Geoffrey J. Markowitz

TGF-β-miR-34a-CCL22 Signaling-Induced Treg Cell Recruitment Promotes Venous Metastases of HBV-Positive Hepatocellular Carcinoma

Portal vein tumor thrombus (PVTT) is strongly correlated to a poor prognosis for patients with hepatocellular carcinoma (HCC). In this study, we uncovered a causative link between hepatitis B virus (HBV) infection and development of PVTT. Mechanistically, elevated TGF-β activity, associated with the persistent presence of HBV in the liver tissue, suppresses the expression of microRNA-34a, leading to enhanced production of chemokine CCL22, which recruits regulatory T (Treg) cells to facilitate immune escape. These findings strongly suggest that HBV infection and activity of the TGF-β-miR-34a-CCL22 axis serve as potent etiological factors to predispose HCC patients for the development of PVTT, possibly through the creation of an immune-subversive microenvironment to favor colonization of disseminated HCC cells in the portal venous system.

MiR-148a functions to suppress metastasis and serves as a prognostic indicator in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) presents a major challenge in the clinic due to its lack of reliable prognostic markers and targeted therapies. Accumulating evidence strongly supports the notion that microRNAs (miRNAs) are involved in tumorigenesis and could serve as biomarkers for diagnostic purposes. To identify miRNAs that functionally suppress metastasis of TNBC, we employed a concerted approach with selecting miRNAs that display differential expression profiles from bioinformatic analyses of breast cancer patient databases and validating top candidates with functional assays using breast cancer cell lines and mouse models. We have found that miR-148a exhibits properties as a tumor suppressor as its expression is inversely correlated with the ability of both human and mouse breast cancer cells to colonize the lung in mouse xenograft tumor models. Mechanistically, miR-148a appears to suppress the extravasation process of cancer cells, likely by targeting two genes WNT1 and NRP1 in a cell non-autonomous manner. Importantly, lower expression of miR-148a is detected in higher-grade tumor samples and correlated with increased likelihood to develop metastases and poor prognosis in subsets of breast cancer patients, particularly those with TNBC. Thus, miR-148a is functionally defined as a suppressor of breast cancer metastasis and may serve as a prognostic biomarker for this disease.

Distinct Receptor Tyrosine Kinase Subsets Mediate Anti-HER2 Drug Resistance in Breast Cancer.

Targeted inhibitors of the human epidermal growth factor receptor 2 (HER2), such as trastuzumab and lapatinib, are among the first examples of molecularly targeted cancer therapy and have proven largely effective for the treatment of HER2-positive breast cancers. However, approximately half of those patients either do not respond to these therapies or develop secondary resistance. Although a few signaling pathways have been implicated, a comprehensive understanding of mechanisms underlying HER2 inhibitor drug resistance is still lacking. To address this critical question, we undertook a concerted approach using patient expression data sets, HER2-positive cell lines, and tumor samples biopsied both before and after trastuzumab treatment. Together, these methods revealed that high expression and activation of a specific subset of receptor tyrosine kinases (RTKs) was strongly associated with poor clinical prognosis and the development of resistance. Mechanistically, these RTKs are capable of maintaining downstream signal transduction to promote tumor growth via the suppression of cellular senescence. Consequently, these findings provide the rationale for the design of therapeutic strategies for overcoming drug resistance in breast cancer via combinational inhibition of the limited number of targets from this specific subset of RTKs.

Inflammation-Dependent IL18 Signaling Restricts Hepatocellular Carcinoma Growth by Enhancing the Accumulation and Activity of Tumor-Infiltrating Lymphocytes

Chronic inflammation in liver tissue is an underlying cause of hepatocellular carcinoma. High levels of inflammatory cytokine IL18 in the circulation of patients with hepatocellular carcinoma correlates with poor prognosis. However, conflicting results have been reported for IL18 in hepatocellular carcinoma development and progression. In this study, we used tissue specimens from hepatocellular carcinoma patients and clinically relevant mouse models of hepatocellular carcinoma to evaluate IL18 expression and function. In a mouse model of liver fibrosis that recapitulates a tumor-promoting microenvironment, global deletion of the IL18 receptor IL18R1 enhanced tumor growth and burden. Similarly, in a carcinogen-induced model of liver tumorigenesis, IL18R1 deletion increased tumor burden. Mechanistically, we found that IL18 exerted inflammation-dependent tumor-suppressive effects largely by promoting the differentiation, activity, and survival of tumor-infiltrating T cells. Finally, differences in the expression of IL18 in tumor tissue versus nontumor tissue were more predictive of patient outcome than overall tissue expression. Taken together, our findings resolve a long-standing contradiction regarding a tumor-suppressive role for IL18 in established hepatocellular carcinoma and provide a mechanistic explanation for the complex relationship between its expression pattern and hepatocellular carcinoma prognosis. Cancer Res; 76(8); 2394-405. ©2016 AACR.

TGF-β-Regulated MicroRNAs and Their Function in Cancer Biology

The transforming growth factor-β (TGF-β) is known to regulate a large number of biological processes and is involved in various aspects of tumor development. Recent studies have shown that the biogenesis of miRNAs can be regulated by TGF-β signaling directly via Smad-dependent mechanisms and/or other unknown mechanisms, which may induce autoregulatory feedback loops in response to the activation of TGF-β signaling, influencing the fate of tumor cells. In this chapter, we summarize the currently described mechanisms underlying TGF-βs regulation of miRNA biogenesis, and the functional role of TGF-β-regulated miRNAs in tumor initiation, epithelial-mesenchymal transition, and tumor microenvironment modulation. Finally, we introduce methods to study TGF-β-regulated miRNAs and their functions in tumor progression and metastasis using an example of publication from our lab demonstrating the presence of a TGF-β-miR-34a-CCL22 signaling axis, which serves as a potent etiological pathway for the development of hepatocellular carcinoma venous metastases.

