A Novel Approach to Treat Trastuzumab Resistant HER2+ Breast Cancer: Activation PKCδ

In the United States, approximately one in eight women will be diagnosed with invasive breast cancer in her lifetime and one in forty will die from breast cancer. From among all the different subtypes of breast cancer, HER2+ can be found in approximately 20% of breast cancer tumors. HER2 overexpression has been associated with an aggressive form of breast cancer, increasing the risk of metastasis or recurrent tumors in 50% of patients. The standard of care for HER2 positive breast cancer, trastuzumab, has been effective for some patients; however, others develop resistance over time, leading to disease progression and metastasis. Drug resistance and tumor recurrence remain significant clinical challenges, limiting treatment efficacy. To address these issues, it is crucial to identify new mechanisms of action and develop novel therapeutic approaches to prevent or treat resistant disease. Previous work in the Osipo lab has shown a correlation between levels of PKCδ activity and resistance to trastuzumab therapy. It was hypothesized that by a TPA-mediated activation of PKCδ, trastuzumab resistant HER2+ breast cancer could be re-sensitized to trastuzumab. Surprisingly, TPA-mediated activation of PKCδ was sufficient to inhibit both proliferation and cancer stem cells survival of trastuzumab resistant cells lines. The addition of trastuzumab had little effect. Due to the nature of TPA to potentially cause cancer in other tissues, safer alternatives are needed to activate PKCδ. The hypothesis drawn to describe these findings is that: Activation of PKCδ is a novel therapeutic approach to treat trastuzumab resistant breast cancer using new compounds that mimic TPA. Two aims will be employed in order to test this hypothesis. Specific Aim 1: Test other PKC agonists as alternatives to TPA for inhibition of cell proliferation and cancer stem cell survival. After screening seven compounds raging between cyclic lactams, DAG lipids and Byostratin-1; a non-phorbol ester known as TPPB was the most potent inhibitor of cell proliferation and cancer stem cell survival using HCC1954 and BT474 resistant cell lines. Results showed that TPPB inhibited proliferation and cancer stem cell survival similarly to TPA. PKCδ was knocked down using an RNAi approach to show that the TPPB was on target for PKCδ. Furthermore, a pilot animal study using HCC1954 cells yielded information about potential toxicity. The rate of tumor growth with 1mg/Kg dose of TPPB was not significantly changed compared to the vehicle control group, suggesting that the dose may have been too low to elicit a measurable effect. Currently, a maximal tolerated dose (MTD) study is being performed using 5mg/Kg and 10 mg/Kg TPPB in HCC1954-harboring mice. The MTD study will provide enough information for larger scale in vivo studies in regard to toxicity and tumor growth. Specific Aim 2: Determine the mechanism by which activation of PKCδ inhibits growth of trastuzumab resistant HER2+ breast cancer. RNA sequencing was performed to determine the mechanism by which TPA-mediated activation of PKCδ inhibits proliferation and cancer stem cell survival of trastuzumab resistant breast cancer cell lines. The most significant pathway activated by TPA was the TNFα- NFKβ pathway. However, through several experimental approaches, it was determined that neither the canonical nor non-canonical NF-κB pathways were not required for cell proliferation and CSC survival inhibition. Alternative mechanisms, like JNK signaling and cellular senescence were also explored, but none of them were able to rescue the proliferative phenotype. After reinterrogating the RNA sequencing data, other signaling pathways such as p53 or cell differentiation emerged as possible mechanisms to explore. These results suggest that even though TPA has been shown to be an effective agent to activate PKCδ, TPPB could possibly be a safer alternative. Future studies will determine mechanisms by which activating PKCδ selectively inhibits growth of trastuzumab resistant breast cancer in vivo and if PKCδ is a novel vulnerability for trastuzumab resistant disease.