Sonal J. Desai

Interleukin-8 confers androgen-independent growth and migration of LNCaP: differential effects of tyrosine kinases Src and FAK

Interleukin-8 (IL-8), a chemokine implicated in the metastasis and angiogenesis of a variety of cancers, has been reported to be overexpressed in prostate cancer. In this study, we ascribe a new role for IL-8 in prostate cancer progression using LNCaP cells. We demonstrate that IL-8 activates the androgen receptor and confers androgen-independent growth, while serving as a potent chemotactic factor. Our evaluation of the possible signal pathways involved in androgen-independence and cell migration shows that the tyrosine kinases Src and FAK (focal adhesion kinase) are involved in IL-8-induced signaling. Pharmacological and genetic inhibitors of Src and FAK interfere with IL-8-induced cell migration, while only the Src inhibitor was able to repress androgen-independent growth. This suggests that both growth and migration depend on the activity of Src, whereas cell migration also requires the activation of FAK. Our evidence that IL-8-induced androgen-independent growth is, at least in part, due to androgen receptor activation includes (1) an inhibitor of androgen receptor activity diminishes cell growth; (2) androgen receptor transactivation potential is augmented by IL-8 and (3) androgen receptor is recruited to the promoter of prostate specific antigen (PSA) upon IL-8 treatment, based on chromatin immunoprecipitation experiments. Taken together, our data suggest that in addition to its role in metastasis and angiogenesis, IL-8 may also serve as a facilitator for androgen-independent transition of prostate cancers. To our knowledge, this is the first report about the tyrosine kinase signals and androgen receptor activation induced by IL-8 in prostate cancer cells. The observation that IL-8 mediates its growth and chemotactic effects via Src and FAK suggests the potential use for tyrosine kinase inhibitors at early stage of prostate cancer development.

Large-Scale Screening and Molecular Characterization of EML4-ALK Fusion Variants in Archival Non–Small-Cell Lung Cancer Tumor Specimens Using Quantitative Reverse Transcription Polymerase Chain Reaction Assays

Introduction: The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non–small-cell lung cancer (NSCLC) patients. Methods: We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients. Results: Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23–89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001). Conclusions: This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.

Male Germ Cell–Associated Kinase, a Male-Specific Kinase Regulated by Androgen, Is a Coactivator of Androgen Receptor in Prostate Cancer Cells

Androgen receptor (AR) is a ligand-induced transcriptional factor, which plays an important role in the normal development of prostate as well as in the progression of prostate cancer. Numerous coactivators, which associate with AR and function to remodel chromatin and recruit RNA polymerase II to enhance the transcriptional potential of AR, have been identified. Among these coactivators, few are protein kinases. In this study, we describe the characterization of a novel protein kinase, male germ cell-associated kinase (MAK), which serves as a coactivator of AR. We present evidence, which indicates that (a) MAK physically associates with AR (MAK and AR are found to be coprecipitated from cell extracts, colocalized in nucleus, and corecruited to prostate-specific antigen promoter in LNCaP as well as in transfected cells); (b) MAK is able to enhance AR transactivation potential in an androgen- and kinase-dependent manner in several prostate cancer cells and synergize with ACTR/steroid receptor coactivator-3 coactivator; (c) small hairpin RNA (shRNA) knocks down MAK expression resulting in the reduction of AR transactivation ability; (d) MAK-shRNA or kinase-dead mutant, when introduced into LNCaP cells, reduces the growth of the cells; and (e) microarray analysis of LNCaP cells carrying kinase-dead MAK mutant showed a significant impediment of AR signaling, indicating that endogenous MAK plays a general role in AR function in prostate cancer cells and likely to be a general coactivator of AR in prostate tissues. The highly restricted expression of this kinase makes it a potentially useful target for intervention of androgen independence.

