Sudheer Kumar Gara

Germline HABP2 Mutation Causing Familial Nonmedullary Thyroid Cancer

Familial nonmedullary thyroid cancer accounts for 3 to 9% of all cases of thyroid cancer, but the susceptibility genes are not known. Here, we report a germline variant of HABP2 in seven affected members of a kindred with familial nonmedullary thyroid cancer and in 4.7% of 423 patients with thyroid cancer. This variant was associated with increased HABP2 protein expression in tumor samples from affected family members, as compared with normal adjacent thyroid tissue and samples from sporadic cancers. Functional studies showed that HABP2 has a tumor-suppressive effect, whereas the G534E variant results in loss of function.

TGF-β Signaling in Myeloid Cells Is Required for Tumor Metastasis

TGF-β is overexpressed in advanced human cancers. It correlates with metastasis and poor prognosis. However, TGF-β functions as both a tumor suppressor and a tumor promoter. Here, we report for the first time that genetic deletion of Tgfbr2 specifically in myeloid cells (Tgfbr2(MyeKO)) significantly inhibited tumor metastasis. Reconstitution of tumor-bearing mice with Tgfbr2(MyeKO) bone marrow recapitulated the inhibited metastasis phenotype. This effect is mediated through decreased production of type II cytokines, TGF-β1, arginase 1, and inducible nitric oxide synthase, which promoted IFN-γ production and improved systemic immunity. Depletion of CD8 T cells diminished the metastasis defect in the Tgfbr2(MyeKO) mice. Consistent with animal studies, myeloid cells from patients with advanced-stage cancer showed increased TGF-β receptor II expression. Our studies show that myeloid-specific TGF-β signaling is an essential component of the metastasis-promoting puzzle of TGF-β. This is in contrast to the previously reported tumor-suppressing phenotypes in fibroblasts, epithelial cells, and T cells.

Gr‐1+CD11b+ cells are responsible for tumor promoting effect of TGF‐β in breast cancer progression

One great challenge in our understanding of TGF‐β cancer biology and the successful application of TGF‐β‐targeted therapy is that TGF‐β works as both a tumor suppressor and a tumor promoter. The underlying mechanisms for its functional change remain to be elucidated. Using 4T1 mammary tumor model that shares many characteristics with human breast cancer, particularly its ability to spontaneously metastasize to the lungs, we demonstrate that Gr‐1+CD11b+ cells or myeloid derived suppressor cells are important mediators in TGF‐β regulation of mammary tumor progression. Depletion of Gr‐1+CD11b+ cells diminished the antitumor effect of TGF‐β neutralization. Two mechanisms were involved: first, treatment with TGF‐β neutralization antibody (1D11) significantly decreased the number of Gr‐1+CD11b+ cells in tumor tissues and premetastatic lung. This is mediated through increased Gr‐1+CD11b+ cell apoptosis. In addition, 1D11 treatment significantly decreased the expression of Th2 cytokines and Arginase 1. Interestingly, the number and property of Gr‐1+CD11b+ cells in peripheral blood/draining lymph nodes correlated with tumor size and metastases in response to 1D11 treatment. Our data suggest that the efficacy of TGF‐β neutralization depends on the presence of Gr‐1+CD11b+ cells, and these cells could be good biomarkers for TGF‐β‐targeted therapy.

miR30a Inhibits LOX Expression and Anaplastic Thyroid Cancer Progression

Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies, but its genetic drivers remain little understood. In this study, we report losses in expression of the miRNA miR30a, which is downregulated in ATC compared with differentiated thyroid cancer and normal tissue. miR30a downregulation was associated with advanced differentiated thyroid cancer and higher mortality. Mechanistically, we found miR30a decreased cellular invasion and migration, epithelial-mesenchymal transition marker levels, lysyl oxidase (LOX) expression, and metastatic capacity. LOX was identified as a direct target of miR30a that was overexpressed in ATC and associated with advanced differentiated thyroid cancer and higher mortality rate. Consistent with its role in other cancers, we found that LOX inhibited cell proliferation, cellular invasion, and migration and metastasis in vitro and in vivo. Together, our findings establish a critical functional role for miR30a downregulation in mediating LOX upregulation and thyroid cancer progression, with implications for LOX targeting as a rational therapeutic strategy in ATC.

