The biological and therapeutic role of DRIM (Down Regulated in Metastasis) in cancer and metastatic cancers

Dong, Xuefei 2024. The biological and therapeutic role of DRIM (Down Regulated in Metastasis) in cancer and metastatic cancers. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Introduction. DRIM (Down-Regulated In Metastasis Protein), subsequently known as UTP20, was initially discovered as a protein from a coding gene in metastatic breast cancer cell lines. It was subsequently thought to be a molecule related to the metastatic potential of cancer cells and as a putative nuclear protein regulating certain gene expression in cells, including cancer cells. However, the overall biological role, its biochemical interaction partners, cellular location and its clinical impact in cancer are largely not characterised. The present study investigated the expression and clinical value of DRIM in human breast and colon cancer and explored the biological function of the protein, the cellular location and its biochemical implications in cancer cells. Materials and methods. DRIM expression at transcript and protein levels was assessed in human breast cancer and colon cancer, in order to establish its expression pattern in relation to clinical and pathological factors, as well as patient survival. The value of DRIM in patient response to drug therapies was also evaluated. The study created cancer cell models with differential expression of DRIM, by way of genetic knockdown. Using the cell models, the responsive proteins after DRIM knockdown, as well as proteins interacting with the DRIM protein, were evaluated by way of protein microarray-based proteomics platforms. A range of cellular functions including cell-matrix adhesion, cellular migration and response to drugs were tested using in vitro cell models. The impact of DRIM expression on tumour growth was also evaluated using in vivo tumour modelling. Finally, the cellular location of the DRIM protein was explored using fractionated proteins from human cells and immunofluorescence. Results. Tumour tissues from patients with breast cancer expressed significantly higher levels of DRIM transcripts and protein than normal mammary tissues. Patients with high levels of DRIM transcript had significantly shorter disease-free survival (DFS) and overall survival (OS), although the latter was weaker. The survival relationship had a connection with the ER hormone receptor and HER2 status in breast cancer tissues. In a public dataset of breast cancer (TCGA), high levels of DRIM were seen to confer chemoresistance. The resistance was particularly significant in ER positive, HER2 negative, ER(+)/HER2(-) and luminal-B breast cancer molecular subtypes. A series of breast cancer cell models were created with DRIM knockdown. Cells with knockdown exhibited changes in their rate of growth and degree of cell-matrix adhesion and also in the pace of cell migration. Knockdown also rendered cells with changed sensitivities to a range of chemotherapeutic drugs used in breast cancer treatment, indicating an acquisition of drug sensitivity following loss of DRIM, a finding supported by clinical, in vitro and in vivo tumour models. Using proteins generated from the cell models on proteomics platforms, the study identified a number of proteins and related pathways in response to DRIM modification. Using proteins extracted from different cell compartments, the study further revealed that DRIM protein in cancer cells is primarily located in the x cytoplasmic fraction, in the cytoskeletal fraction and more weakly, in the chromatin bind fractions of the cells, although not in the membrane nor in the cytosolic fractions. These findings from proteomics were further validated by immunofluorescence visualisation. The study further identified a small number of proteins that were colocalised (by way of immunofluorescence) and co-immunoprecipitated with the DRIM protein. Amongst the co-localised proteins was paxillin, a focal adhesion regulator, further implicating DRIM as a cytoskeletal associated protein that regulates cell behaviour including migration, growth and drug responses, possibly via paxillin. It was also shown that nucleus-located DRIM also interacted with paxillin located to the nucleus, a plausible explanation of the contribution found for the reduction of SRC, a cell migration regulator. The identification of the DRIM interactive proteins from proteomics allowed further identification of a molecular signature from the breast cancer cohort, that is made of a small group of DRIM interactive molecules, a signature that has a significant prognostic value, superior to that of DRIM alone, in predicting clinical outcome in the patients. In a brief parallel study, we observed an opposite role of DRIM in human colon cancer, in that high levels of DRIM in colon cancer tissues were associated with a favourable clinical outcome. In contrast to breast cancer, high levels of DRIM in cancer cells and in clinical colon cancer indicated sensitive status to chemotherapy drugs and chemotherapies, with mechanisms to be further explored. Conclusion. The present study has revealed that DRIM is an important clinical biomarker for disease progression and clinical outcome in breast cancer and colon cancer patients. DRIM protein is located in the nucleus and cytoplasm in association with the cytoskeleton. The later contributes to the biological regulation of cancer cell functions, particularly in cell-matrix adhesion and cellular migration seen with DRIM, an aspect at least partly attributable to the connection with paxillin. DRIM is a biomarker for breast cancer and is a regulator of drug responses in patients, a hormone receptor dependent phenomenon. The nucleus and cytoskeletal location of DRIM, via its interactive proteins, including paxillin, form the potential mechanism of action by DRIM.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Medicine
Date of First Compliant Deposit:	17 October 2024
Last Modified:	17 Oct 2024 15:17
URI:	https://orca.cardiff.ac.uk/id/eprint/173068

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