Role of RIN1 on telomerase activity driven by EGF-Ras mediated signaling in breast cancer

Significance 

Breast cancer is the most frequently diagnosed cancer in women and one of the leading causes of cancer death for women. The mortality due to breast cancer has been declining in the developed countries and the continuing decrease in mortality from breast cancer has been attributed to early detection due to screening and improved adjuvant therapy. Despite the decreased incidence and mortality, breast cancer remains a major cause of cancer mortality for women and accounts for 15% of all cancer deaths in women in western countries.

Identifying the key features of the Ras interference 1 (RIN1) protein is critical in understanding the dynamics of the epidermal growth factor (EGF)-induced cellular proliferation. This multidomain and multifunctional protein is a unique modifier of the EGF-induced signal transduction. RIN1 interacts with the activated EGF-receptor tail and the active form of Ras, consequently activating Rab5. Expression of RIN1 has been reported to block cell proliferation while that of Rab5 has been reported to accelerate cell proliferation. Overexpression of RIN1 is associated with progression in some forms of cancers. However, in breast cancer cells, its silencing could lead to breast cancer progression.

Telomerase activity, which several studies suggest to be down-regulated by cellular conditions that inhibit cell proliferation, is associated with cell proliferation. Some growth factors, such as EGF, control telomerase activity and suggest the involvement of signal molecules like RIN1 in telomerase activity regulation.

Florida International University Researchers Dr. W. Zhang, Dr. Maria-Luisa Veisaga, and Professor Manuel Barbieri investigated EGF effects of many signaling molecule activities in breast cancer cell lines expressing RIN1 constructs in an attempt to understand the molecular mechanism of the growth-factor-dependent RIN1 regulation. They also examined the signal transduction pathway responsible for telomerase activation. They were able to show that the expression of RIN1 is responsible for blocking EGF-induced telomerase. The original research article is published in the journal, Experimental Cell Research.

The investigators performed molecular biology experiments where they infected cell lines with the retrovirus encoding RIN1 and green fluorescent protein, cultured in the growth medium, and then serum-starved. After starvation, the cells were allowed to bind EGF. RNA was extracted from normal breast and breast tumor cell lines and real-time PCR was performed.

Expression of RIN1 completely inhibited MCF-7 and MCF-12A breast cancer cell proliferation and partially inhibited MDA-MB-231 cell proliferation after EGF stimulation. They reported that the expression of the RIN1 C-terminal was fundamental in blocking the proliferation of the MDA-MB-231 cells, although the inhibition was as a result of independent expression of RIN1:RA and RIN1:Vsp9 domains in particular.

The authors also found that the endogenous level of RIN1 dropped in the metastatic MDA-MB-231 cells as opposed to the non-tumorigenic MCF-12A cells. The expression of RIN1:R629A mutants was observed to inhibit the proliferation of MDA-MB-231 cells, while that of RIN1:R629A and RIN1:Y561F mutants reversed the inhibitory effect of RIN1:WT.

Further studies in Professor Manuel Barbieri’s laboratory revealed that the expression of RIN1:WT in MDA-MB-231 cells eliminated activities of both kinase 1/2 (ERK1/2), which is extracellular-signal-regulated, and protein kinase B (AKT). It further downregulated cellular-myelocytomatosis (c-Myc), erythroblast transformation specific, and signal transducer and activator of transcription 3 activities. However, p38 protein kinases and kinase (SAPK)/c-Jun N-terminal kinase (JNK) remained unchanged. The expression of high-mobility group box 1 (HMGB1) as blocked but that of forkhead box transcription factor 1 (FOXO1) increased in cells expression RIN1.

The expression of RIN1 inhibited telomerase activity as well as the expression of human telomerase reverse transcriptase (hTERT), and this correlated with downregulations of Stat3, c-Myc, and Ets-2 activation. In all, the findings of the study show that RIN1 plays a critical role in telomerase activity modulation and hTERT expression in MDA-MB-231 cells after EGF stimulation.

Triple negative breast cancer (TNBC) represents approximately 10–15% of all breast cancers and patients with TNBC have a poor outcome compared to the other subtypes of breast cancer. MDA MB-231 is commonly used cell line in pre-clinical research to better understand TNBC cells behaviors. According to the authors it is possible to inhibit proliferation of MDA MB-231 cells that express RIN1 and trigger telomerase inhibitory pathway via a downregulation of c-Myc and HMBG1 expression and upregulation of FOXO1 expression.

About the author

Professor Manuel A. Barbieri  is the Molecular and Cellular Research Group Leader and the technical director of the Health Auxiliary Laboratory at Florida International University. He is interested in addressing fundamental questions concerning cellular function and behavior, and encompasses the study of cell surface receptors, biology of the cancer cell, and plant secondary metabolites in drug discovery. Understanding how cells respond to stimuli requires an elucidation of pathways that control the crucial programs of cell proliferation, survival and differentiation.

Professor Barbieri is dissecting the role of Ras interference 1 (RIN1) protein in controlling these processes, which can be investigated and assessed in both in vitro and in vivo systems by applying biochemical, cellular, and molecular biological methods in normal and diseased cells.

Professor Barbieri also has active research in the isolation, characterization, and the effect of Natural Products (secondary metabolites), which are evolutionarily optimized as drug-like small molecules from plants that represent the richest sources of novel molecular compounds for drug discovery. Thus, the discovery of new natural products promises significant advances not only in biology but also in biochemistry and medicine.

Reference

W. Zhang, M.L. Veisaga, and M.A. Barbieri. Role of RIN1 on telomerase activity driven by EGF-Ras mediated signaling in breast cancer. Experimental Cell Research, issue 396 (2020) 112318

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