Phase I trial of systemic intravenous infusion of interleukin-13-Pseudomonas exotoxin in patients with metastatic adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare but lethal malignancy without effective current therapy for metastatic disease. IL‐13‐PE is a recombinant cytotoxin consisting of human interleukin‐13 (IL‐13) and a truncated form of Pseudomonas exotoxin A (PE). The main objectives of this Phase I dose‐escalation trial were to assess the maximum‐tolerated dose (MTD), safety, and pharmacokinetics (PK) of IL‐13‐PE in patients with metastatic ACC. Eligible patients had confirmed IL‐13 receptor alpha 2 (IL‐13Rα2) expressions in their tumors. IL‐13‐PE at dose of 1–2 μg/kg was administered intravenously (IV) on day 1, 3, and 5 in a 4‐week cycle. Six patients received 1 μg/kg and two patients received 2 μg/kg of IL‐13‐PE. Dose‐limiting toxicity was observed at 2 μg/kg, at which patients exhibited thrombocytopenia and renal insufficiency without requiring dialysis. PK analysis demonstrated that at MTD, the mean maximum serum concentration (Cmax) of IL‐13‐PE was 21.0 ng/mL, and the terminal half‐life of IL‐13‐PE was 30–39 min. Two (25%) of the eight patients had baseline neutralizing antibodies against PE. Three (75%) of the remaining four tested patients developed neutralizing antibodies against IL‐13‐PE within 14–28 days of initial treatment. Of the five patients treated at MTD and assessed for response, one patient had stable disease for 5.5 months before disease progression; the others progressed within 1–2 months. In conclusion, systemic IV administration of IL‐13‐PE is safe at 1 μg/kg. All tested patients developed high levels of neutralizing antibodies during IL‐13‐PE treatment. Use of strategies for immunodepletion before IL‐13‐PE treatment should be considered in future trials.

Integrated genome-wide analysis of genomic changes and gene regulation in human adrenocortical tissue samples

To gain insight into the pathogenesis of adrenocortical carcinoma (ACC) and whether there is progression from normal-to-adenoma-to-carcinoma, we performed genome-wide gene expression, gene methylation, microRNA expression and comparative genomic hybridization (CGH) analysis in human adrenocortical tissue (normal, adrenocortical adenomas and ACC) samples. A pairwise comparison of normal, adrenocortical adenomas and ACC gene expression profiles with more than four-fold expression differences and an adjusted P-value < 0.05 revealed no major differences in normal versus adrenocortical adenoma whereas there are 808 and 1085, respectively, dysregulated genes between ACC versus adrenocortical adenoma and ACC versus normal. The majority of the dysregulated genes in ACC were downregulated. By integrating the CGH, gene methylation and expression profiles of potential miRNAs with the gene expression of dysregulated genes, we found that there are higher alterations in ACC versus normal compared to ACC versus adrenocortical adenoma. Importantly, we identified several novel molecular pathways that are associated with dysregulated genes and further experimentally validated that oncostatin m signaling induces caspase 3 dependent apoptosis and suppresses cell proliferation. Finally, we propose that there is higher number of genomic changes from normal-to-adenoma-to-carcinoma and identified oncostatin m signaling as a plausible druggable pathway for therapeutics.

Dual Inhibition of HDAC and tyrosine kinase signaling pathways with CUDC-907 inhibits thyroid cancer growth and metastases

Purpose: There is currently no standard therapy for anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC), which account for two-thirds of thyroid cancer–related deaths. Driver mutations in the PI3K/AKT and RAF/RAS/MEK/ERK pathways are common in ATC and PDTC. Histone deacetylases (HDAC) regulate cancer initiation and progression. Our aim was to determine the therapeutic efficacy of simultaneously targeting these pathways in thyroid cancer with a single agent and to evaluate biomarkers of treatment response. Experimental Design: CUDC-907 is a first-in-class compound, functioning as a dual inhibitor of HDACs and the PI3K/AKT pathway. We investigated its antiproliferative effect in vitro and in vivo. Results: CUDC-907 significantly inhibited cellular proliferation in thyroid cancer cell lines, induced G2–M arrest with decreased levels of the checkpoint regulators cyclin B1, AURKA, AURKB, PLK1, and increased p21 and p27. Treatment induced apoptosis with increased caspase-3/7 activity and decreased survivin levels and decreased cellular migration and invasion. CUDC-907 treatment caused H3 hyperacetylation and decreased HDAC2 expression. HDAC2 was upregulated in ATC and other thyroid cancer histologic subtypes. CUDC-907 treatment reduced both p-AKT and p-ERK1/2 levels. Finally, CUDC-907 treatment, in a metastatic mouse model of thyroid cancer, showed significant inhibition of growth and metastases, and tumors from treated mice had decreased HDAC2 expression, suggesting that this may be a useful biomarker of response. Conclusions: Dual inhibition of HDAC and the tyrosine kinase signaling pathways with CUDC-907 is a promising treatment strategy for advanced, metastatic thyroid cancer. Clin Cancer Res; 23(17); 5044–54. ©2017 AACR.

