RESEARCH
Many harmful chemicals in the environment challenge human DNA. Cells have evolved exquisite surveillance mechanisms known as DNA damage response (DDR), which maintain genome integrity following DNA damage. The aberrant regulation of DDR leads to genome instability and various diseases including cancer.
My laboratory has been trying to dissect out the process by which DNA damage and deregulation of cell cycle checkpoint or anti-apoptotic genes can lead to cellular transformation of breast epithelial cells.
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We have shown that alkylation damage leads to transformation of breast epithelial cells grown as three-dimensional acinar cultures. An increase in protein expression was observed and we are currently two such proteins, Api5 and TopBP1.
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Api5 is an anti-apoptotic protein and its overexpression in the breast acinar cultures led to tumorigenesis with an increase in proliferation and disruption of cell polarity. Currently, we are investigating the molecular mechanism by which Api5 deregulation leads to tumorigenesis. Api5 overexpression leads to the up regulation of FGF2 during the early days of morphogenesis. There is activation of Akt signalling during the early stages and ERK signalling during the late stages of acinar morphogenesis. Hence, we hypothesise that overexpression of Api5 may result in the early activation of FGF2/FGFR1/PDK1/Akt axis, promoting polarity disruption and higher proliferation. This signalling later switches to the Ras/MAPK/ERK pathway during the later stages of acinar morphogenesis, thereby, reducing apoptosis and supporting sustained proliferation.
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We have demonstrated that overexpression of Api5 leads to partial epithelial-mesenchymal transition (EMT) in non-tumorigenic breast epithelial cells grown as three-dimensional acinar cultures. Currently, we are investigating the mechanism of Api5 mediated partial EMT as well as Api5's potential to confer cancer stem cell like properties to non-tumorigenic breast epithelial cells grown as mammospheres.
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Api5 is reported to regulate apoptosis by various strategies. Studies have also shown that it can regulate key molecules involved in breast morphogenesis. This involves growth factor receptor signalling and the pro-apoptotic proteins. Identifying whether Api5 plays a role in breast morphogenesis can explain the regulation of several signalling cascades. By altering the expression levels and pattern of Api5, we expect to identify the molecular signalling associated with the protein during the morphogenesis. To elucidate the signalling events and the effect on morphometry, breast epithelial cell line with altered expression of Api5 will be grown on 3D cultures. Using in vivo model system, the expression pattern and regulation of Api5 will be analysed by selectively altering expression in breast epithelial cells. This study would also provide a better understanding on breast morphogenesis in general. Using multiple model systems, this study can uncover new molecular players involved in gland development. These learnings can then be extended to breast cancer to identify biomarkers and possible drug targets for better treatment regimes.
​Api5 interacts with DNA through its C-terminal end and is predicted to possess a transactivation domain. Using in silico analysis we identified a potential DNA binding region despite lacking a canonical DNA binding domain. Api5 demonstrated increased chromatin association in metastatic cancer cell lines. Interaction analysis suggested a role in ATR activation during UV and replication stress. Knockdown of Api5 increased susceptibility to UV-induced DNA damage, indicating involvement in single-strand DNA break repair and replication stress. We are currently investigating Api5's role in the DNA damage response pathways, with implications for cancer therapeutics.
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The effect of DNA damage on transformation of non-tumorigenic breast epithelial cells resulted in the activation of the sensor kinase, DNA-PK leading to aberrant Golgi morphology, thereby transforming the breast epithelial cells to a mesenchymal phenotype. In addition to Golgi dispersal, a dramatic reorganisation of the cytoskeleton resulting in parallel array of microtubules and loss of actin stress fibres were observed. Currently we are studying the effects of DNA damage on cytoskeleton dynamics.​