Costa Frangou

Characterization of TAZ domains important for the induction of breast cancer stem cell properties and tumorigenesis

The Hippo pathway is an evolutionarily conserved regulator of tissue growth and cell fate during development and regeneration. Conversely, deregulation of the Hippo pathway has been reported in several malignancies. Here, we used integrative functional genomics approaches to identify TAZ, a transcription co-activator and key downstream effector of the Hippo pathway, as an essential driver for the propagation of TNBC malignant phenotype. We further showed in non-transformed human mammary basal epithelial cells that expression of constitutively active TAZ confers cancer stem cell (CSC) traits that are dependent on the TAZ and TEAD interacting domains. In addition, to gain a better understanding of how TAZ functions, we performed genetic-function analysis of TAZ. Significantly, we identified that both the WW and transcriptional activation domains of TAZ are critical for the induced CSC properties as well as tumorigenic potential as manifested in vitro and in human breast cancer xenograft in vivo. Collectively, our data suggest that pharmacological inhibition of TAZ activity may provide a novel means of targeting and eliminating breast CSCs.

Phosphorylation of Tyr188 in the WW domain of YAP1 plays an essential role in YAP1-induced cellular transformation.

ABSTRACT The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP1, a transcriptional co-activator amplified in mouse and human cancers where it promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. The Hippo tumor suppressor pathway has been suggested to inhibit the YAP1 function through serine phosphorylation-induced cytoplasmic retention and degradation. Here we report that the tyrosine188 (Y188) site of YAP1 isoform with 2 WW domains (known as YAP1-2) plays an important role in YAP1-induced cellular transformation. IP-Mass Spectrometry analysis of YAP1 identified the phosphorylation of Y188 but not other tyrosine residues. In contrast to the aberrant 3D acinus formation observed in YAP1-WT transduced cells, overexpression of YAP1-Y188F (non-phosphorylated mimic) displayed normal 3D structures. In addition, knockdown of the endogenous YAP1 in MDA-MB231 breast cancer cells inhibited cell proliferation and migration, which were then successfully rescued by the exogenous YAP1-WT and YAP1-Y188E but not Y188F. Mechanistically, we also demonstrated that YAP1-Y188F had a higher affinity to the upstream negative regulator PTPN14 and was extensively localized in the cytoplasm. Since the Y188 is located in the conserved aromatic core of the WW domain of YAP1, our finding has a wide implication for WW domain signaling in general, where Y phosphorylation may act as a common positive regulator of the complex formation via WW domains. In summary, our results indicate that tyrosine 188 plays an important role in the YAP1-induced cellular transformation and its phosphorylation may intriguingly serve as a positive indicator of YAP1 activation.

VGLL4 Selectively Represses YAP-Dependent Gene Induction and Tumorigenic Phenotypes in Breast Cancer

Members of the mammalian Vestigial-like (VGLL) family of transcriptional cofactors activate genes in response to a wide variety of environmental cues. Recently, VGLL proteins have been proposed to regulate key signaling networks involved in cancer development and progression. However, the biological and clinical significance of VGLL dysregulation in human breast cancer pathogenesis remains unknown. Here, we report that diminished VGLL4 expression, but not VGLL1-3, correlated with both shorter relapse-free survival and shorter disease-specific survival of cancer patients with different molecular subtypes of breast cancer. Additionally, we further demonstrate that overexpression of VGLL4 reduces breast cancer cell proliferation, migration, intravasation/extravasation potential, favors cell death, and suppresses tumor growth in vivo. Mechanistically, VGLL4 negatively regulates the TEAD1-YAP1 transcriptional complex and exerts its growth inhibitory control through its evolutionary conserved TDU2 domain at its C-terminus. The results suggest that VGLL4 is a candidate tumor suppressor gene which acts by selectively antagonizing YAP-dependent tumor growth. VGLL4 may be a promising therapeutic target in breast cancer.

