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    Research Grants Awarded

    Role Of Autophagy In Breast Cancer Cell Death In Response To Chemotherapy

    Grant Mechanism:
    Career Catalyst Research

    Scientific Abstract:
    Advanced triple negative (estrogen receptor (ER) negative, progesterone receptor (PgR) negative and HER-2/neu negative) breast cancer presents a significant clinical challenge, for which only limited treatment options exist. The high frequency of TP53 and PTEN mutations in this breast cancer subtype indicates pharmacological approaches that target defects in cell cycle checkpoints and phosphoinositol-3-kinase (PI3K) signaling may be effective. UCN-01 (7-hydroxystaurosporine), a broad-spectrum, serine-threonine protein kinase inhibitor targets the checkpoint kinase Chk1 and the PI3K signaling mediator PDK1 and therefore, may be useful in treating triple negative breast cancers. Indeed, while carrying out a phase I study of UCN-01 in combination with irinotecan in solid tumors, we observed responses in 4 out of 4 metastatic triple negative breast cancer patients. Partial responses were observed in 2 of the four patients and stable disease was observed in the other two. As a result, the Cancer Therapy Evaluation Program approved an extension of the study to determine the efficacy, tolerability and pharmacodynamic parameters of irinotecan/UCN-01 in the triple negative breast cancer population. We will test the hypothesis that the success of combining irinotecan (DNA damaging agent) with UCN-01 (protein kinase inhibitor) in triple negative breast cancers is due to the ability of UCN-01 to induce checkpoint bypass specifically in p53 deficient tumors (through Chk1 inhibition) and/or to inhibit PI3K signaling (through PDK1 inhibition). These studies will be carried out using a novel mouse model in which pre-therapy patient tumor specimens are engrafted into the ?humanized? mammary pad of NOD/SCID mice and the initial xenograft tumor is then transplanted into additional mice for experimental treatment and pathway studies that parallel the human studies. This approach, named HAMLET for Human and Mouse Linked Evaluation of Tumors, will be used to model the irinotecan/UCN-01 clinical trial, which is currently enrolling patients with triple negative breast cancer. To date, we have had an ~75% success rate in establishing the initial tumor xenograft in mice and a 100% success rate in subsequent tumor passages. Importantly, expression profiling demonstrated that the characteristics of the human tumor are preserved in the mouse xenograft suggesting that it is a valid model with which to study triple negative disease. Finally, Chk1 inhibitors that have a narrower spectrum of activity will be assessed using the HAMLET model. Similarly, inhibitors of the PI3K pathway may be investigated in these models if our initial evaluation of UCN-01 activity suggests that PI3K pathway inhibition is a critical component of this agent's therapeutic effect. The triple negative breast cancer HAMLET model will allow a preclinical evaluation of newer generations of Chk1- and PI3K-inhibitors, increasing the chances of future clinical trial success in humans. AIM 1. Subdivide Triple Negative Breast Tumors with respect to p53 status and integrity of the PI3K pathway. AIM 2. Develop robust pharmacodynamic biomarkers to address the mechanism of anti-tumor effect of UCN-01 and irinotecan either alone or in combination in HAMLET models. AIM 3. Combine irinotecan with more selective Chk1 inhibitor (Go6976) in HAMLET model. AIM 4. Perform preclinical trial in HAMLETs using optimal dosing and scheduling parameters determined in Aim 3 to assess anti-tumor effect of irinotecan and Go6976.

    Lay Abstract:
    A major strategy for treating cancer is to target the fundamental difference between cancerous and normal cells. Tumor cells display an abnormal propensity for growth and proliferation, thus are in net need for nutrient to support their growth and propagation. Moreover, solid tumor cells such as breast cancer cells develop mechanisms to adapt to nutrient-poor environments. The abnormal bioenergetic status may render the cancerous cells more susceptible to the perturbation of cell metabolism. Our previous studies indicated that DNA alkylating agents, the most widely used chemotherapy in clinic, can induce tumor cell specific death by inhibiting glycolysis. Targeting cell metabolism as an anti-tumor strategy is currently under pre-clinical and clinical investigations. Autophagy is an evolutionarily conserved process where in response to nutrient poor conditions, cells start to digest own contents. By doing so, autophagy serves to 1) recycle the damaged intracellular molecules or organelles; and 2) provide cells with nutrients under stressed conditions. The role of autophagy in tumorigenesis and anti-cancer treatment is gaining attention. Deficiency of an autophagy essential gene Atg 6 (Beclin 1) is observed in 40-75% of sporadic ovarian and breast cancers. It has been reported that autophagy is activated in cancer cells in response to several anticancer therapies. However, the biological function of autophagy in tumor response to anti-cancer treatment remains unclear and controversial. Some reports suggest that in the absence of apoptosis, autophagy may work to kill cancer cells. In contrast, evidence also exists supporting the theory that autophagy may help cancer cells to survive. Thus autophagy may on one hand promote cancer cell death, and on the other hand, may promote cancer cell survival and resistance to therapy. Hence, while it is clear that targeting autophagy may be developed as a novel approach for clinical oncology, it remains unclear whether autophagy should be enhanced or suppressed, in the context of different genomic or biochemical backgrounds of breast cancer types. I this proposal, we hypothesize that autophagy plays an important role in tumor cell response to anti-cancer treatment, and that the ability of cells to die by another form of cell death, apoptosis, has critical impact on the outcome of manipulating autophagy as a potential clinical approach for adjuvant treatment. We will use genetically defined apoptosis-competent and apoptosis-deficient baby mouse kidney (BMK) cells with epithelial origin, as well as human breast cancer lines to block apoptosis, to first determine whether autophagy is induced, and its role in promoting cell death or survival in cultured cells, in response to DNA damaging agents and inhibition of cancer cell metabolism. We will then take this study to a more clinically relevant setting using mice bearing grafted tumors. These studies will be performed by using pharmacological compounds or genetic tools to inhibit autophagy. The possible findings will have significant impact on targeting autophagy as an effective mean for adjuvant treatment of breast cancer patients. Understanding the role of autophagy in promoting cancer cell death or survival in the context of cells? ability to die by other forms of cell death will help to determine whether autophagy should be inhibited or enhanced, in order to achieve more specific and effective outcomes in anti-cancer therapy.