In silico VHL Gene Mutation Analysis and Prognosis of Pancreatic Neuroendocrine Tumors in von Hippel–Lindau Disease

Context Patients with von Hippel-Lindau (vHL) disease caused by a missense VHL mutation have a more severe phenotype compared with other VHL mutation types. Objective To define pancreatic neuroendocrine tumor (PNET) aggressiveness according to VHL genotype. Design A prospective natural history study. Setting The National Institutes of Health clinical center. Patients Patients with vHL disease, pancreatic manifestations, and germline missense VHL gene mutations. Intervention In-silico prediction of VHL mutation via five computational prediction models. Patients with >80% prediction for disease-causing mutations in all models [high predicted risk (HPR)] were compared with others [low predicted risk (LPR)]. Main Outcome Measure Rates of metastases, surgical intervention, and disease progression. Results Sixty-nine patients were included: 2 developed metastases, 12 needed surgery, and 31 had disease progression during a median follow-up of 60 months (range 13 to 84 months). Thirteen patients were excluded for low prediction reliability. In the remaining 56 patients (45 with PNETs, 11 with pancreatic cysts), the HPR group (n = 13) had a higher rate of disease progression than the LPR group (n = 43) in multivariable analysis (hazard ratio 3.6; 95% confidence interval, 1.1 to 11.9; P = 0.037). The HPR group also had a higher risk of developing metastases (P = 0.015). Among patients with codon 167 hotspot mutations (n = 26), those in the HPR group had a higher risk for disease progression (P = 0.03) than other patients. Conclusions Computational models for predicting the impact of missense VHL gene mutations may be used as a prognostic factor in patients with PNETs in the context of vHL disease.

Do patients with familial nonmedullary thyroid cancer present with more aggressive disease? Implications for initial surgical treatment

Background: There are conflicting reports on whether familial nonmedullary thyroid cancer is more aggressive than sporadic nonmedullary thyroid cancer. Our aim was to determine if the clinical and pathologic characteristics of familial nonmedullary thyroid cancer are different than nonmedullary thyroid cancer. Methods: We compared patients with familial nonmedullary thyroid cancer to a cohort of 53,571 nonmedullary thyroid cancer patients from the Surveillance, Epidemiology, and End Results database. Results: A total of 78 patients with familial nonmedullary thyroid cancer from 31 kindreds presented at a younger age (P = .04) and had a greater rate of T1 disease (P = .019), lymph node metastasis (P = .002), and the classic variant of papillary thyroid cancer on histology (P < .001) compared with the Surveillance, Epidemiology, and End Results cohort. Patients with ≥3 affected family members presented at a younger age (P = .04), had a lesser female‐to‐male ratio (P = .04), and had a greater rate of lymph node metastasis (P = .009). Compared with the Surveillance, Epidemiology, and End Results cohort, we found a higher prevalence of lymph node metastasis in familial nonmedullary thyroid cancer index cases (P = .003) but not in those diagnosed by screening ultrasonography (P = .58). Conclusion: Patients with familial nonmedullary thyroid cancer present at a younger age and have a greater rate of lymph node metastasis. The treatment for familial nonmedullary thyroid cancer should be more aggressive in patients who present clinically and in those who have ≥3 first‐degree relatives affected.

Synergistic combination of flavopiridol and carfilzomib targets commonly dysregulated pathways in adrenocortical carcinoma and has biomarkers of response

Drug repurposing is an effective approach to identify active drugs with known toxicity profiles for rare cancers such as ACC. The objective of this study was to determine the anticancer activity of combination treatment for ACC from previously identified candidate agents using quantitative high-throughput screening (qHTS). In this study, we evaluated the anticancer activity of flavopiridol and carfilzomib in three ACC cell lines in vitro and in vivo. Human ACC samples were analyzed for drug-target analysis, and cancer-related pathway arrays were used to identify biomarkers of treatment response. Because flavopiridol is a potent cyclin-dependent kinase (CDK) inhibitor, we found significantly higher CDK1 and CDK2 mRNA expression in three independent cohorts human ACC (p<0.01) and CDK1 protein by immunohistochemistry (p<0.01) in human ACC samples. In vitro treatment with flavopiridol and carfilzomib in all three ACC cell lines resulted in a dose-dependent, anti-proliferative effect, and the combination had synergistic activity as well as in three-dimensional tumor spheroids. We observed increased G2M cell-cycle arrest and apoptosis with combination treatment compared to other groups in vitro. The combination treatment decreased XIAP protein expression in ACC cell lines. Mice with human ACC xenografts treated with flavopiridol and carfilzomib had significantly lower tumor burden, compared to other groups (p<0.05). We observed increased cleaved-caspase expression and decreased XIAP in tumor xenografts of mice treated with combined agents. Our preclinical data supports the evaluation of combination therapy with flavopiridol and carfilzomib in patients with advanced ACC.