Yanis Boumber

Musashi RNA-Binding Proteins as Cancer Drivers and Novel Therapeutic Targets

Aberrant gene expression that drives human cancer can arise from epigenetic dysregulation. Although much attention has focused on altered activity of transcription factors and chromatin-modulating proteins, proteins that act posttranscriptionally can potently affect expression of oncogenic signaling proteins. The RNA-binding proteins (RBP) Musashi-1 (MSI1) and Musashi-2 (MSI2) are emerging as regulators of multiple critical biological processes relevant to cancer initiation, progression, and drug resistance. Following identification of Musashi as a regulator of progenitor cell identity in Drosophila, the human Musashi proteins were initially linked to control of maintenance of hematopoietic stem cells, then stem cell compartments for additional cell types. More recently, the Musashi proteins were found to be overexpressed and prognostic of outcome in numerous cancer types, including colorectal, lung, and pancreatic cancers; glioblastoma; and several leukemias. MSI1 and MSI2 bind and regulate the mRNA stability and translation of proteins operating in essential oncogenic signaling pathways, including NUMB/Notch, PTEN/mTOR, TGFβ/SMAD3, MYC, cMET, and others. On the basis of these activities, MSI proteins maintain cancer stem cell populations and regulate cancer invasion, metastasis, and development of more aggressive cancer phenotypes, including drug resistance. Although RBPs are viewed as difficult therapeutic targets, initial efforts to develop MSI-specific inhibitors are promising, and RNA interference–based approaches to inhibiting these proteins have had promising outcomes in preclinical studies. In the interim, understanding the function of these translational regulators may yield insight into the relationship between mRNA expression and protein expression in tumors, guiding tumor-profiling analysis. This review provides a current overview of Musashi as a cancer driver and novel therapeutic target. Clin Cancer Res; 23(9); 2143–53. ©2017 AACR.

A Proteomics Analysis Reveals 9 Up-Regulated Proteins Associated with Altered Cell Signaling in Colon Cancer Patients

Colorectal cancer is the second most common cancer in women and third most common cancer in men. Cell signaling alterations in colon cancer, especially in aggressive metastatic tumors, require further investigations. The present study aims to compare the expression pattern of proteins associated with cell signaling in paired tumor and non-tumor samples of patients with colon cancer, as well as to define the cluster of proteins to differentiate patients with non-metastatic (Dukes’ grade B) and metastatic (Dukes’ grade C&D) colon cancer. Frozen tumor and non-tumor samples were collected after tumor resection from 19 patients with colon cancer. The Panorama™ Antibody Microarray-Cell Signaling kits were used for the analyses. The expression ratios of paired tumor/non-tumor samples were calculated for the each protein. We employed R packages ‘samr’, ‘gplots’, ‘supclust’ (pelora, wilma algorithms), ‘glmnet’ for the differential expression analysis, supervised clustering and penalized logistic regression. Significance analysis of microarrays revealed 9 significantly up-regulated proteins, including protein kinase C gamma, c-Myc, MDM2, pan cytokeratin, and 1 significantly down-regulated protein (GAP1) in tumoral mucosa. Pan-cytokeratin and APP were up-regulated in tumor versus non-tumor tissue, and were selected in the predictive cluster to discriminate colon cancer type. Higher levels of S-100b and phospho-Tau-pSer199/202 were confirmed as the predictors of non-metastatic colon cancer by all employed regression/clustering methods. Deregulated proteins in colon cancer are involved in oncogenic signal transduction, cell cycle control, and regulation of cytoskeleton/transport. Further studies are needed to validate potential protein markers of colon cancer development and metastatic progression.

The convergent roles of NF-κB and ER stress in sunitinib-mediated expression of pro-tumorigenic cytokines and refractory phenotype in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common form of kidney cancer. While cure remains exceptionally infrequent in RCC patients with systemic or recurrent disease, current targeted molecular strategies, including multi-targeted tyrosine kinase inhibitors (TKIs), notably changed the treatment paradigm of advanced renal cancer. Yet, complete and durable responses have been noted in only a few cases. Our studies reveal that sunitinib triggers two resistance-promoting signaling pathways in RCC cells, which emanate from the endoplasmic reticulum (ER) stress response: a PERK-driven ER stress response that induces expression of the pro-tumorigenic cytokines IL-6, IL-8, and TNF-α, and a TRAF2-mediated NF-κB survival program that protects tumor cells against cell death. PERK blockade completely prevents sunitinib-induced expression of IL-6, IL-8 and TNF-α, whereas NF-κB inhibition reinstates sensitivity of RCC cells to sunitinib both in vitro and in vivo. Taken together, our findings indicate that ER stress response may contribute to sunitinib resistance in RCC patients.

