Rapamycin synergizes cisplatin sensitivity in basal-like breast cancer cells through up-regulation of p73.

Recent gene expression profiling studies have identified five breast cancer subtypes, of which the basal-like subtype is the most aggressive. Basal-like breast cancer poses serious clinical challenges as there are currently no targeted therapies available to treat it. Although there is increasing evidence that these tumors possess specific sensitivity to cisplatin, its success is often compromised due to its dose-limiting nephrotoxicity and the development of drug resistance. To overcome this limitation, our goal was to maximize the benefits associated with cisplatin therapy through drug combination strategies. Using a validated kinase inhibitor library, we showed that inhibition of the mTOR, TGFβRI, NFκB, PI3K/AKT, and MAPK pathways sensitized basal-like MDA-MB-468 cells to cisplatin treatment. Further analysis demonstrated that the combination of the mTOR inhibitor rapamycin and cisplatin generated significant drug synergism in basal-like MDA-MB-468, MDA-MB-231, and HCC1937 cells but not in luminal-like T47D or MCF-7 cells. We further showed that the synergistic effect of rapamycin plus cisplatin on basal-like breast cancer cells was mediated through the induction of p73. Depletion of endogenous p73 in basal-like cells abolished these synergistic effects. In conclusion, combination therapy with mTOR inhibitors and cisplatin may be a useful therapeutic strategy in the treatment of basal-like breast cancers

Arginine Deprivation, Autophagy, Apoptosis (AAA) for the Treatment of Melanoma

The majority of melanoma cells do not express argininosuccinate synthetase (ASS), and hence cannot synthesize arginine from citrulline. Their growth and proliferation depend on exogenous supply of arginine. Arginine degradation using arginine deiminase (ADI) leads to growth inhibition and eventually cell death while normal cells which express ASS can survive. This notion has been translated into clinical trial. Pegylated ADI (ADI-PEG20) has shown antitumor activity in melanoma. However, the sensitivity to ADI is different among ASS(−) melanoma cells. We have investigated and reviewed the signaling pathways which are affected by arginine deprivation and their consequences which lead to cell death. We have found that arginine deprivation inhibits mTOR signaling but leads to activation of MEK and ERK with no changes in BRAF. These changes most likely lead to autophagy, a possible mechanism to survive by recycling intracellular arginine. However apoptosis does occur which can be both caspase-dependent or independent. In order to increase the therapeutic efficacy of this form of treatment, one should consider adding other agent(s) which can drive the cells toward apoptosis or inhibit the autophagic process

Arginine Deprivation as a Targeted Therapy for Cancer

Certain cancers may be auxotrophic for a particular amino acid and amino acid deprivation is one method to treat these tumors. Arginine deprivation is a novel approach to target tumors which lack argininosuccinate synthetase (ASS) expression. ASS is a key enzyme which converts citrulline to arginine. Tumors which usually do not express ASS include melanoma, hepatocellular carcinoma, some mesotheliomas and some renal cell cancers. Arginine can be degraded by several enzymes including arginine deiminase (ADI). Although ADI is a microbial enzyme from mycoplasma, it has high affinity to arginine and catalyzes arginine to citrulline and ammonia. Citrulline can be recycled back to arginine in normal cells which express ASS, whereas ASS(−) tumor cells cannot. A pegylated form of ADI (ADI-PEG20) has been formulated and has shown in vitro and in vivo activity against melanoma and hepatocellular carcinoma. ADI-PEG20 induces apoptosis in melanoma cell lines. However, arginine deprivation can also induce ASS expression in certain melanoma cell lines which can lead to in-vitro drug resistance. Phase I and II clinical trials with ADI-PEG20 have been conducted in patients with melanoma and hepatocellular carcinoma and antitumor activity has been demonstrated in both cancers. This article reviews our laboratory and clinical experience as well as others with ADI-PEG20 as an antineoplastic agent. Future direction in utilizing this agent is also discussed

Abstract LB-222: Identifying cFLIP as a marker and also a potentially “druggable” target of SAHA+TRAIL (TNF-Related Apoptosis Inducing Ligand), cytotoxicity in malignant pleural mesothelioma (MPM)

