Victoria A. Elliott

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.

Cotargeting the PI3K and RAS Pathways for the Treatment of Neuroendocrine Tumors

Background: The precise involvement of the PI3K/mTOR and RAS/MEK pathways in carcinoid tumors is not well defined. Therefore, the purpose of our study was to evaluate the role these pathways play in carcinoid cell proliferation, apoptosis, and secretion and to determine the effects of combined treatment on carcinoid tumor inhibition. Methods: The human neuroendocrine cell lines BON (pancreatic carcinoid), NCI-H727 (lung carcinoid), and QGP-1 (somatostatinoma) were treated with either the pan-PI3K inhibitor, BKM120, or the dual PI3K–mTOR inhibitor, BEZ235, alone or in combination with the MEK inhibitor, PD0325901; proliferation, apoptosis, and protein expression were assessed. Peptide secretion was evaluated in BON and QGP-1 cells. The antiproliferative effect of BEZ235, alone or combined with PD0325901, was then tested in vivo. Results: Both BKM120 and BEZ235 decreased proliferation and increased apoptosis; combination with PD0325901 significantly enhanced the antineoplastic effects of either treatment alone. In contrast, neurotensin peptide secretion was markedly stimulated with BKM120 treatment, but not BEZ235. The combination of BEZ235 + PD0325901 significantly inhibited the growth of BON xenografts without systemic toxicity. Conclusions: Both BKM120 and BEZ235 effectively inhibited neuroendocrine tumor (NET) cell proliferation and stimulated apoptosis. However, inhibition of the PI3K pathway alone with BKM120 significantly stimulated neurotensin peptide secretion; this did not occur with the dual inhibition of both PI3K and mTOR using BEZ235 suggesting that this would be a more effective treatment regimen for NETs. Moreover, the combination of BEZ235 and the MEK inhibitor PD0325901 was a safe and more effective therapy in vivo compared with single agents alone. Clin Cancer Res; 20(5); 1212–22. ©2014 AACR.

Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model

Background Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized. Methods HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice. Results CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF) stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance. Conclusions Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.

Novel small interfering RNA cotargeting strategy as treatment for colorectal cancer

BACKGROUND RNA interference has the potential to be more selective than small molecule inhibitors and can be used to target proteins, such as Ras, that are currently undruggable. The purpose of our study was to determine the optimal cotargeting strategy of the commonly mutated PI3K/AKT/mTOR and Ras pathways by a selective RNA interference approach in colorectal cancer cell lines possessing coexistent PIK3CA and KRAS mutations. METHODS Human colorectal cancer cell lines HCT116 and DLD-1 were treated with a panel of small interfering RNAs directed against the PI3K/AKT/mTOR and Ras pathways; proliferation, apoptosis, and protein expression were assessed. Combined treatment with small interfering RNA and 5-fluorouracil was then evaluated. RESULTS PIK3CA and KRAS small interfering RNAs were most effective as single treatments; combined treatments with PIK3CA and KRAS small interfering RNA resulted in a more pronounced inhibition of colorectal cancer cell proliferation. Either KRAS small interfering RNA alone or combined PIK3CA and KRAS small interfering RNA treatments increased apoptosis in HCT116 cells but not in the DLD-1 cell line. Inhibition of 4E-BP1 phosphorylation correlated with increased apoptosis. In addition, small interfering RNA treatment combined with 5-fluorouracil further inhibited colorectal cancer cell proliferation. CONCLUSION Combined PIK3CA and KRAS small interfering RNA treatments offer an effective therapy against colorectal cancer cells with coexisting mutations in both pathways. Decreased 4E-BP1 phosphorylation correlates with increased apoptosis and may provide a biomarker indicative of treatment success. In addition, small interfering RNA directed to PIK3CA and KRAS may be used to enhance the effects of current chemotherapy.

Murine portal vein catheterization to analyze liver-directed therapies

BACKGROUND Small interfering RNA (siRNA) provides a highly selective method to target mutated pathways; however, its use is complicated by specific delivery to tumor cells. The aims of the present study were to develop a novel murine model of portal vein catheterization for the chronic delivery of therapeutic agents to liver metastases, determine the benefits of local delivery of siRNA to liver metastases, and determine the utility of epithelial cell adhesion molecule (EpCAM) as a selective target for siRNA delivery to colorectal cancer (CRC) metastases. MATERIALS AND METHODS First, portal vein catheterization was performed through a midline laparotomy in 2 mo-old Balb/C mice. Second, the portal venous flow distribution and catheter patency were evaluated using fluorescent-labeled microspheres. Metastatic studies were performed by splenic injection of CT26 murine colon cancer cells. Uptake of DY-547-labeled siRNA was assessed by IVIS imaging, with delivery to the metastases confirmed using fluorescent microscopy. Finally, EpCAM expression was evaluated using immunohistochemical staining of human tissue microarrays. RESULTS Successful portal vein catheterization was confirmed by saline injection and ultrasound. Fluorescent imaging of microspheres confirmed excellent distribution and catheter patency. Portal venous injection of DY547-labeled siRNA demonstrated a high level of fluorescence throughout the liver, with siRNA also identified within the liver metastases. Also, all primary CRCs and liver metastases stained strongly for EpCAM, with no expression in normal hepatocytes. CONCLUSIONS Liver-directed therapy can provide the selective delivery of siRNA to CRC metastases. EpCAM expression in CRC, but not normal liver, could further selectively target hepatic metastases of epithelial origin.

