Tim N. Beck

Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis

Significance The evolutionarily conserved RNA-binding protein Musashi-2 (MSI2) regulates mRNA translation and influences multiple biological processes, including maintenance of stem cell identity. This work for the first time, to our knowledge, identifies that more aggressive patient tumors have higher MSI2 levels. We define a critical role for MSI2 in supporting non-small cell lung cancer (NSCLC) invasiveness and further define claudins 3, 5, and 7 (CLDN3, CLDN5, and CLDN7), TGF-β receptor 1 (TGFβR1), and the small mothers against decapentaplegic homolog 3 (SMAD3) as targets through which MSI2 regulates this process. The observation that MSI2 expression is progressively elevated from an early stage in human NSCLC tumors suggests that this protein may play an essential role in the reprogramming of TGF-β signaling from growth-inhibiting to invasion-promoting during oncogenesis. Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of KrasLA1/+;P53R172HΔG/+ (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial–mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial–mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

Genomic insights into head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is frequently impervious to curative treatment efforts. Similar to other cancers associated with prolonged exposure to carcinogens, HNSCCs often have a high burden of mutations, contributing to substantial inter- and intra-tumor heterogeneity. The heterogeneity of this malignancy is further increased by the rising rate of human papillomavirus (HPV)-associated (HPV+) HNSCC, which defines an etiological subtype significantly different from the more common tobacco and alcohol associated HPV-negative (HPV-) HNSCC. Since 2011, application of large scale genome sequencing projects by The Cancer Genome Atlas (TCGA) network and other groups have established extensive datasets to characterize HPV- and HPV+ HNSCC, providing a foundation for advanced molecular diagnoses, identification of potential biomarkers, and therapeutic insights. Some genomic lesions are now appreciated as widely dispersed. For example, HPV- HNSCC characteristically inactivates the cell cycle suppressors TP53 (p53) and CDKN2A (p16), and often amplifies CCND1 (cyclin D), which phosphorylates RB1 to promote cell cycle progression from G1 to S. By contrast, HPV+ HNSCC expresses viral oncogenes E6 and E7, which inhibit TP53 and RB1, and activates the cell cycle regulator E2F1. Frequent activating mutations in PIK3CA and inactivating mutations in NOTCH1 are seen in both subtypes of HNSCC, emphasizing the importance of these pathways. Studies of large patient cohorts have also begun to identify less common genetic alterations, predominantly found in HPV- tumors, which suggest new mechanisms relevant to disease pathogenesis. Targets of these alterations including AJUBA and FAT1, both involved in the regulation of NOTCH/CTNNB1 signaling. Genes involved in oxidative stress, particularly CUL3, KEAP1 and NFE2L2, strongly associated with smoking, have also been identified, and are less well understood mechanistically. Application of sophisticated data-mining approaches, integrating genomic information with profiles of tumor methylation and gene expression, have helped to further yield insights, and in some cases suggest additional approaches to stratify patients for clinical treatment. We here discuss some recent insights built on TCGA and other genomic foundations.

Bioinformatic approaches to augment study of epithelial-to-mesenchymal transition in lung cancer

Bioinformatic approaches are intended to provide systems level insight into the complex biological processes that underlie serious diseases such as cancer. In this review we describe current bioinformatic resources, and illustrate how they have been used to study a clinically important example: epithelial-to-mesenchymal transition (EMT) in lung cancer. Lung cancer is the leading cause of cancer-related deaths and is often diagnosed at advanced stages, leading to limited therapeutic success. While EMT is essential during development and wound healing, pathological reactivation of this program by cancer cells contributes to metastasis and drug resistance, both major causes of death from lung cancer. Challenges of studying EMT include its transient nature, its molecular and phenotypic heterogeneity, and the complicated networks of rewired signaling cascades. Given the biology of lung cancer and the role of EMT, it is critical to better align the two in order to advance the impact of precision oncology. This task relies heavily on the application of bioinformatic resources. Besides summarizing recent work in this area, we use four EMT-associated genes, TGF-β (TGFB1), NEDD9/HEF1, β-catenin (CTNNB1) and E-cadherin (CDH1), as exemplars to demonstrate the current capacities and limitations of probing bioinformatic resources to inform hypothesis-driven studies with therapeutic goals.

EGFR and RB1 as Dual Biomarkers in HPV-Negative Head and Neck Cancer.

