title: Investigation of the role of Col4a1 and Itga11 in enzalutamide resistance of prostate cancer cells
Creators: name: Ciszek, Aleksandra
Publisher: name: The University of Edinburgh
Year: 2022
Subjects: subject: Prostate carcinoma
subject: Androgen Deprivation Therapy (ADT)
subject: Castration-Resistant Prostate Cancer (CRPC)
subject: Metastatic CRPC (mCRPC)
subject: Tumour-associated macrophages (TAMs)
subject: cell-derived matrix (CDM)

description: Prostate carcinoma is the most commonly diagnosed non-cutaneous cancer in males in the Western World. Androgen Deprivation Therapy (ADT) is initially effective in patients with localized prostate cancer (PCa), but eventually, cancer cells develop resistance to therapy and progress into Castration-Resistant Prostate Cancer (CRPC). CRPC is very likely to metastasise to bone, which worsens morbidity and mortality. Metastatic CRPC (mCRPC) is currently incurable and accounts for over 90% of PCa-related deaths. Tumour-associated macrophages (TAMs) can promote ADT resistance in bone metastasis, but the mechanism is yet unknown. Recent studies in our lab have identified that macrophages regulate the expression of a set of extracellular matrix genes that are dysregulated in prostate cancer cells resistant to ADT. Among them, our preliminary data suggest that Col4a1 may interact with Itga11 to promote therapy resistance growth. This study focused on the role of Col4a1 and Itga11 in the development of resistance to antiandrogen Enzalutamide in murine prostate cancer bone metastasis cell line Myc-CaP Bo 6943, which is susceptible to treatment. It also aimed to determine the potential effect of tumour cell-derived matrix (CDM) on macrophage polarization. Myc-Cap Bo 6943 cell lines with knockdown of Col4a1 or Itga11 were developed using shRNAs. Four clones with Col4a1 knockdown and four clones with Itga11 knockdown showed a decrease in mRNA levels in quantitative reverse-transcription PCR (RT-qPCR) (P&lt;0.05). Knockouts of Col4a1 or Itga11 were developed using CRISPR-Cas9 technology. Two Col4a1 and two Itga11 knockout clones were confirmed with Sanger sequencing. Immunocytochemistry (ICC) confirmed the knockouts at a protein level in both Col4a1 knockout clones, and in one Itga11 clone. Growth of the cells with the established knockout or knockdown was assessed by MTT assay following the removal of androgens or treatment with Enzalutamide. All clones with the knockout or knockdown of the genes of interest showed a decrease in growth following the treatments. No statistically significant difference was found between the growth of knockout/knockdown cells and wild type cells under treatment. To determine the influence of tumour CDM on polarisation of macrophages, murine bone marrow-derived macrophages (BMMs) were incubated with tumour CDM from Myc-CaP Bo 6943 cells. Flow cytometry analysis showed a slight increase in the expression of M2 macrophages markers, CD204 and IL4R on the surface. Further studies should be undertaken to determine the role of Col4a1 and Itga11 in the development of therapy resistance. Potentially, their knockout or knockdown could be established in a castration-resistant cell line to see if lack of these genes has a potency of rendering resistant cells susceptible to treatment. The successfully generated cell lines can be used for in vivo experiments for further validation. Additional experiments can be performed to determine the downstream signalling of COL IV-ITGA11 interaction in tumour cells.