Loss of KIBRA function activates EGFR signaling by inducing AREG

The Hippo signaling pathway is a central regulator of organ size, tissue homeostasis, and tumorigenesis. KIBRA is a member of the WW domain-containing protein family and has recently been reported to be an upstream protein in the Hippo signaling pathway. However, the clinical significance of KIBRA deregulation and the underlying mechanisms by which KIBRA regulates breast cancer (BC) initiation and progression remain poorly understood. Here, we report that KIBRA knockdown in mammary epithelial cells induced epithelial-to-mesenchymal transition (EMT) and increased cell migration and tumorigenic potential. Mechanistically, we observed that inhibiting KIBRA induced growth factor-independent cell proliferation in 2D and 3D culture due to the secretion of amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand. Also, we show that AREG activation in KIBRA-knockdown cells depended on the transcriptional coactivator YAP1. Significantly, decreased expression of KIBRA is correlated with recurrence and reduced BC patient survival. In summary, this study elucidates the molecular events that underpin the role of KIBRA in BC. As a result, our work provides biological insight into the role of KIBRA as a critical regulator of YAP1-mediated oncogenic growth, and may have clinical potential for facilitating patient stratification and identifying novel therapeutic approaches for BC patients.

Abstract A059: Targeted RNA expression profiling for biomarker discovery in complex biologic samples

New rapid and robust transcriptome-based methods for cellular characterization of the tumor microenvironment and biomarker discovery are required to improve prognosis and treatment of cancer and other diseases. However, challenges with current approaches for the above applications include high sample requirements, poor sensitivity, low dynamic range, and limited throughput. To address these limitations, we have developed the DriverMap targeted RNA expression profiling assay using a genome-wide set of 19,000 validated primer pairs that leverages the sensitivity of multiplex RT-PCR with the throughput and digital readout depth of next-generation sequencing (NGS). Starting from just 10pg (single-cell) to 100ng (10,000 cells) of total RNA is sufficient to quantify over 5 orders of magnitude variation in gene expression levels with performance similar to conventional qRT-PCR. Further, the use of gene-specific primers enables direct analysis of total RNA isolate and obviates the need for globin and rRNA depletion from whole blood samples. In this study, we present the performance of the assay for immunophenotyping of immune cells in whole blood samples from sepsis patients and assess the immune responses to complex immunomodulatory stimuli in an ex vivo model system. We will also present profiling results that demonstrate how this assay can be used to analyze the level of immune cell infiltration in tumor samples, and identify active pathways in tumor and xenograft samples. Preliminary studies demonstrate the assay’s unparalleled specificity and sensitivity, resulting in better detection of low-abundance mRNA transcripts as well as an improved cost-effectiveness for high-throughput clinical applications. Citation Format: Alex Chenchik, Costa Frangou, Mikhail Makhanov. Targeted RNA expression profiling for biomarker discovery in complex biologic samples [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A059.

Abstract B094: Targeted expression and molecular profiling assay for tumor microenvironment

New rapid and robust transcriptome-based methods for cellular characterization of the tumor microenvironment and biomarker discovery are required to improve cancer prognosis and treatment. However, challenges with current approaches for the above applications include high sample requirements, poor sensitivity, low dynamic range, and limited throughput. To address these limitations, we have developed a unique approach for targeted transcriptome profiling using validated targeted primers that leverages the sensitivity of multiplex RT-PCR with the throughput of Next-Generation Sequencing (NGS) technology. By combining these methods, just 10-100 ng of total RNA is sufficient to quantify over 5 orders of magnitude variation in gene expression levels. Further, the use of targeted primers enables direct analysis of total RNA isolate and obviates the need for globin depletion from whole blood samples. Finally, using a defined set of amplicons to assess expression levels of all protein-coding genes facilitates and simplifies data analysis and allows more precise sample-to-sample normalization. We will present profiling results that demonstrate how this assay can be used to analyze the level of immune cell infiltration, assess intact and deficient immune mechanisms, and generally elucidate the tumor microenvironment of breast cancer samples. Citation Format: Alex Chenchik, Andrey Komarov, Michael Makhanov, Sunitha Sastry, Costa Frangou. Targeted expression and molecular profiling assay for tumor microenvironment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B094.

