum. The association between IL6Rα expression and LY75 expression has not been previously reported, and the promotion of cellular adherence is a novel role for LY75. These studies indicate that overexpression of LY75 may be an additional mechanism by which IL6 signaling influences the progression of ovarian cancer, and suggests that blocking LY75 could be a valuable clinical strategy for reducing the early metastasis of ovarian cancer.

Identification of Akt-selective cytotoxic compounds that enhance cytotoxic responses to rapamycin.

We performed a high-throughput screen to identify compounds with a selective ability to induce apoptosis in Akt-expressing cells without disrupting Bcl-xL-dependent survival. Results showed that a screening strategy based on Alamar Blue underrepresented the viability of Bcl-xL-expressing cells relative to Akt-expressing cells, possibly due to metabolic differences between the two cell survival programs. Using an alternative screen based on plasma membrane integrity, we identified several compounds that target Akt-dependent survival without toxic effect to Bcl-xL-dependent survival. These compounds enhanced the cytotoxic potential of rapamycin, a chemotherapeutic that inhibits survival signaling downstream of Akt. The results demonstrate a screening method and the subsequent identification of two compounds with selective activity in counteracting Akt-dependent cell survival.

Abstract LB-256: S6K1 mediates glycolysis and apoptosis resistance in Pten deficient cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pten loss and subsequent Akt activation promotes cell survival in human cancer cells through activation of glycolytic metabolism and repression of pro-apoptotic factors. Because sustained glycolysis is required for Akt dependent apoptosis resistance, we investigated the downstream signaling components that mediate Akt dependent increases in glycolysis in Pten-deficient cells. Loss of the ribosomal protein S6K1 in Pten-deficient cells prevented Akt-induced glycolysis and triggered Bax translocation and commitment to apoptosis. Inactivation of S6K1 was associated with decreased expression of the pro-glycolytic HIF1α transcription factor. Restoring HIF1α expression was sufficient to restore both glycolysis and cell survival in S6K1-deficient cells. Conversely, inhibiting HIF1α expression in Pten deficient cells resulted in decreased glycolysis and cell survival, mimicking the loss of S6K1. In vivo , S6K1 deficiency delayed the development of lethal disease in the Pten fl/fl Mx-1 Cre+ model of leukemia. Thus, together the data suggest that S6K1 is a useful target for counteracting the metabolic program that supports apoptosis resistance in Pten-deficient cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-256. doi:10.1158/1538-7445.AM2011-LB-256