Saravanan Thiyagarajan

Role of GLI2 Transcription Factor in Growth and Tumorigenicity of Prostate Cells

Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including prostate cancer. The GLI2 transcription factor is a primary mediator of Hh signaling. However, its relative contribution to development of prostate tumors is poorly understood. To establish the role of GLI2 in maintaining the tumorigenic properties of prostate cancer cells, we developed GLI2-specific small hairpin RNA. Knockdown of GLI2 in these cells resulted in significant down-regulation of the Hh signaling pathway, followed by inhibition of colony formation, anchorage-independent growth, and growth of xenografts in vivo. Conversely, ectopic expression of Gli2 in nontumorigenic prostate epithelial cells resulted in accelerated cell cycle progression, especially transition through G(2)-M, and augmented proliferation. Altogether, our findings suggest that GLI2 plays a critical role in the malignant phenotype of prostate cancer cells, and GLI2 may potentially become an attractive therapeutic target for the treatment of prostate cancer.

Twist1 Suppresses Senescence Programs and Thereby Accelerates and Maintains Mutant Kras-Induced Lung Tumorigenesis

KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with KrasG12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

The Twist box domain is required for Twist1-induced prostate cancer metastasis

Twist1, a basic helix-loop-helix transcription factor, plays a key role during development and is a master regulator of the epithelial–mesenchymal transition (EMT) that promotes cancer metastasis. Structure–function relationships of Twist1 to cancer-related phenotypes are underappreciated, so we studied the requirement of the conserved Twist box domain for metastatic phenotypes in prostate cancer. Evidence suggests that Twist1 is overexpressed in clinical specimens and correlated with aggressive/metastatic disease. Therefore, we examined a transactivation mutant, Twist1-F191G, in prostate cancer cells using in vitro assays, which mimic various stages of metastasis. Twist1 overexpression led to elevated cytoskeletal stiffness and cell traction forces at the migratory edge of cells based on biophysical single-cell measurements. Twist1 conferred additional cellular properties associated with cancer cell metastasis including increased migration, invasion, anoikis resistance, and anchorage-independent growth. The Twist box mutant was defective for these Twist1 phenotypes in vitro. Importantly, we observed a high frequency of Twist1-induced metastatic lung tumors and extrathoracic metastases in vivo using the experimental lung metastasis assay. The Twist box was required for prostate cancer cells to colonize metastatic lung lesions and extrathoracic metastases. Comparative genomic profiling revealed transcriptional programs directed by the Twist box that were associated with cancer progression, such as Hoxa9. Mechanistically, Twist1 bound to the Hoxa9 promoter and positively regulated Hoxa9 expression in prostate cancer cells. Finally, Hoxa9 was important for Twist1-induced cellular phenotypes associated with metastasis. These data suggest that the Twist box domain is required for Twist1 transcriptional programs and prostate cancer metastasis. Implications: Targeting the Twist box domain of Twist1 may effectively limit prostate cancer metastatic potential. Mol Cancer Res; 11(11); 1387–400. ©2013 AACR.

Inhibition of TWIST1 Leads to Activation of Oncogene-Induced Senescence in Oncogene Driven Non-Small Cell Lung Cancer

A large fraction of non–small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. Although targeted agents exist for EGFR- and EML4-ALK–driven NSCLCs, no therapies target the most frequently found driver mutation, KRAS. Furthermore, acquired resistance to the currently targetable driver mutations is nearly universally observed. Clearly a novel therapeutic approach is needed to target oncogene-driven NSCLCs. We recently showed that the basic helix-loop-helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in transgenic mouse models and that inhibition of Twist1 in these models led to Kras-induced senescence. In the current study, we examine the role of TWIST1 in oncogene-driven human NSCLCs. Silencing of TWIST1 in KRAS-mutant human NSCLC cell lines resulted in dramatic growth inhibition and either activation of a latent oncogene-induced senescence program or, in some cases, apoptosis. Similar effects were observed in EGFR mutation–driven and c-Met–amplified NSCLC cell lines. Growth inhibition by silencing of TWIST1 was independent of p53 or p16 mutational status and did not require previously defined mediators of senescence, p21 and p27, nor could this phenotype be rescued by overexpression of SKP2. In xenograft models, silencing of TWIST1 resulted in significant growth inhibition of KRAS-mutant, EGFR-mutant, and c-Met–amplified NSCLCs. Remarkably, inducible silencing of TWIST1 resulted in significant growth inhibition of established KRAS-mutant tumors. Together these findings suggest that silencing of TWIST1 in oncogene driver–dependent NSCLCs represents a novel and promising therapeutic strategy. Mol Cancer Res; 11(4); 329–38. ©2013 AACR.

