B.M. Evers

Inhibition of Fatty Acid Synthase Attenuates CD44-Associated Signaling and Reduces Metastasis in Colorectal Cancer

Fatty acid synthase (FASN) and ATP-citrate lyase, key enzymes of de novo lipogenesis, are significantly upregulated and activated in many cancers and portend poor prognosis. Even though the role of lipogenesis in providing proliferative and survival advantages to cancer cells has been described, the impact of aberrant activation of lipogenic enzymes on cancer progression remains unknown. In this study, we found that elevated expression of FASN is associated with advanced stages of colorectal cancer (CRC) and liver metastasis, suggesting that it may play a role in progression of CRC to metastatic disease. Targeted inhibition of lipogenic enzymes abolished expression of CD44, a transmembrane protein associated with metastases in several cancers including CRC. In addition, inhibition of lipogenic enzymes and reduced expression of CD44 attenuated the activation of MET, Akt, FAK, and paxillin, which are known to regulate adhesion, migration, and invasion. These changes were consistent with an observed decrease in migration and adhesion of CRC cells in functional assays and with reorganization of actin cytoskeleton upon FASN inhibition. Despite the modest effect of FASN inhibition on tumor growth in xenografts, attenuation of lipogenesis completely abolished establishment of hepatic metastasis and formation of secondary metastasis. Together, our findings suggest that targeting de novo lipogenesis may be a potential treatment strategy for advanced CRC.

The deubiquitination enzyme USP46 functions as a tumor suppressor by controlling PHLPP-dependent attenuation of Akt signaling in colon cancer

PH domain leucine-rich-repeats protein phosphatase (PHLPP) is a family of Ser/Thr protein phosphatases that serve as tumor suppressors by negatively regulating Akt. Our recent studies have demonstrated that the ubiquitin proteasome pathway has an important role in the downregulation of PHLPP in colorectal cancer. In this study, we show that the deubiquitinase USP46 stabilizes the expression of both PHLPP isoforms by reducing the rate of PHLPP degradation. USP46 binds to PHLPP and directly removes the polyubiquitin chains from PHLPP in vitro and in cells. Increased USP46 expression correlates with decreased ubiquitination and upregulation of PHLPP proteins in colon cancer cells, whereas knockdown of USP46 has the opposite effect. Functionally, USP46-mediated stabilization of PHLPP and the subsequent inhibition of Akt result in a decrease in cell proliferation and tumorigenesis of colon cancer cells in vivo. Moreover, reduced USP46 protein level is found associated with poor PHLPP expression in colorectal cancer patient specimens. Taken together, these results indentify a tumor suppressor role of USP46 in promoting PHLPP expression and inhibiting Akt signaling in colon cancer.

Noggin attenuates cerulein-induced acute pancreatitis and impaired autophagy

Objectives To investigate the role of bone morphogenetic protein (BMP) signaling in acute pancreatitis (AP) by administration of noggin, an endogenous BMP antagonist, in a cerulein-induced AP model. Methods Acute pancreatitis was induced by 9 hourly intraperitoneal injections of cerulein (50 &mgr;g/kg). Control mice received phosphate-buffered saline injections. In a separate group, noggin (0.5 mg/kg) was given intraperitoneally at 1 hour before and 2, 4, and 6 hours after AP induction. The mice were euthanized at 1 hour after completion of AP induction. The blood samples and the pancreas were harvested for analysis. Isolated pancreatic acini from normal mice and AR42J cells were treated with BMP2 and cerulein. AR42J cells were also treated with noggin. Phosphorylation of Smad1/5/8 was measured. Results Bone morphogenetic protein signaling was up-regulated in AP mouse pancreas. Bone morphogenetic protein 2 and cerulein-induced phosphorylation of Smad1/5/8 in the acinar cells in vitro, which was blocked by noggin. Noggin administration in vivo attenuated AP induction, decreased vacuole formation in acinar cells, blocked LC3-II levels, and partially restored Beclin-1 and lysosomal-associated membrane protein 2 levels. Conclusions Bone morphogenetic protein signaling seems to promote AP induction and autophagy, as suggested by our study showing that noggin ameliorates AP and partially restores autophagic homeostasis.

W1991 Upregulation of Epithelial Mesenchymal Transition in Colorectal Cancer After PTEN Knockdown

Background: Colonic carcinogenesis is driven by complex interacting signals including epidermal growth factor receptor signals between transforming colonocytes and supporting stromal cells. EGFR effectors include cyclin D1 and cyclooxygenase-2 (Cox-2). Cyclin D1 regulates proliferation by controlling G1 to S cell cycle transition and is over-expressed in colonic epithelial cells during colonic carcinogenesis. Cox-2 is the rate-limiting enzyme for prostaglandin biosynthesis and is initially increased in stromal fibroblasts and later in colonocytes during malignant progression. In order to begin to dissect the cross talk between these cell types, we measured cyclin D1 and Cox-2 expression in colonic fibroblasts and colon cancer cells grown under monoculture or co-culture conditions. Methods: Colonic embryonic fibroblasts CCD-18Co and Caco-2 colon cancer cells were plated under identical conditions alone, or together in close contact on opposing sides of the transwell membrane. Cells were untreated (basal conditions), stimulated with EGF, an important growth factor, or IL-1β, an important pro-inflammatory cytokine secreted by these cells. After 4 or 24 hrs cells were harvested and lysates probed for cyclin D1 and Cox 2 expression by Western blotting. Results: Compared to cells in monoculture, co-culture conditions that facilitated close interaction of these cells significantly increased basal Cox-2 (>5-fold in CCD18-Co and >2-fold in Caco-2 cells) and cyclin D1 (>2-fold in Caco-2 cells). When added to upper and lower transwell chambers, EGF and IL 1β further enhanced expression of Cox 2 (Caco2 and CCD-18Co) and cyclin D1 (Caco-2 cells) over basal co-culture conditions by > 2fold that persisted for up to 24 hrs (p 70% (p<0.05). Conclusion: We have demonstrated potentially important tumor-promoting effector responses In Vitro that emanate from cross talk between colonic fibroblasts and malignant colonocytes. EGFR-dependent mechanisms appear to mediate some of these responses. Using bioengineered Caco-2 cells that selectively up or down-regulate EGFR signals, we are further dissecting paracrine and autocrine signals of this important growth factor receptor that controls effector responses in stromal cells and malignant colonocytes.