Clinical decision making for human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is predominantly guided by disease stage and anatomic location, with few validated biomarkers. The epidermal growth factor receptor (EGFR) is an important therapeutic target, but its value in guiding therapeutic decision making remains ambiguous. We integrated analysis of clinically annotated tissue microarrays with analysis of data available through the TCGA, to investigate the idea that expression signatures involving EGFR, proteins regulating EGFR function, and core cell-cycle modulators might serve as prognostic or drug response–predictive biomarkers. This work suggests that consideration of the expression of NSDHL and proteins that regulate EGFR recycling in combination with EGFR provides a useful prognostic biomarker set. In addition, inactivation of the tumor suppressor retinoblastoma 1 (RB1), reflected by CCND1/CDK6-inactivating phosphorylation of RB1 at T356, inversely correlated with expression of EGFR in patient HNSCC samples. Moreover, stratification of cases with high EGFR by expression levels of CCND1, CDK6, or the CCND1/CDK6-regulatory protein p16 (CDKN2A) identified groups with significant survival differences. To further explore the relationship between EGFR and RB1-associated cell-cycle activity, we evaluated simultaneous inhibition of RB1 phosphorylation with the CDK4/6 inhibitor palbociclib and of EGFR activity with lapatinib or afatinib. These drug combinations had synergistic inhibitory effects on the proliferation of HNSCC cells and strikingly limited ERK1/2 phosphorylation in contrast to either agent used alone. In summary, combinations of CDK and EGFR inhibitors may be particularly useful in EGFR and pT356RB1-expressing or CCND1/CDK6-overexpressing HPV-negative HNSCC. Mol Cancer Ther; 15(10); 2486–97. ©2016 AACR.

Integrating In Silico Resources to Map a Signaling Network

The abundance of publicly available life science databases offers a wealth of information that can support interpretation of experimentally derived data and greatly enhance hypothesis generation. Protein interaction and functional networks are not simply new renditions of existing data: they provide the opportunity to gain insights into the specific physical and functional role a protein plays as part of the biological system. In this chapter, we describe different in silico tools that can quickly and conveniently retrieve data from existing data repositories and we discuss how the available tools are best utilized for different purposes. While emphasizing protein-protein interaction databases (e.g., BioGrid and IntAct), we also introduce metasearch platforms such as STRING and GeneMANIA, pathway databases (e.g., BioCarta and Pathway Commons), text mining approaches (e.g., PubMed and Chilibot), and resources for drug-protein interactions, genetic information for model organisms and gene expression information based on microarray data mining. Furthermore, we provide a simple step-by-step protocol for building customized protein-protein interaction networks in Cytoscape, a powerful network assembly and visualization program, integrating data retrieved from these various databases. As we illustrate, generation of composite interaction networks enables investigators to extract significantly more information about a given biological system than utilization of a single database or sole reliance on primary literature.

EGFR Inhibitors as Therapeutic Agents in Head and Neck Cancer

Squamous cell carcinoma of the head and neck (SCCHN) is one of the more challenging cancers to treat. Although great progress has been made over the years, available treatment options are still far from ideal, as epitomized by a 5-year survival rate of only 30–40 %. A unique feature of SCCHN is that elevated expression of the epidermal growth factor receptor (EGFR), a member of the ErbB receptor tyrosine kinase (RTK) family and highly relevant to oncogenic proliferation, occurs in a significant number of cases, which has prompted great interest in utilizing EGFR-targeted therapies to treat this devastating disease. Significant advances in the treatment of SCCHN have been made using EGFR-targeting monoclonal antibodies. Another class of EGFR-targeting inhibitors, tyrosine kinase inhibitors (TKIs), has also shown promise as a potential treatment option. In order to appreciate how these therapeutic agents work and why they fail when they do, it is crucial to explore the biology of the ErbB family members, the signaling pathways that are associated with them, and how they interact with each specific therapeutic agent. This chapter discusses the biology of EGFR and other ErbB family members in SCCHN, and summarizes the current status of the application of EGFR and ErbB inhibitors.

Head and neck squamous cell carcinoma: Ambiguous human papillomavirus status, elevated p16, and deleted retinoblastoma 1

Head and neck squamous cell carcinoma (HNSCC) is potentially curable, but treatment planning remains a challenge. Oncogenic human papillomavirus (HPV)‐positive disease is often associated with a good prognosis compared with HPV‐negative disease. However, some HPV‐positive HNSCC recurs, often with distant metastases and significant treatment resistance.