Abstract Background: Despite expressing adequate levels of receptors for TRAIL significant percentages of cancer cells are resistant to TRAIL-induced apoptosis. We have previously reported that histone deacetylase inhibitor SAHA (vorinostat) + TRAIL combination induces profound supra-additive cytotoxicity in MPM in vitro. We observe that only MPM cells with some TRAIL sensitivity are very susceptible to SAHA+TRAIL cytotoxicity. We hypothesize that TRAIL-initiated death signal at the membrane level determine cellular susceptibility to SAHA+TRAIL. The objective of this study is to identify the molecular marker that predicts TRAIL and thus TRAIL+SAHA sensitivity in MPM cells. Materials and methods: Intrinsic sensitivity of 8 MPM and primary normal endothelial cells to TRAIL and SAHA+TRAIL is determined by cell viability assay; basal expression of cFLIP, caspase 8, DR4/DR5, FADD, RIP, TRADD in these cells are evaluated by western blots. Selective knockdown of FLIP is achieved by siRNA. FLIP mRNA levels were determined by quantitative RT-PCR. Results: 5/8 MPM cells are very suceptible to SAHA+TRAIL cytotoxicity (combination-sensitive cells) while 3 others are classified as combination-resistant. Western blot analysis identifies an inverse relationship between cFLIP as well as procaspase 8 expression and sensitivity to SAHA+TRAIL with only the difference in cFLIP levels as quantified by densitometric analysis being distinctive between two groups: 0.87±0.02 for 5 combination-sensitive MPM cells versus 0.15±0.05 for 3 combination-resistant MPM cells. siRNA-mediated partial cFLIP knockdown restores DISC activity in high cFLIP expressing cells as evidenced by caspase 8 and 3 catalytic processing with stronger caspase activation being noted in combination-treated cells. Quantitative RT-PCR demonstrates high level of cFLIP messenger RNA in high cFLIP expressors indicating that cFLIP is transcriptionally upregulated in these cells. Selective cFLIP downregulation in high cFLIP expressing cells restores susceptibility to TRAIL and strongly sensitizes them to SAHA+TRAIL cytotoxicity. Additionally, selective complete cFLIP knockdown abrogates the need for SAHA to achieve profound TRAIL-mediated cell death in MPM cells regardless of their intrinsic cFLIP epxression. Conclusdion: Our study identifies cFLIP expression as a marker of cellular sensitivity to SAHA+TRAIL in MPM in vitro. More importantly, gene knockdown experiments provide the proof of concept that cFLIP is a pontential “druggable” target and downregulation of which sensitizes resistant cancer cells to TRAIL and SAHA+TRAIL. Ongoing works aim to validate this observation in a larger panel of novel MPM cells and to define treatments strategies to downregulate cFLIP in tumor cells expressing high levels of this antiapoptotic protein. 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-222. doi:10.1158/1538-7445.AM2011-LB-222</jats:p

Phase II trial of SOM230 (pasireotide LAR) in patients with unresectable hepatocellular carcinoma

Lynn G Feun,&sup1; Medhi Wangpaichitr,&sup2; Ying-Ying Li,&sup1; Deukwoo Kwon,&sup3; Stephen P Richman,&sup1; Peter J Hosein,&sup1; Niramol Savaraj&sup1;,&sup2; &sup1;Department of Medicine, Medical Oncology, Sylvester Comprehensive Cancer Center, University of Miami, &sup2;Department of Surgery, Miami VA Healthcare System, Research Service, &sup3;Biostatistics and Bioinformatics Core, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA Background: A phase II trial of pasireotide was performed to assess its efficacy and safety in advanced or metastatic hepatocellular carcinoma (HCC).Patients and methods: Patients with advanced HCC and Child&ndash;Pugh score &le;7 received pasireotide LAR 60 mg intramuscularly every 28 days. Primary endpoint was disease control rate. Secondary endpoints were time to tumor progression, response rate, treatment-related adverse events, and overall survival. Serum insulin growth factor-1 was measured before and after pasireotide.Results: Twenty patients were treated and evaluable. Eighteen patients (90%) had prior therapy; 16 patients (80%) had multiple therapies. Median age was 65, 75% had Barcelona Clinic Liver Cancer stage C, and 55% had metastatic disease. The main toxicity was hyperglycemia. Rare adverse effects included reversible grade 4 elevation in alanina transaminase/aspartate transaminase in one patient. The best response was stable disease in 9 patients (45%). Median time to tumor progression for the 20 patients was 3 months, and median survival was 9 months.Conclusion: Pasireotide had limited clinical benefit as second-line or third-line treatment in patients with advanced or metastatic HCC. Low baseline insulin growth factor-1 level may be indicative when SOM230 treatment may be ineffective, and decreasing levels after treatment may be indicative of disease control. Keywords: pasireotide, hepatocellular carcinoma, insulin growth factor-1&nbsp

CS-03 * UTILIZING COLLAGEN AND DISCOIDIN DOMAIN RECEPTOR SIGNALING AS A NEWTHERAPEUTIC STRATEGY IN HUMAN MALIGNANT GLIOMA

We have previously shown that certain gliomas expressed procollagen type1 alpha1(1α1). Procollagen is synthesized in the endoplasmic reticulum, modified and packaged in the Golgi body and transported out of the cells. Once exited, the N-and C-terminal are cleaved by peptidase and fibril is formed. The collagen can interact with integrin, Endo180, or discoindin domain receptor(DDR). DDR is a class of receptor tyrosine kinase. The binding to collagen triggers autophosphorylation of DDR and initiation of other signaling cascades which are poorly understood, but have been implicated for cellular proliferation and invasion. DDR is comprised of 2 members(DDR1 and DDR2). Thus far, the clinical relevance of this pathway is not known. However, DDR1 has been reported in glioblastoma multiforme, both in cell lines and tissue and correlates with poor prognosis. In this report, we investigated the expression of procollagen1α1, DDR1 and DDR2 in 6 glioma cell lines. Silencing procollagen1α1, DDR1 or DDR2 resulted in impairment of cell migration. Silencing procollagen1α1 resulted in cell cycle arrest at G2/M. This effect is less when silencing DDR1 or DDR2. However, silencing DDR1 or DDR2 resulted in attenuation of pERK and pAKT. Since there is no inhibitor of collagen or DDRs, we have used Brefeldin A, which can block procollagen secretion out of the cell. Treatment with Brefeldin A resulted in retention of procollagen in the ER. Thus, less procollagen was secreted and hence there was less ligand to bind DDRs. As a result, less DDRs were activated. Our data suggest that there is a definite relationship between collagen and DDRs in gliomas which can affect cellular proliferation and migration. The downstream of this signaling is not yet understood but it can be explored for future treatment of glioma which possess both the ligand and receptor