Abstract A010: Cancer immunotherapy biomarker profiling assay

Introduction: Selecting patients predisposed to respond to existing and novel immunotherapy treatments requires the development of novel prognostic and predictive biomarkers. Recent reports have identified several gene expression signatures specific for immunity status and immune contexture in solid tumor microenvironments, and enable predictions of efficacy of a number of chemo- and immunotherapeutics. We used these expression signatures to develop a robust standardized assay—the Cancer Immunotherapy 2000 (CI2000) panel—that would dissect immunosurveillance mechanisms and discover novel prognostic and predictive immune response biomarkers. Material and Method: The CI2000 panel provides signatures for approximately 400 immunity-related genes from 16 predictive and prognostic core genes that have been validated in recent chemo- and immunotherapy clinical trials across several tumor types, including melanoma, colorectal, breast, and lung cancers. In addition, using computation analysis of immunotherapy network model, we predicted key nodes in pathways specific for antigen presentation and recognition, inhibition, activation and motility of immune cells, adhesion, and apoptosis of cancer cells. This computational analysis produced approximately 400 more targets beyond the core panel. The CI2000 panel also includes a comprehensive set of 1,200 genes specific for detection and quantitative profiling in the tumor microenvironment of different types of activated immune cells of adaptive and innate immunity. Further, an additional set of housekeeping genes with constant expression between different cancer types was included in the assay for data normalization. Results and Discussion: The CI2000 assay quantitatively profiles the expression levels of approximately 2,000 key cancer genes from 10-100ng of total RNA using multiplex RT-PCR amplification followed by next-generation HT sequencing (NGS). Built-in standards for each gene target enable sample-to-sample calibration of the NGS data and provide a reference to adjust for background noise which often depends on the quality of samples. Control studies have shown that the CI2000 assay quantifiably measures 4 orders of magnitude variation in the expression levels of 2,000 key cancer immune-related genes from as few as 100 cells in whole lysate, and profiles from frozen biopsies, surgically-removed tumor samples, PBMCs, and FFPE clinical samples show a comparable range and sensitivity. We will present profiling data from infiltrating immune cells and key intact and deficient immune mechanisms in the tumor microenvironment of breast cancer samples to demonstrate the performance and utility of the CI2000 assay. Conclusions: Comprehensive profiling of tumor-associated immune cells with the CI2000 gene panel provides a sensitive, standardized, and quantitative method to investigate cancer immunoediting mechanisms. Robust methods for molecular characterizations of the immune mechanism in the tumor microenvironment are essential to meet the imminent need for diagnostic approaches that identify patient population responsive to the growing number of immunotherapeutic treatments. Toward this goal, the described CI2000 assay provides a platform for the discovery prognostic and predictive immune response biomarker signatures. Citation Format: Alex Chenchik, Leonid Iakoubov, Michael Makhanov, Costa Frangou. Cancer immunotherapy biomarker profiling assay. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A010.

Abstract 744: Cancer immunotherapy biomarker profiling assay

Selecting patients predisposed to respond to existing and novel immunotherapy treatments requires the development of novel prognostic and predictive biomarkers. Recent reports have identified several gene expression signatures specific for immunity status and immune contexture in solid tumor microenvironments, and these enable predictions of efficacy of a number of chemo- and immunotherapeutics. Robust methods for molecular characterizations of the immune mechanism in the tumor microenvironment are essential to meet the imminent need for diagnostic approaches that identify patient populations responsive to the growing number of immunotherapeutic treatments. Toward this goal, we developed the Cancer ImmuneNet 2500 (CIN2500) assay for profiling cellular composition in the tumor microenvironment and discovery of novel prognostic and predictive immune response biomarkers. The CIN2500 panel provides signatures for approximately 400 immunity-related genes from 16 predictive and prognostic core genes that have been validated in recent chemo- and immunotherapy clinical trials across several tumor types, including melanoma, colorectal, breast, and lung cancers. In addition, using computational analysis of an immunotherapy network model, we predicted and included in the assay approximately 300 key nodes in pathways specific for antigen presentation and recognition, inhibition, activation and motility of immune cells, and adhesion and apoptosis of cancer cells. The CIN2500 panel also includes a comprehensive set of 1,700 genes specific for detection and quantitative profiling in the tumor microenvironment of different types of activated immune cells of adaptive and innate immunity, and stromal, fibroblast, cancer, endothelial and adipose cell types. The CIN2500 assay quantitatively profiles the expression levels of approximately 2,500 key cancer genes from 10-100ng of total RNA using multiplex RT-PCR amplification followed by Next-Generation Sequencing (NGS). Built-in standards for each gene target enable sample-to-sample calibration of the NGS data and provide a reference to adjust for background noise that often depends on the quality of samples. Control studies have shown that the CIN2500 assay quantifiably measures 4 orders of magnitude variation in the expression levels of 2,500 immune-related genes from as few as 100 cells in whole lysate, and profiles from frozen biopsies, surgically-removed tumor samples, PBMCs, and FFPE clinical samples show a comparable range and sensitivity. We will present profiling data from infiltrating immune cells and key intact and deficient immune mechanisms in the tumor microenvironment of breast cancer samples to demonstrate the performance and utility of the CIN2500 assay. Citation Format: Alex Chenchik, Leonid Iakoubov, Mikhail Makhanov, Costa Frangou. Cancer immunotherapy biomarker profiling assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 744.