Autophagy Pathway Is Required for IL-6 Induced Neuroendocrine Differentiation and Chemoresistance of Prostate Cancer LNCaP Cells

Prostate cancer (PCa) cells undergoing neuroendocrine differentiation (NED) are clinically relevant to the development of relapsed castration-resistant PCa. Increasing evidences show that autophagy involves in the development of neuroendocrine (NE) tumors, including PCa. To clarify the effect of autophagy on NED, androgen-sensitive PCa LNCaP cells were examined. Treatment of LNCaP cells with IL-6 resulted in an induction of autophagy. In the absence of androgen, IL-6 caused an even stronger activation of autophagy. Similar result was identified in NED induction. Inhibition of autophagy with chloroquine (CQ) markedly decreased NED. This observation was confirmed by beclin1 and Atg5 silencing experiments. Further supporting the role of autophagy in NED, we found that LC3 was up-regulated in PCa tissue that had relapsed after androgen-deprivation therapy when compared with their primary tumor counterpart. LC3 staining in relapsed PCa tissue showed punctate pattern similar to the staining of chromogranin A (CgA), a marker for NED cells. Moreover, autophagy inhibition induced the apoptosis of IL-6 induced NE differentiated PCa cells. Consistently, inhibition of autophagy by knockdown of beclin1 or Atg5 sensitized NE differentiated LNCaP cells to etoposide, a chemotherapy drug. To identify the mechanisms, phosphorylation of IL-6 downstream targets was analyzed. An increase in phospho-AMPK and a decrease in phospho-mTOR were found, which implies that IL-6 regulates autophagy through the AMPK/mTOR pathway. Most important to this study is the discovery of REST, a neuronal gene-specific transcriptional repressor that is involved in autophagy activation. REST was down-regulated in IL-6 treatment. Knockdown experiments suggest that REST is critical to NED and autophagy activation by IL-6. Together, our studies imply that autophagy is involved in PCa progression and plays a cytoprotective role when NED is induced in PCa cells by IL-6 treatment. These results reveal the potential of targeting autophagy as part of a combined therapeutic regime for NE tumors.

MammaPrint for individualized recurrence risk assessment and treatment recommendations for early-stage breast cancer patients

Chapter 14 MammaPrint for Individualized Recurrence Risk Assessment and Treatment Recommendations for Early-Stage Breast Cancer Patients Sonal J. Desai and Tianhong Li University of California Davis Comprehensive Cancer Center, Division of Hematology & Oncology, Sacramento, CA 95817, USA sonal.desai@ucdmc.ucdavis.edu, tianhong.li@ucdmc.ucdavis.edu 14.1 Introduction Breast cancer is the second most common cancer and the second leading cause of cancer-related deaths in women worldwide. In 2013 an estimated 232,340 women are expected to be diagnosed with new cases of invasive breast cancers in the United States, and 39,620 women are expected to die from the disease [1, 2]. The majority of these deaths are due to disease recurrence or distant metastasis after initial treatment. Adjuvant systemic therapy with either endocrine therapy and/or chemotherapy has been shown to reduce the risk of distant recurrence and death from invasive breast cancer after local treatment with surgery with or without radiation therapy. To save lives, existing guidelines, aimed at avoiding under use of adjuvant Handbook of Therapeutic Biomarkers in Cancer Edited by Sherry X. Yang and Janet E. Dancey Copyright

