The matricellular protein CYR61 interferes with normal pancreatic islets architecture and promotes pancreatic neuroendocrine tumor progression

The significance of matricellular proteins during development and cancer progression is widely recognized. However, how these proteins actively contribute to physiological development and pathological cancer progression is only partially elucidated. In this study, we investigated the role of the matricellular protein Cysteine-rich 61 (CYR61) in pancreatic islet development and carcinogenesis. Transgenic expression of CYR61 in beta cells (Rip1CYR mice) caused irregular islets morphology and distorted sorting of a cells, but did not alter islets size, number or vascularization. To investigate the function of CYR61 during carcinogenesis, we crossed Rip1CYR mice with Rip1Tag2 mice, a well-established model of beta cell carcinogenesis. Beta tumors in Rip1Tag2CYR mice were larger, more invasive and more vascularized compared to tumors in Rip1Tag2 mice. The effect of CYR61 on angiogenesis was fully abrogated by treating mice with the anti-VEGFR2 mAb DC101. Results from in vitro assays demonstrated that CYR61 modulated integrin alpha(6)beta(1)-dependent invasion and adhesion without altering its expression. Taken together, these results show that CYR61 expression in pancreatic beta cells interferes with normal islet architecture, promotes islet tumor growth, invasion and VEGF/VERGFR-2-dependent tumor angiogenesis. Taken together, these observations demonstrate that CYR61 acts as a tumor-promoting gene in pancreatic neuroendocrine tumors.

Roles of Epigenetic Dysregulation in Cancer Progression and Metastasis

Mohammad Sadegh Saghafinia

Nearly all the cells of an organism share the same DNA sequence or genome, and yet they show different phenotypes and carry out different functions. This diversity is made possible by a verity of molecular modifications acting on the DNA sequence that collectively define the cell's epigenome. Failure in the proper regulation of epigenome can lead to abnormal activation or inhibition of vital signaling pathways, which contributes to the initiation and progression of multiple diseases, including cancer. In this thesis, I used a combination of in silico, in vitro, and in vivo techniques to explore the roles of epigenetic dysregulation in tumor progression and metastasis. In the first part, I performed a systematic and unbiased investigation of cancer-associated DNA methylation and gene expression changes across human cancers. I designed a novel algorithmic approach (RESET) to identify aberrant DNA methylation and associated gene expression changes across >6,000 human tumors. We identified a DNA Methylation Instability (DMI) phenotype in tumors acquiring numerous hyper and hypomethylated transcription start sites, which is associated with mutations of chromatin remodeling factors and Wnt-signaling. We showed that silenced genes coalesced in specific pathways including apoptosis, transcriptional regulation, and cell metabolism. The majority of the enhanced genes belonged to cancer-germline antigens (CG), whose expression correlated with response to anti-PD-1 in melanoma patients. Finally, we demonstrated the potential of RESET to explore aberrant DNA methylation in pediatric tumors; pediatric Wilms tumors with diffuse anaplasia exhibit DMI, and specific silencing events predict a worse outcome in cases with favorable histology. These results established a new approach to explore pan-cancer epigenetic modifications and provided a resource of candidate oncogenic events for future functional and therapeutic studies. In the second part, I studied the dynamics of cancer progression and metastasis in Pancreatic Neuroendocrine Tumors (PanNET). This rare tumor is the second most common form of pancreatic cancer. Two principal subtypes of PanNET has been identified: insulinomas (IT) and metastasis-like primaries (MLP), corresponding to the low and high grade of human PanNETs, respectively. From the mouse model of PanNET (RT2), we profiled the single-cell, bulk mRNA and miRNA transcriptomes, and the proteomes of primary and metastasis specimens. We demonstrated that the tumor progression from IT to MLP follows the reverse embryonic and postnatal developmental path. Transcriptomic data from human patients showed that aggressive human PanNETs also follow the same reverse developmental trajectory of dedifferentiation. We established one possible mechanism underlying IT-to-MLP transition. Over-expression of the miR-181cd cluster in IT-like cancer cell-lines resulted in acquisition of the MLP phenotypes. miR-181cd mediated its effect through suppressing the expression of Meis2 and, indirectly, inducing the expression of Hmgb3 and Mycn transcription factors. Inhibiting the expression of Hmgb3 in MLP-like cancer cell-lines resulted in a significant growth decrease both in vitro and in vivo, demonstrating the importance of Hmgb3 in the maintenance of MLP-like cell state. These data presented dedifferentiation as a mechanism by which malignant neuroendocrine cancer cells acquire progenitor-like features, enabling them to become more aggressive and metastatic.