Molecular mechanisms of the preventable causes of cancer in the United States

Annually, there are 1.6 million new cases of cancer and nearly 600,000 cancer deaths in the United States alone. The public health burden associated with these numbers has motivated enormous research efforts into understanding the root causes of cancer. These efforts have led to the recognition that between 40% and 45% of cancers are associated with preventable risk factors and, importantly, have identified specific molecular mechanisms by which these exposures modify human physiology to induce or promote cancer. The increasingly refined knowledge of these mechanisms, which we summarize here, emphasizes the need for greater efforts toward primary cancer prevention through mitigation of modifiable risk factors. It also suggests exploitable avenues for improved secondary prevention (which includes the development of therapeutics designed for cancer interception and enhanced techniques for noninvasive screening and early detection) based on detailed knowledge of early neoplastic pathobiology. Such efforts would complement the current emphasis on the development of therapeutic approaches to treat established cancers and are likely to result in far greater gains in reducing morbidity and mortality.

Abstract 34: AMH and AMHR2 regulate survival signaling, epithelial-mesenchymal transition (EMT) and resistance to HSP90 inhibition in non-small cell lung cancer (NSCLC)

In spite of encouraging advances in the treatment of lung cancer, the five-year survival rate has remained below 20 percent. Much of the mortality is associated with uncontrolled metastasis. Members of the TGF-s/BMP superfamily have long been known to regulate progression and metastasis in NSCLC. In this study, we identify unexpected inputs into TGF-s/BMP superfamily signaling that significantly modulate intrinsic tumor properties and drug response in NSCLC. Anti-Mullerian Hormone (AMH) and its specific type II receptor, AMHR2, share common type I receptors with BMP, and regulate 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. However, 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 for the first time confirmed autocrine expression of AMH and AMHR2 in this non-gonadal tumor environment. TGF-s and BMP signaling is important for the regulation of epithelial-mesenchymal transition (EMT). Strikingly, depletion of AMH/AMHR2 induced EMT-like features, including downregulation of cadherins, assumption of a mesenchymal morphology, and expression of mesenchymal markers, which 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 down regulation of mesenchymal markers such as vimentin and α-smooth muscle actin. 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. These data suggested an unusual relationship between ganetespib control of cell growth and differentiation status. We further established that inhibition of HSP90 increased expression of AMH and AMHR2 as well as several of the type 1 receptors (ALK2, ALK3, ALK6) that are necessary for signaling by BMP. Mechanistically, AMH and AMHR2 depletion increased phosphorylation of SMAD proteins, but depressed activity of NF-kB and AKT, both critical regulators of EMT and survival. These results for the first time indicate the presence of an AMH-AMHR2-NFkB-AKT signaling axis of therapeutic relevance in NSCLC. 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. Citation Format: Tim Beck, Emmanuelle Nicolas, Yan Zhou, Ilya Serebriiskii, Erica Golemis. AMH and AMHR2 regulate survival signaling, epithelial-mesenchymal transition (EMT) and resistance to HSP90 inhibition in non-small cell lung cancer (NSCLC). [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 34. doi:10.1158/1538-7445.AM2015-34

The Role of HGF/c-MET in Head and Neck Squamous Cell Carcinoma

Head and Neck Squamous Cell Carcinoma (HNSCC) remains a formidable challenge to physicians, scientists, and patients. New targets that can be exploited to improve the outcome of patients afflicted with this dreadful disease are desperately needed: one such potential target is c-MET. The c-MET receptor tyrosine kinase, also known as hepatocyte growth factor receptor (HGFR), is robustly overexpressed and sometimes mutated or amplified in head and neck cancer cells, while overexpression of its ligand, hepatocyte growth factor/scatter factor (HGF/SF), often occurs in tumor-adjacent mesenchymal cells, providing paracrine signals that support tumor growth. Activation of c-MET stimulates numerous downstream signaling pathways that contribute to tumor growth, including GRB2/RAS, PI3K, STAT3, SRC, β-catenin, and Notch. Overexpression or anomalous activation of c-MET is often associated with resistance to targeted therapies inhibiting receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), that communicate to similar growth factor cascades. In this review, we emphasize the role of c-MET/HGF in HNSCC as well as the potential for therapeutic targeting of this receptor.