Investigation of BCL-2 interacting killer (BIK) as a breast cancer biomarker and its role in failed apoptosis

  • Author / Creator
    Pandya, Vrajesh K
  • Breast cancer is the number one cause of cancer-associated deaths in women worldwide. It can be broadly classified into estrogen receptor (ER) positive or -negative subtypes which form the basis of treatment. Despite the therapeutic advancements, more than half a million women annually succumb to the illness suggesting critical gaps in our understanding of the disease. Chemo-, radiation- and hormonal therapies are used to reduce tumor burden work by inducing apoptosis. Hence investigation of players in the apoptotic program can uncover the mechanisms of cancer aggression. Apoptosis is regulated by the BCL-2 family of proteins, which sense and relay the death signals to the downstream machinery that executes cell death. The BH3-only proteins, a subgroup of the BCL-2 family, act as the sensors of a variety of cell-intrinsic stressors and determine therapeutic response against cancer. Thus, it is believed that if the BH3-only proteins are absent or downregulated it may create a permissive environment for the development of therapeutic resistance leading to cancer aggression. In order to decipher how BH3 proteins affect breast cancer pathophysiology, I interrogated the gene expression levels of five BH3-only members in breast cancer tumors with respect to patient survival outcomes. Interestingly, we found that the BH3-only member BCL-2 interacting killer (BIK) was associated with worse patient survival and patients with high levels of BIK transcript were twice as likely to die from the disease compared to the BIK-low patients. This finding was surprising, given the pro-apoptotic nature of BIK. Hence, we validated these findings in a different cohort of patients by quantitating BIK protein levels using immunohistochemistry. Strikingly, BIK protein levels were also associated with worse survival outcomes of the patients. Thus, high BIK expression in two independent patient cohorts (total n=327) was associated with poor patient survival. Further interrogation revealed that high BIK mRNA levels in association with high levels of ATG5, a regulator of the cytoprotective autophagic pathway, predicted worse prognostic outcomes. This provided a clue that BIK might have a tumor-promoting effect through enhanced cell survival in the face of tumor-associated stress. In order to test this, I ectopically expressed BIK in breast cancer cells and tested their autophagic response to nutrient starvation or mammalian target of rapamycin complex 1 (mTORC1) inhibition. While there was some autophagic response, BIK did not stimulate robust autophagy. Furthermore, MTT assays revealed that BIK expression did not confer a growth or survival benefit on the cells when they were nutrient starved or rapamycin-treated. Together these observations showed that BIK did not strongly regulate autophagy in the cell lines tested yet hinted that BIK may have tumor-promoting effects in patients. More than 70% of all breast cancers are ER-positive driven by the female sex hormone estrogen. This type of cancer is treated with anti-estrogen therapy that utilizes drugs such as tamoxifen to induce cancer cell apoptosis and prevent relapse. Recent studies have identified an aberration of apoptosis called “failed apoptosis” in which apoptosis is initiated but does not go to completion, causes DNA double-strand breaks and mutations leading to tumorigenesis. Importantly, BIK is upregulated in response to anti-estrogen treatment of the ER-positive breast cancer which prompted me to interrogate BIK’s involvement in this patient subgroup. Interestingly, we discovered that high BIK levels predicted poor prognosis of anti-estrogen treated ER-positive but not alternatively treated ER-negative patients. I hypothesized that BIK facilitated tumor aggression in the ER-positive patients through failed apoptosis. Indeed, BIK expression in breast cancer cell lines led to minimal cell death but caused caspase activation, and double-strand DNA damage dependent on caspase activated DNAse (CAD). Importantly, tamoxifen-mediated BIK expression also induced genomic damage in cells, suggesting a link between anti-estrogen stimulated DNA damage-mediated mutagenesis. Finally, I explored whether transient expression of BIK altered cell phenotypes after long-term culture. Interestingly, cells surviving this treatment produced progeny with aggressive phenotypes characterized by high clonogenic survival, elevated anchorage-independent growth, and enrichment of cancer stem cells. These results suggest that poor survivals of BIK-high ER-positive patients could be partly due to failed apoptosis which may provoke mutations and provide a means of evolution for cancer cells. Thus, in this thesis, I identified BIK as a novel biomarker of breast cancer and discovered a potential mechanism of BIK-mediated tumor evolution.

  • Subjects / Keywords
  • Graduation date
    Spring 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
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