EPFL2020

Anti-angiogenic immunotherapy for lung cancer

Ece Kadioglu

Lung cancer is the leading cause of cancer-associated deaths worldwide. Platinum-based chemotherapy is the most common therapeutic approach in advanced non-small cell lung cancer (NSCLC), particularly for tumors that carry non-druggable activating mutations. However, these tumors are generally not eradicated when diagnosed at an advanced stage and are frequently resistant to chemotherapy. Therefore, there is an urgent medical need for new treatments for this frequent cancer type. Immunotherapies are treatments based on increasing the strength of immune responses against cancer. In particular, monoclonal antibodies that block inhibitory "immune-checkpoints" expressed on T cells (e.g., programmed cell death protein 1, or PD1) show clinical efficacy in increasing tumor types. However, these therapies improve survival in only a minority of NSCLC patients, and ongoing efforts are being made for increasing their efficacy through combinatorial treatments. In this regard, growing evidence indicates that tumor angiogenesis is closely associated with immunosuppression. Pro-angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (ANG2), cause abnormalities and dysfunctions in tumor blood vessels that impair T cell extravasation and anti-tumoral activity. Recent findings in preclinical cancer models show that reprogramming the features of tumor-associated blood vessels by anti-angiogenic drugs may help to increase intratumoral T cell abundance and sensitize refractory tumors to immunotherapy. Nevertheless, the potential of anti-angiogenics in the context of lung cancer immunotherapy is poorly understood. Therefore, the main objective of my project was to audit combinations of anti-angiogenic therapy and immune checkpoint blockade in a genetically engineered mouse model of NSCLC. I first studied the anti-tumoral activity of dual VEGFA and ANG2 inhibition using a bispecific antibody (A2V) in the KrasLSL-G12D/+;p53fl/fl (KP) NSCLC model. I found that A2V, but not single VEGFA or ANG2 blockade, had robust tumor-inhibitory activity in KP mice, which was at least comparable to standard-of-care chemotherapy. However, A2V only moderately increased cytotoxic T cells in the tumors. To potentially increase the immunogenicity of KP tumors, I also generated two KP mouse variants by either co-expressing a surrogate neoantigen or genetically deleting a DNA mismatch repair enzyme in the cancer cells. However, the therapeutic efficacy of A2V was not further improved in spite of a slightly more activated immune response. I next investigated combination therapies involving A2V and PD1 blockade. In contrast to previous findings in other cancer types, the addition of PD1 antibodies deteriorated the efficacy of A2V, likely through enhancing immunosuppressive mechanisms sustained, at least in part, by regulatory T cells and T cell-antagonizing cytokines. In summary, my work supports the potential clinical efficacy of combined VEGFA and ANG2 blockade for lung cancer therapy but provided little evidence for its synergy in association with anti-PD1 therapy.

EPFL2019

Role of RIP4 in lung adenocarcinoma differentiation

Jawahar Kopparam

Various studies have attributed NF-kB activation to promote KRAS mediated non-small cell lung cancer progression. In order to identify key genes regulating NF-kB activation and cell survival in lung adenocarcinoma, a synthetic lethal partner screen for KRAS was conducted in cell lines expressing mutant or wildtype KRAS. This screen identified an important role of Receptor-interacting serine/threonine protein kinase 4 (RIP4) in KRAS mediated oncogenesis. But altering RIP4 expression did not affect the viability of KRAS mutant cells, however a novel role of RIP4 in maintaining tumor differentiation was observed. Loss of epithelial differentiation and extracellular matrix remodeling are known to facilitate cancer progression and are associated with poor prognosis in patients with lung cancer. Bioinformatics analyses of human lung adenocarcinoma samples showed that poorly differentiated tumors express low levels of RIP4, whereas high levels are associated with better overall survival. Interleukin 6 (IL6) signaling is activated in lung adenocarcinoma expressing oncogenic KRAS and contributes to cancer invasiveness. Knockdown of RIP4 using shRNA in vitro enhanced activation of IL-6 signaling and also the ability of the cells to invade through collagen. In contrast, overexpression of RIP4 inhibited IL6-mediated Signal Transducer and activator of Transcription 3 (STAT3) activation, which abrogated IL6-dependent induction of lysyl oxidase, a collagen cross-linking enzyme. Tumor-specific Rip4 knockdown in an autochthonous mouse model of lung adenocarcinoma initiated by Kras(G12D) expression with loss of p53, progressed to a poorly differentiated state marked by an increase in Hmga2, reduced Ttf1 and enrichment of genes regulating extracellular matrix (ECM) remodeling and JAK-STAT signaling. Activated STAT3 can enhance NF-kB activation by interacting with NF-kB and promoting its nuclear retention. Tumors with RIP4 knockdown also exhibited enhanced STAT3- NF-kB interaction suggesting a cross talk between the two pathways. Tail vein injections of cells overexpressing RIP4 showed a reduced potential to invade and form tumors. Analysis of factors regulating RIP4 expression revealed that RIP4 is induced in a p53 dependent manner in response to chemotherapy. Altering the induced RIP4 levels in vitro had a modest effect on chemotherapy induced cell death. Our work has identified that loss of RIP4 enhances IL6 signaling in lung cancer cells, promoting the expression of ECM remodeling genes and cancer dedifferentiation.

EPFL2016