Isolation of Glioma-Initiating Cells for Biological Study

Glioblastoma multiforme (GBM, WHO grade IV astrocytoma) is the most common and lethal primary brain tumor in adults, with an average survival of slightly more than 1 year after initial diagnosis. GBMs display significant heterogeneity within the tumor mass, among which a subpopulation of cells called glioma-initiating cells (GICs) is responsible for tumorigenesis and resistance to conventional therapies. Therefore, understanding the mechanism underlying the biological properties of GICs would help develop better therapies to target this population for GBM treatment. This protocol provides detailed procedures to isolate GICs and non-GICs from patients specimen and glioma xenografts, which serves as the first step for the biological studies of GICs. Upon separation of GICs and non-GICs, a series of studies, such as expression profiling and functional screen, etc., can be performed to identify signal pathways responsible for the malignant nature of GICs. Besides, translational studies can also be conducted to examine drug responses of GICs. In sum, isolation of GICs with reliable methods will provide the basis for the further biological studies.

Immune reprogramming via PD-1 inhibition enhances early-stage lung cancer survival

Success of immune checkpoint inhibitors in advanced non-small-cell lung cancer (NSCLC) has invigorated their use in the neoadjuvant setting for early-stage disease. However, the cellular and molecular mechanisms of the early immune responses to therapy remain poorly understood. Through an integrated analysis of early-stage NSCLC patients and a Kras mutant mouse model, we show a prevalent programmed cell death 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) axis exemplified by increased intratumoral PD-1+ T cells and PD-L1 expression. Notably, tumor progression was associated with spatiotemporal modulation of the immune microenvironment with dominant immunosuppressive phenotypes at later phases of tumor growth. Importantly, PD-1 inhibition controlled tumor growth, improved overall survival, and reprogrammed tumor-associated lymphoid and myeloid cells. Depletion of T lymphocyte subsets demonstrated synergistic effects of those populations on PD-1 inhibition of tumor growth. Transcriptome analyses revealed T cell subset-specific alterations corresponding to degree of response to the treatment. These results provide insights into temporal evolution of the phenotypic effects of PD-1/PD-L1 activation and inhibition and motivate targeting of this axis early in lung cancer progression.

Abstract 4498: Targeting IRE1a-XBP1 signaling in lung cancer

Every year 1.5 million people die from lung cancer worldwide. Approximately ~15-30% of non small cell lung cancers (NSCLC) are driven by KRAS mutations and currently possess no viable therapeutic options. As such the discovery of effective therapeutic alternatives for the treatment of KRAS driven NSCLC are sorely needed. One prospect being explored in other solid tumors, but currently unexplored in NSCLC, is targeting the endoplasmic reticulum (ER) stress response. Adverse conditions in tumors such as hypoxia, nutrient starvation and reactive oxygen species disrupt protein folding, and induce ER stress. The most evolutionarily conserved pathway by which this insult is alleviated is the IRE1α-XBP1 axis, which responds by increasing the expression of a broad range of protein folding and quality control genes. Previous work has demonstrated that several cancers utilize this pathway to promote tumorigenesis, progression, epithelial to mesenchymal transition and metastasis. A preliminary analysis of a tissue microarray containing 304 human NSCLC samples, 254 demonstrated at least some positivity and >50% were strongly positive for the active isoform of XBP1. Given these preliminary findings we investigated the status of IRE1a-XB1 in our murine orthotopic model and confirmed the activation of IRE1a-XBP1 signaling in tumor cells in the context of the tumor microenvironment. Furthermore, in both cell types we can see the activation of canonical downstream targets of XBP1. Utilizing the CRISPR-CAS9 system, we knocked out IRE1a in the orthotopic tumor cell line. IRE1a abrogation was confirmed by western blot, evaluating the fraction of active XBP1 after induced ER stress in vitro, and qPCR of canonical downstream targets. Additionally, this knockout did not alter proliferation or morphology in vitro. However, when tumors were grown orthotopically, Cas9-IRE1a knockouts failed to grow past 10 days, whereas Cas9-Scramble controls and the parental tumor line grew comparably until end stage. Given these preliminary findings and data from prior studies, we hypothesize that the abnormal activation of the IRE1α-XBP1 axis may influence KRAS NSCLC progression and aggressiveness and serve as a potential novel avenue for the treatment of NSCLC. After probing the impact of the underlying mechanism on tumor growth we will finally evaluate the translational utility of targeting the ER stress pathway with a small molecule inhibitor against IRE1a. Citation Format: Michael J. Crowley, Shira Yomtoubian, Geoffrey Markowitz, Nasser K. Altorki, Dingcheng Gao, Juan Cubillos-Ruiz, Vivek Mittal. Targeting IRE1a-XBP1 signaling in lung cancer [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 4498. doi:10.1158/1538-7445.AM2017-4498

Noncoding RNAs Regulating Cancer Signaling Network

The cellular signaling network plays a fundamental role during development and disease, especially cancer progression. By deregulating signaling pathways, cancer cells acquire hallmarks of the disease including uncontrolled proliferation, evasion from cell death, activation of angiogenesis, invasion, and metastasis. Noncoding RNAs make substantial contributions to regulating signal transduction in cancer, thereby promoting or suppressing different biological processes during tumorigenesis. This chapter provides an overview on the regulatory functions of noncoding RNAs in the signaling network in cancer cells. It summarizes examples of noncoding RNAs that act as oncogenes or tumor-suppressing genes involved in key signal pathways as well as signal crosstalk in cancer cells.