Abstract 336: Genomic annotation of non-small cell lung cancer patient-derived xenograft models for personalized cancer therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Recent studies have characterized non-small cell lung cancer (NSCLC) as one of the most genomically deranged of all cancers, necessitating that both new drug development and patient therapy account for intra- and inter-patient tumor heterogeneity. Clinically annotated NSCLC patient-derived xenograft (PDX) models represent a novel approach to integrate this genomic complexity into a clinically relevant pre-clinical platform. We here describe molecular characterization to profile all currently “druggable” oncogenes for NSCLC in paired PDXs and original patient NSCLC tumor (PT). Method: Genomic DNA from archival formalin-fixed, paraffin-embedded (FFPE) PT and fresh first human-to-mouse (P0) NSCLC PDX tumors were isolated and subjected to oncogene mutational profiling using Sequenoms OncoCarta Panel v1. This panel detects 238 mutations in 19 genes commonly altered in cancer. RT-PCR-based molecular analyses of EGFR and KRAS mutations, EML4-ALK fusion transcripts, and RNA expression levels of ERCC1, RRM1 and TS genes were performed by Response Genetics, Inc. Genomic DNA from 3 serially passaged NSCLC PDX tumors (2 KRAS and 1 EGFR mutation models) up to 5 passages were also analyzed. Results: In the first 7 of 9 patient-PDX NSCLC models tested, oncogene mutational fidelity was preserved between PDX and PT with a good correlation of molecular biomarker expression (p<0.01). Two paired models had discrepancies in genotyping: from harboring 2 or 3 oncomutations at a frequency of 5-17% in PT to no mutation detected in P0 tumors), likely due to intra-patient tumor heterogeneity from clonal evolution. Of 3 models that have serial passaged tumors, the frequencies of oncomutation in each model were similar among the same passage (P0) or serial passage (P0 to P5) tumors. In several models tested for in vivo drug efficacy based on the molecular biomarker expression, results matched treatment outcome of the original patients., Conclusion: Our results validate the overall genomic fidelity of PDX tumors compared to original PT. Molecular characterization of individual tumor results in a clinically and genomically annotated PDX model with potential utility for selecting and validating clinically relevant drug target(s) for personalized cancer therapy. Acknowledgement: Supported by UC Davis Comprehensive Cancer Center Developmental Award (NIH/NCI P30CA093373), UL1 RR024146 from the National Center for Research Resources, the Jackson Laboratory, Response Genetics Inc., and the Addario Foundation. Citation Format: Sonal J. Desai, Neal Goodwin, Regina Gandour-Edwards, Royce F. Calhoun, David T. Cooke, Laurel A. Beckett, Martin K.H. Maus, Stephanie H. Astrow, Philip C. Mack, Ralph deVere White, David R. Gandara, Tianhong Li. Genomic annotation of non-small cell lung cancer patient-derived xenograft models for personalized cancer therapy. [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 336. doi:10.1158/1538-7445.AM2013-336

Abstract 5040: Dysregulation of the mTOR/AKT pathway in serum tumor DNA correlates with primary and acquired resistances to erlotinib in advanced NSCLC patients

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Non-small cell lung cancer (NSCLC) is the most common and lethal cancer worldwide. Erlotinib, an oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is the first molecularly targeted agent that has demonstrated modest yet comparable survival benefit to chemotherapy in unselected patients with advanced NSCLC after failing first-line chemotherapy. However, all patients eventually die from disease progression and a cure does not currently exist, even in patients with NSCLC tumors harboring EGFR TKI-sensitive EGFR mutations and achieving an initially dramatic tumor response. We previously demonstrated that dysregulation of the EGFR/mTOR/AKT signaling pathway contributes primarily to the acquired resistance to erlotinib in an erlotinib-resistant clone H3255R#2 (EGFRWT/WT), which was derived from the parental, erlotinib-sensitive human lung adenocarcinoma cell line H3255 (EGFRL858R/WT). To gain further molecular insights into the resistance mechanisms, we explored genome-wide alterations in genomic DNA (gDNA) from erlotinib-resistant cells as compared to parental cells using the Affymetrix Human SNP 6.0 array platform, which contains approximately one million probes for the detection of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). We found that significant alterations were simultaneously present in several core functional pathways, including ErbB, mTOR/AKT, apoptosis, and cell cycle, as well as EMT, stem cell and metastasis related genes. Selected targets have been validated in vitro. Serum tumor DNA has emerged as an easily accessible and stable source of tumor biospecimens for advanced NSCLC patients and has been used for detecting somatic mutations in EGFR and K-Ras genes either directly or amplified by high fidelity whole genome amplification technology. We further determined whether this genome-wide array-based platform could be used to clinically assess the drug resistance mechanisms in advanced NSCLC patients. We analyzed the genomic alterations in two paired serum tumor DNA samples obtained before treatment and at treatment failure from NSCLC patients with primary or secondary resistance to erlotinib on a clinical trial (clinicaltrials.gov, identifier: [NCT00950365][1]). Of particular interest, we found that there were similar changes in the gDNAs from erlotinib-resistant patients’ sera and gDNA from R#2 cells within the mTOR/AKT pathway, including amplification of PI3K, AMPK, and VEGF genes, and deletion of RSK genes. Analyses on more banked, paired serum tumor DNA samples from patients enrolled in the same trial are ongoing. Our results support targeting the mTOR/AKT and/or VEGF pathway as a strategy to overcome clinical resistance to EGFR TKIs and serum tumor DNA could be a useful surrogate to assessing resistance mechanisms to therapy in advanced NSCLC patients. 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 5040. doi:10.1158/1538-7445.AM2011-5040 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00950365&atom=%2Fcanres%2F71%2F8_Supplement%2F5040.atom