MAGE-C2 Promotes Growth and Tumorigenicity of Melanoma Cells, Phosphorylation of KAP1, and DNA Damage Repair

Melanoma-associated antigen-encoding (MAGE) genes are expressed in melanoma and other cancers but not in normal somatic cells. MAGE expression is associated with aggressive tumor growth, poor clinical outcome, and resistance to chemotherapy, but the mechanisms have not been completely elucidated. In this study, we show that downregulation of MAGE-C2 in A375 melanoma cells and low-passage cultures from human metastatic melanomas (MRA cells) results in increased apoptosis and decreased growth of tumor xenografts in athymic nude mice. Previously, we showed that MAGE-C2 binds KAP1, a scaffolding protein that regulates DNA repair. Phosphorylation of KAP1-Serine 824 (Ser824) by ataxia-telangiectasia-mutated (ATM) kinase is necessary for repair of DNA double-strand breaks (DSBs); now we show that MAGE-C2 knockdown reduces, whereas MAGE-C2 overexpression increases, ATM kinase-dependent phosphorylation of KAP1-Ser824. We demonstrate that MAGE-C2 increases co-precipitation of KAP1 with ATM and that binding of MAGE-C2 to KAP1 is necessary for increased KAP1-Ser824 phosphorylation. Furthermore, ectopic expression of MAGE-C2 enhances repair of I-SceI endonuclease-induced DSBs in U-2OS cells. As phosphorylation of KAP1-Ser824 facilitates relaxation of heterochromatin, which is necessary for DNA repair and cellular proliferation, our results suggest that MAGE-C2 can promote tumor growth by phosphorylation of KAP1-Ser824 and by enhancement of DNA damage repair.

Nelfinavir induces radiation sensitization in pituitary adenoma cells

Pituitary adenomas with local invasion and high secretory activity remain a therapeutic challenge. The HIV protease inhibitor nelfinavir is a radiosensitizer in multiple tumor models. We tested nelfinavir as a radiosensitizer in pituitary adenoma cells in vitro and in vivo. We examined the effect of nelfinavir with radiation on in vitro cell viability, clonogenic survival, apoptosis, prolactin secretion, cell cycle distribution, and the PI3K-AKT-mTOR pathway. We evaluated tumor growth delay and confirmed nelfinavirs effect on the PI3K-AKT-mTOR pathway in a hind-flank model. Nelfinavir sensitized pituitary adenoma cells to ionizing radiation as shown by viability assays and clonogenic assay with an enhancement ratio of 1.2 (p

Inhibition of Rapamycin-Induced AKT Activation Elicits Differential Antitumor Response in Head and Neck Cancers

The PI3K/AKT/mTOR pathway is an important signaling axis that is perturbed in majority of cancers. Biomarkers such as pS6RP, GLUT1, and tumor FDG uptake are being evaluated in patient stratification for mTOR pathway inhibitors. In the absence of a clear understanding of the underlying mechanisms in tumor signaling, the biomarker strategy for patient stratification is of limited use. Here, we show that no discernible correlation exists between FDG uptake and the corresponding Ki67, GLUT1, pS6RP expression in tumor biopsies from patients with head and neck cancer. Correlation between GLUT1 and pS6RP levels in tumors was observed but elevated pS6RP was noticed even in the absence of concomitant AKT activation, suggesting that other downstream molecules of PI3K/AKT and/or other pathways upstream of mTOR are active in these tumors. Using an ex vivo platform, we identified putative responders to rapamycin, an mTOR inhibitor in these tumors. However, rapamycin did not induce antitumor effect in the majority of tumors with activated mTOR, potentially attributable to the observation that rapamycin induces feedback activation of AKT. Accordingly, treatment of these tumors with an AKT inhibitor and rapamycin uniformly resulted in abrogation of mTOR inhibition-induced AKT activation in all tumors but failed to induce antitumor response in a subset. Phosphoproteomic profiling of tumors resistant to dual AKT/mTOR inhibitors revealed differential activation of multiple pathways involved in proliferation and survival. Collectively, our results suggest that, in addition to biomarker-based segregation, functional assessment of a patients tumor before treatment with mTOR/AKT inhibitors may be useful for patient stratification.