Sequential occurrence of small cell and non-small lung cancer in a male patient: Is it a transformation?

ABSTRACT Lung cancer is one of the leading causes of cancer-related mortality and is categorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). We present a patient with epidermal growth factor receptor (EGFR)-mutant-NSCLC who developed metastatic SCLC after initial therapy with second-generation EGFR-tyrosine kinase inhibitor, afatinib. A 65-year-old male non-smoker was diagnosed with adenocarcinoma of the right lung, stage IVA (M1a). Due to tumor positivity for EGFR-Exon 19 deletion, the patient was started on oral afatinib, which resulted in a partial response. After ten months of treatment, he presented in the office with abdominal pain, distension, weight loss and jaundice. He had diffuse skeletal and hepatic metastases on PET/CT scan with interval progression of his cancer. Although the recurrence of lung adenocarcinoma was suspected, the patient was diagnosed with SCLC on liver biopsy. He received two cycles of chemotherapy and died due to pneumonia and sepsis.

Abstract B25: Anti-Mullerian hormone (AMH) supports epithelial identity and survival signaling in lung cancer

Abstracts: Fourth AACR International Conference on Frontiers in Basic Cancer Research; October 23-26, 2015; Philadelphia, PA The purpose of this study is to characterize resistance mechanisms of non-small cell lung cancer (NSCLC) to heat shock protein 90 (HSP90) inhibition. Unexpectedly, we identified anti-Mullerian hormone (AMH) and its type II receptor (AMHR2) as two resistance associated genes. AMH and AMHR2 are TGF-s/BMP superfamily members that share common type I receptors with BMP, resulting in overlapping signaling outputs. AMH and AMHR2 have thus far predominantly been studied in the context of gonadal development, in regulation of the female reproductive cycle and in gynecological malignancies, and AMH is not known to be expressed outside of these tissues. Using a focused RNAi library designed to detect genes associated with resistance to the HSP90 inhibitor ganetespib, siRNAs against AMH and AMHR2 sensitized 4 out of 5 and 3 out of 5 NSCLC cell lines to ganetespib, respectively. We then for the first time confirmed expression of AMH and AMHR2 in this non-gonadal tumor environment. Furthermore, analysis of data from The Cancer Genome Atlas (TCGA) indicated that AMH and AMHR2 are significantly upregulated in a subpopulation of NSCLC. Strikingly, depletion of AMH/AMHR2 induced EMT-like features, including downregulation of cadherins, a mesenchymal morphology, increased invasion, and expression of mesenchymal markers: features that are generally associated with resistance to chemotherapy. In contrast, inhibition of HSP90 selected for a more epithelial-like population of cells, as evident by increased E-cadherin or P-cadherin, and downregulation of mesenchymal markers such as VIM and ZEB1. To confirm that mesenchymal-like cells are indeed more responsive to HSP90 inhibition, we depleted E-cadherin or P-cadherin and again observed sensitization to ganetespib. We further found that AMH and AMHR2 did not confer sensitization to cisplatin. Interestingly, depletion of cadherins, and the associated induction of EMT-like properties, also resulted in downregulation of AMH and AMHR2, further supporting the link between AMH/AMHR2 expression and epithelial identity. In vivo studies confirmed that AMH depletion sensitizes NSCLC cells to ganetespib and showed that it also significantly reduces overall tumor volume. Reduction of AMH or AMHR2 also decreased phosphorylation of direct effector SMAD proteins, and depressed activity of NFκB and AKT, both regulators of EMT and survival. These results for the first time indicate the presence of an AMH-AMHR2-NFκB-AKT signaling axis of therapeutic relevance in NSCLC. To further explore the therapeutic potential of this observation, we treated NSCLC with combinations of ganetespib and the proteasome inhibitor bortezomib- an inhibitor previously shown to disrupt NFκB signaling in some cancers - and observed significant synergy between the two drugs. In conclusion, our work for the first time characterizes AMH and AMHR2 in NSCLC. We go on to show that AMH and AMHR2 regulated canonical-SMAD signaling as well as non-canonical AKT-NFκB signaling, in addition to regulating the epithelial identify of cancer cells. Importantly, our results also suggest that AMH and AMHR2 may serve as biomarkers to predict resistance to HSP90 inhibitors, and that it may be beneficial to prime lung tumors with HSP90 inhibitors, to reverse EMT and decrease survival signaling, prior to treatment with chemotherapy. Note: This abstract was not presented at the conference. Citation Format: Tim N. Beck, Vladislav Korobeynikov, Alexander Kudinov, Rachel Georgeopoulos, Emmanuelle Nicolas, Margret B. Einarson, Yan Zhou, Yanis Boumber, David A. Proia, Ilya G. Serebriiskii, Erica A. Golemis. Anti-Mullerian hormone (AMH) supports epithelial identity and survival signaling in lung cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B25.