Abstract 4354: CRISPR/Cas9 genome-wide gRNA library for target identification

Genome-wide loss-of-function screening is a fundamental method to identify genes responsible for driving biological responses, and complex pooled lentiviral-based libraries expressing large numbers of genetic disruptors, such as shRNAs, make large-scale cell screening practical. While RNAi-based approaches have proven to be an effective strategy for these screens, recent work suggests CRISPR technology offers an effective alternative. Although shRNA and sgRNA pooled library screens are similar in concept, the gene interruption with the two techniques occurs by a very different mechanism so some divergence may be expected when comparing results obtained using one method versus the other. To investigate the potential difference in the two methodologies, we performed parallel dropout viability screens to identify essential genes in a pair of primary isogenic CML cell lines using a CRISPR/Cas9 knockout library and an RNA interference (RNAi) library targeting the same set of 6,300 genes with the same number of targeted effectors (sgRNA or shRNA) for each gene. The results showed significant, but not complete, overlap in the essential genes identified by each assay in each cell line indicating that both approaches are effective to identify the majority of essential genes in a cell system. However, analysis did indicate that a small number of essential targets were only identified with CRISPR and certain unique targets seemed to show up only in the RNAi screen results. By combining data from the two screening methodologies, a consistent number of viability genes and pathways could be identified and subsequently validated by independent cell based assays at a very high confirmation rate. Citation Format: Donato Tedesco, Paul Diehl, Mikhail Makhanov, Sylvain Baron, Dmitry Suchkov, Costa Frangou, Alex Chenchik. CRISPR/Cas9 genome-wide gRNA library for target identification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4354.

Abstract A23: CancerNet biomarker profiling panel

Background: Next-generation sequencing (NGS) is playing a transformational role in cancer discovery research; providing a powerful way to study DNA and/or RNA from clinical specimens. Unfortunately, the cost and complexity of whole genome sequencing approaches represent major barriers to use of these methodologies in routine molecular diagnostic testing. Nonetheless, a comprehensive catalog of all types of mutations in cancer opens unique opportunities for understanding the mechanism of cancer onset or progression and facilitates a more personalized approach to clinical care, including improved risk stratification and treatment selection. Methodology: Recently developed targeted approaches reduce NGS data complexity and generate qualitative sequencing information by measurement of a subset of targets per technical replicate with minimal sample usage. To this end, we have developed a novel, multiplex PCR target enrichment-based NGS pipeline (Driver-Map) designed to comprehensively assess the genomic landscape of both solid tumors and hematologic malignancies from clinical samples. Importantly, CancerCore analyzes the genomic alterations for 569 pan-cancer driver genes. Simultaneous quantitative target gene expression analysis using multiplex quantitative RNA-Seq (Q-RNA-Seq) for ∼2,000 additional genes (CancerNet) and computational network modeling facilitates the rapid identification of cancer gene and/or pathway models to identify commonalities across tumor type(s) and supporting pharmacogenetics information. Results: To determine the broad utility of this methodology and determine whether Driver-Map captures biologically relevant information, we apply it to clinical samples from triple-negative breast cancer (TNBC) patients and demonstrate that our approach identifies novel therapeutically tractable genes. Conclusion: In summary, Driver-Map provides both strand-specific sequencing at single-base resolution and ‘digital’ gene expression profiling, resulting in better detection of both rare genetic variants and low abundance mRNA transcripts. Moreover, Driver-Map results in unparalleled specificity and sensitivity while increasing the cost-effectiveness for high-throughput clinical Next-Gen applications. Citation Format: Alex Chenchik, Paul Diehl, Leonid Iakoubov, Costa Frangou. CancerNet biomarker profiling panel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A23.