Rationally co-targeting divergent pathways in KRAS wild-type colorectal cancers by CANscript technology reveals tumor dependence on Notch and Erbb2

KRAS mutation status can distinguish between metastatic colorectal carcinoma (mCRC) patients who may benefit from therapies that target the epidermal growth factor receptor (EGFR), such as cetuximab. However, patients whose tumors harbor mutant KRAS (codons 12/13, 61 and 146) are often excluded from EGFR-targeted regimens, while other patients with wild type KRAS will sometimes respond favorably to these same drugs. These conflicting observations suggest that a more robust approach to individualize therapy may enable greater frequency of positive clinical outcome for mCRC patients. Here, we utilized alive tumor tissues in ex-vivo platform termed CANscript, which preserves the native tumor heterogeneity, in order to interrogate the antitumor effects of EGFR-targeted drugs in mCRC (n = 40). We demonstrated that, irrespective of KRAS status, cetuximab did not induce an antitumor response in a majority of patient tumors. In the subset of non-responsive tumors, data showed that expression levels of EGFR ligands contributed to a mechanism of resistance. Transcriptomic and phosphoproteomic profiling revealed deregulation of multiple pathways, significantly the Notch and Erbb2. Targeting these nodes concurrently resulted in antitumor efficacy in a majority of cetuximab-resistant tumors. These findings highlight the importance of integrating molecular profile and functional testing tools for optimization of alternate strategies in resistant population.

Predicting tumor-immune response to checkpoint inhibitors using a novel patient-derived live tumor explant model.

e20035Background: Immunotherapy has emerged as a powerful treatment paradigm wherein therapies primarily target immune components. For example, blockade of PD-1 and PD-L1 offers effective treatment...

Abstract 3681: A patient derivedex vivoplatform CANScript™ predicts distinct therapeutic outcomes to multiple PD-1 checkpoint inhibitors in single tumor biopsies

Background: Emerging clinical evidence using immunotherapy in recent years has demonstrated its power to suppress tumor growth by releasing the brakes on the immune system. For example, blockade of immune checkpoints, such as PD-1, has revolutionized treatment options for patients with aggressive cancers such as head and neck squamous cell carcinoma (HNSCC). However, clinical responses to PD-1 inhibition vary widely among patients while majority of them do not show any anti-tumor response. Multiple FDA-approved drugs against the same immune checkpoints have resulted in globally distinct outcomes in the clinic. There is a huge unmet need to understand these disparities at the individual patient level and to maximize the clinical benefits of these agents. Methods: Here, we employed a patient-derived ex vivo model, CANScript™ (Majumder B et al. Nature Commun 2015 Feb 27;6:6169 and Goldman A et al. Nature Commun 2015 Feb 11;6:6139), which recreates the native 3D tumor microenvironment, autocrine-paracrine dynamic and response to therapy by incorporating fresh tumor tissue and autologous immune cells with immunotherapy agents. Utilizing late stage HNSCC (N=50) we interrogated phenotypic response to two FDA-approved PD-1 inhibitors, Pembrolizumab (KEYTRUDA) and Nivolumab (OPDIVO). To do this, we used a comprehensive panel of immunological assays to evaluate changes in the immune compartments by flowcytometry and immunohistochemistry (primarily CD8, CD45, FOXP3, CXCR4, CD68, PDL1, PD1), multiplex cytokine profiling (IL6, IL8, IFN-g, IL10, IL12, Perforin, GranzymeB), along with functional/phenotypic effects including tumor proliferation, histological changes and cell death. Results: The data demonstrated that CANScript™ preserves the tumor-immune contexture and native heterogeneity across different clinical stages and patients. Importantly, we observed that PD-1 blockade resulted in patient-specific therapeutic response, which was characterized by differential distribution and maintenance of infiltrating CD8+ and CD4+ lymphocytes, distinct patterning of cytokines linked to functional dysregulation, and changes in tumor proliferation and apoptosis. Interestingly, data suggest that both Pembrolizumab and Nivolumab act on the same immune network axis but trigger functionally diverse phenotypes in the tumor immune compartment and distinct antitumor effects within an individual patient tumor. Conclusion: Together, these findings demonstrate the utility of CANScript™ as an ex vivo platform to predict therapeutic response of immune checkpoint inhibitors at the individual patient level. It also highlights mechanistic variations that could impact clinical outcome of these agents having the same molecular target. Such information can re-shape our understanding of patient selection and rational combinations for novel immune checkpoint inhibitors. Citation Format: Padhma Radhakrishnan, Vasanthakumar Sekar, Nilesh Brijwani, Priyanka Chevour, Babu Balakrishnan, Dency D Pinto, Muthusami Oliyarasi, Debapriya G. Mehrotra, Manjusha Biswas, Sabitha K S, Kodaganur S. Gopinath, Arkasubhra Ghosh, M s Ganesh, Ashok M. Shenoy, Saravanan Thiyagarajan, Biswanath Majumder, Aaron Goldman. A patient derived ex vivo platform CANScript™ predicts distinct therapeutic outcomes to multiple PD-1 checkpoint inhibitors in single tumor biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3681. doi:10.1158/1538-7445.AM2017-3681