Versican and Versican-matrikines in Cancer Progression, Inflammation, and Immunity

Versican is an extracellular matrix proteoglycan with key roles in multiple facets of cancer development, ranging from proliferative signaling, evasion of growth-suppressor pathways, regulation of cell death, promotion of neoangiogenesis, and tissue invasion and metastasis. Multiple lines of evidence implicate versican and its bioactive proteolytic fragments (matrikines) in the regulation of cancer inflammation and antitumor immune responses. The understanding of the dynamics of versican deposition/accumulation and its proteolytic turnover holds potential for the development of novel immune biomarkers as well as approaches to reset the immune thermostat of tumors, thus promoting efficacy of modern immunotherapies. This article summarizes work from several laboratories, including ours, on the role of this central matrix proteoglycan in tumor progression as well as tumor-immune cell cross-talk:

CGRRF1, a growth suppressor, regulates EGFR ubiquitination in breast cancer

Background CGRRF1 is a growth suppressor and consists of a transmembrane domain and a RING-finger domain. It functions as a RING domain E3 ubiquitin ligase involved in endoplasmic reticulum-associated degradation. The expression of CGRRF1 is decreased in cancer tissues; however, the role of CGRRF1 in breast cancer and the mechanism(s) of its growth suppressor function remain to be elucidated. Methods To investigate whether CGRRF1 inhibits the growth of breast cancer, we performed MTT assays and a xenograft experiment. Tumors harvested from mice were further analyzed by reverse phase protein array (RPPA) analysis to identify potential substrate(s) of CGRRF1. Co-immunoprecipitation assay was used to verify the interaction between CGRRF1 and its substrate, followed by in vivo ubiquitination assays. Western blot, subcellular fractionation, and reverse transcription quantitative polymerase chain reaction (qRT-PCR) were performed to understand the mechanism of CGRRF1 action in breast cancer. Publicly available breast cancer datasets were analyzed to examine the association between CGRRF1 and breast cancer. Results We show that CGRRF1 inhibits the growth of breast cancer in vitro and in vivo, and the RING-finger domain is important for its growth-inhibitory activity. To elucidate the mechanism of CGRRF1, we identified EGFR as a new substrate of CGRRF1. CGRRF1 ubiquitinates EGFR through K48-linked ubiquitination, which leads to proteasome degradation. In addition to regulating the stability of EGFR, knockout of CGRRF1 enhances AKT phosphorylation after EGF stimulation. By analyzing the breast cancer database, we found that patients with low CGRRF1 expression have shorter survival. As compared to normal breast tissues, the mRNA levels of CGRRF1 are lower in breast carcinomas, especially in HER2-positive and basal-like breast cancers. We further noticed that CGRRF1 promoter methylation is increased in breast cancer as compared to that in normal breast tissue, suggesting that CGRRF1 is epigenetically modified in breast cancer. Treatment of 5-azactidine and panobinostat restored CGRRF1 expression, supporting that the promoter of CGRRF1 is epigenetically modified in breast cancer. Since 5-azactidine and panobinostat can increase CGRRF1 expression, they might be potential therapies for breast cancer treatment. Conclusion We demonstrated a tumor-suppressive function of CGRRF1 in breast cancer and identified EGFR as its target.

CSIG-29. STRUCTURAL AND FUNCTIONAL STUDIES OF PID1, A NOVEL GROWTH SUPPRESSOR IN BRAIN TUMORS

BACKGROUND Brain cancers, including medulloblastomas and gliomas, are a major cause of death in children and adults. We reported that PID1 (Phosphotyrosine Interaction Domain containing 1) is a growth suppressor in medulloblastomas and gliomas (PMID: 24300787). PID1 also enhances the anti-tumor effects of chemotherapy (PMID: 28400607). We are now seeking to better understand the structure and mechanism of PID1, in order to utilize this knowledge to develop innovative PID1-based therapies. METHODS PID1 mutants were expressed in E. coli as MBP fusion proteins, purified and used for MALS analysis. Additional epitope tagged PID1 constructs (HA, V5, tGFP, eGFP) were generated, expressed in mammalian cells and analyzed by western blots, immunoprecipitation, and functional assays. RESULTS We carried out screening and testing of multiple mutants of purified PID1 and characterized them. Experiments in cell culture supported presence of similar findings in mammalian cells. Colony assays in glioma and medulloblastoma cell lines identified a region in PID1 that confers the most robust growth-inhibitory effect. Experiments are underway to further refine the boundaries and characteristics of this growth-inhibitory region. CONCLUSIONS This project, which is focused on better understanding of the structure and function of PID1, is uncovering novel aspects of its molecular function. Insights gained from this work will guide studies to develop innovative PID1-based therapies for gliomas and medulloblastomas.

Oncogenesis and tumour suppression

Two sets of genes are among the major driver of tumours, both malignant and benign: the oncogenes and the tumour suppressor genes. Oncogene refers to a gene that encodes for a protein (oncoprotein) in which excessive and unregulated activity can transform a normal cell into a cancer cell. As it is necessary for just one of the two gene copies to be abnormal, oncogenesis is defined as dominant. Tumour suppressor genes are known for their roles in inhibiting cell growth and have antitumour effects. According to the classic model, growth suppressor genes are recessive and therefore both copies have to be inactivated in order for an effect to be seen. Exception however occurs! Recently also non-coding mRNAs (i.e. an mRNA that is not translated into a protein) have been found to be able to induce oncogenic and suppressive effects. Finally, both some genes and some non-coding mRNA are able, in certain cellular contexts, to behave both as both an oncogenic and a suppressive factor.