Abstract 4447: Nanoformulation of lapatinib for HER2-positive breast cancer patients with brain metastasis

Despite HER2-targeting agents having a reduced risk of distant relapse in early-stage breast cancer and improved systemic control in metastatic breast cancer, relapse or progression in the brain is a common and serious concern for mortality and morbidity in these HER2-positive breast cancer patients. Currently, radiation is the only effective therapy for these patients with various clinical outcomes. Thus, there is a significant unmet need to develop new therapeutic modalities to improve treatment efficacy for these patients, especially for patients who have failed radiation therapy. The objective of this project is to develop a novel nanoformulation of lapatinib for the treatment of HER2-positive breast cancer patients with brain metastasis. Lapatinib is the only FDA-approved small molecule tyrosine kinase inhibitor targeting both HER2 and epidermal growth factor receptor (EGFR). Lapatinib anecdotally induces objective response in HER2-positive breast cancer patients with brain metastasis. Current preclinical and clinic data suggest that lapatinib has limited and variable penetration through the blood-brain barrier, which is also affected by large inter-patient variations in the bioavailability of oral formulation of lapatinib. Dr. Lam9s laboratory has developed a novel nanoparticle technology using oligocholic acid based micelles (telodendrimers) to load a high dose of hydrophobic drugs for cancer treatment. We have generated a stable nanoformulation of lapatinib with drug loading up to 10 mg per mL. The average particle size is less than 20 nm in diameter, which is desirable for tumor targeting due to the preferential drug accumulation at the tumor site via the enhanced permeation and retention (EPR) effect. We are currently optimizing and characterizing the physicochemical properties of several lapatinib-loaded nanoformulations. The most stable and efficacious nanoformulation of lapatinib will be selected for further preclinical testing using in vitro and in vivo models of HER2+ breast cancer. This nanoformulation of lapatinoib will be suitable for intrathecal injection as it is not affected by individual variations in oral bioavailability and penetration through the blood brain barrier. If successful, we would design a phase I/II clinical study to determine the safety and efficacy of this new nanoformulation of lapatinib for HER2+ breast cancer patients with refractory brain metastasis or meningeal disease. 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 4447. doi:10.1158/1538-7445.AM2011-4447

Abstract 4684: Autophagy blockade sensitizes prostate cancer cells towards Src family kinase inhibitors

There is overwhelming evidence that tyrosine kinases play an important role in cancer development. As a prototype of targeted therapy, tyrosine kinase inhibitors are now successfully applied to cancer treatment. However, as single agents, tyrosine kinase inhibitors have not achieved satisfactory results in the treatment of prostate cancer, principally due to their inability to efficiently kill tumor cells. Our lab has been interested in the role of the Src complex in prostate cancer progression including the induction of androgen independence and metastasis. Previously, we reported that Src inhibitors such as saracatinib and PP2 caused G1 growth arrest and diminished invasiveness in prostate cancer cells, but rarely apoptosis. Here, we have shown that Src family kinase (SFK) inhibitors can induce a high level of autophagy, which protects treated cells from undergoing apoptosis. Src siRNA knockdown experiments confirmed that autophagy was indeed caused by the lack of Src activity. The SFK inhibitor-induced autophagy is accompanied by the inhibition of PI3K (type I)/Akt/mTOR signaling pathway. To test whether autophagy blockade could lead to enhanced cell death, pharmacological inhibitors (3-methyladenine and chloroquine) and a genetic inhibitor (siRNA targeting Atg7) were used in combination with SFK inhibitors. The results showed that autophagy inhibition effectively enhanced cell killing induced by SFK inhibitors. Importantly, we showed a combination of saracatinib with chloroquine in mice significantly reduced prostate cancer (PC3) xenograft growth, compared with the control group. Taken together, these data suggest that 1) autophagy serves a protective role in SFK inhibitor-mediated cell killing; 2) clinically acceptable autophagy modulators may be used beneficially as adjunctive therapeutic agents for SFK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4684.