Abstract 4098: Musashi-2 (MSI2) activates TGF-β signaling and inhibits CLDN7 to promote non-small cell lung cancer (NSCLC) metastasis

Approximately 220,000 new patients were diagnosed with lung cancer and ∼160,000 died from this disease in the United States in 2014. Non-small cell lung cancer (NSCLC) has a 5 year survival rate of approximately 16%, with most deaths associated with distant metastasis. To gain a better insight into the regulation of metastasis, we studied metastasis-competent versus low metastasis-potential cell lines derived from NSCLC tumors of KrasLA1/+; P53R172HG/+ (KP) mice. Initial screening showed consistent and statistically significant upregulation of Musashi-2 (MSI2) in highly metastatic cells on both mRNA and protein level. Furthermore, we established statistically significant elevation of MSI2 protein expression in 123 human NSCLC tumor specimens versus normal lung tissue (p MSI2 is an RNA binding protein that regulates mRNA translation and is upregulated and functionally important in hematologic malignancies including CML and AML. MSI2 knockdown in four independent metastatic murine and human NSCLC cell lines very significantly decreased invasion in vitro but did not significantly change cell proliferation or survival. In orthotropic lung injection of mouse NSCLC cells into the lungs of immunocompetent 129Sv mice, MSI2 depletion dramatically decreased invasion of mediastinal lymph nodes and abrogated metastasis. Reverse-phase protein array (RPPA) screening indicated that Msi2 depletion significantly affected the expression of multiple proteins associated with epithelial-mesenchymal transition (EMT) and control of cell-cell attachments, including the extracellular matrix component fibronectin (FN1) and the tight junction protein claudin-7 (CLDN7). These and additional targets were validated in all four NSCLC model systems. MSI2 dependent control of FN1 expression was mediated at the level of mRNA induction, while CLDN7 expression was strikingly upregulated (10-fold) at the protein but not mRNA level, nominating CLDN7 as a putative direct MSI2 target. Parallel evaluation of a series of candidate direct MSI2 translational targets indicated MSI2 depletion downregulated translation of TGF-β receptor (TGF-βRI) and SMAD3. Morphologically, MSI2 depleted cells were marked by a greater degree of cell-cell attachment, potentially explaining their decreased invasive capacity. Based on this work, we propose that MSI2 supports TGF-βRI dependent EMT signaling and downregulates tight junction controls in a subset of metastatic NSCLC, and may influence tumor response to inhibitors targeting the TGF-β pathway. Citation Format: Alexander Kudinov, Alexander Deneka Deneka, Anna Nikonova, Young-Ho Ahn, Xin Liu Liu, Ilya Serebriiskii, Andrey Efimov, Dong-Hua Yang, Mark Andrake Andrake, Emmanuelle Nicolas, Brian Egleston, Hossein Borghaei, Don Gibbons, Jonathan Kurie, Erica Golemis, Yanis Boumber. Musashi-2 (MSI2) activates TGF-β signaling and inhibits CLDN7 to promote non-small cell lung cancer (NSCLC) metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4098. doi:10.1158/1538-7445.AM2015-4098