Tu1937 Colorectal Cancer Cell Liver Metastasis Is Promoted by C-MET Activation and Upregulation of CD44 Expression

Background: Perceived stigmatization has been associated with poor disease outcomes in adolescents with chronic illnesses. Studies have shown that children diagnosed with inflammatory bowel diseases (IBD) are at greater risk for low self-esteem, poor social functioning and depression. Stressors associated with transitioning from pediatric to adult care could exacerbate these issues. While stigmatization has been studied in adult patients with IBD, little is known about the effects of perceived stigma on adolescent IBD patients or its effect on transitioning. Aims: To explore social determinants of IBD-related stigma, we (a) designed a cross-sectional quantitative questionnaire measuring perceived stigma, health status, quality of life, and transition readiness, and (b) analyzed domains of IBD-related stigma to predict transition readiness.Methods:We developed an adolescent-specific IBD questionnaire modeled on the Perceived Stigma Scale for IBS (PSS-IBS), which assessed three domains of stigma: disclosure, attitude, and feelings of rejection/shame. The questionnaire was administered to pts 15-23 yrs old with established IBD. Descriptive statistics were obtained on all study participants. Logistic regression analyses were used to compare subjects who had healthcare provider (HCP)-initiated interventions regarding care transition with those who had not, against IBD-related social stigma domains. Results: We enrolled 41 IBD pts (mean:17±2 yrs; 54%F, 46%M). Demographic data were as follows:Race/ethnicity (71% Caucasian, 22% African American, 7% Other);Education (63% in high school, 29% in college, 2% in middle school, 5% pursuing GED);Insurance status (71% private, 24% Medicaid/Healthchoice, 5% unsure).12% of pts reported excellent health status, 49% very good, 29% good, 7.5% fair, and 2.5% poor. Logistic regression analyses showed that pts who had not discussed transitioning with their HCP had greater attitude-related stigma regarding their IBD. Subjects who felt that their condition limited their social activities/hobbies were 2.9 times less likely to have discussed transition with the HCP (p ≤0.02). These pts also reported receiving inadequate disease self-management information. Only 45% of those who had not discussed transition, vs 87% of those who had, felt they had enough information to address their medical needs independently. Discussion: Our findings indicate the importance of the pediatric healthcare team in effectively addressing potential psychosocial barriers to health and developing more effective patient education strategies as a means of improving adolescent-adult care transitioning. More specific measures of barriers, such as socioeconomic status, could also be clinically useful in enhancing how we transition adolescent patients to adult care successfully, leading to improved medical outcomes and quality of life for these patients.

Abstract 1467: The potential role of de novo lipogenesis in the regulation of cell motility and invasion in colorectal cancer

The signaling networks that determine progression of cancer to a more aggressive metastatic phenotype include multiple alterations in the oncogenic pathways as well as significant changes in cellular metabolism. ATP-citrate lyase (ACLY) and Fatty Acid Synthase (FASN), key enzymes of lipid biogenesis, are significantly up-regulated and activated in many cancers and their activity is associated with poor prognosis, higher risk of disease recurrence, and death. Even though the role of neoplastic lipogenesis in providing proliferative and survival advantages to cancer cells has been defined, the impact of aberrant activation of lipogenic enzymes on the progression of cancer and development of metastases remains unknown. The purpose of the present study was to determine (i) the expression and/or activity of lipogenic enzymes in human colorectal cancer (CRC) tissues and cells, and (ii) the effect of their inhibition on signaling pathways involved in the regulation of migration and invasion. METHODS: Expression of FASN was analyzed in primary CRCs, matched liver metastasis and normal mucosa using Accumax Tissue Array. Expression of FASN and ACLY was also determined in CRC cell lines by Western blot. KM20, HT29, and HCT116 CRC cells with stable knockdown of ACLY and FASN were established, and downstream signaling pathways were analyzed (Western blot). Effect of inhibition of FASN on cell morphology (immunofluorescence microscopy), invasion (Boyden Chamber Assay), and endothelial cell adhesion was assessed. RESULTS: Expression of FASN was significantly higher in primary tumor and in liver metastases as compared to normal colon mucosa. FASN and ACLY were also highly expressed in CRC cell lines. Inhibition of lipogenic enzymes decreased migration and invasion of CRC cells. Mechanisms underlying these effects included disruption of CD44/c-MET signaling and decreased expression of pAkt, pFAK, RhoA, and RAC1. Furthermore, inhibition of FASN decreased the adherence of CRC cells to HMVEC-L endothelial cells.CONCLUSIONS: Increased expression of FASN and ACLY in both non-invasive and metastatic cell lines and CRC tissues suggest the potential role of de novo lipogenesis in the progression of CRC. Supporting this hypothesis, the current study demonstrates, for the first time, that inhibition of lipogenesis disrupts CD44/c-MET signaling which is well known to promote metastasis in several cancers including CRC. Furthermore, decreased expression of RhoA, Rac1, and pFAK, accompanied by changes in the actin cytoskeleton and the finding that inhibition of FASN attenuates the interaction of cancer cells with endothelial cells, suggests that activation of lipogenic enzymes plays a role in regulation of cancer cell motility and formation of metastasis. Together, these findings suggest that de novo lipogenesis may be a potential therapeutic target for early and advanced stages of CRC. 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 1467. doi:10.1158/1538-7445.AM2011-1467