Browsing by Subject "PROTAC"

The steroid hormone progesterone has complex physiologic effects. In typical development and function, cells respond to progesterone in a dose- and tissue-specific manner. Despite the wide range of physiologic concentrations, canonical effects of progesterone have been characterized in the context of a high physiologic dose (10nM+), relevant during uterine cycling. This narrow focus has produced a gap in knowledge, particularly as it relates to the effects of post-menopausal low concentration progestins (0.1-0.3nM). Given that healthy tissues possess regulatory mechanisms to sense and respond to progesterone in a non-linear dose-specific manner, we hypothesized that breast malignancies would also display discontinuous dose-specific dynamic responses. Our results show that treatment with low dose progestins (0.1-0.3nM) drives proliferation in T47D human breast cancer cells, while high dose progestins (10nM+) inhibit proliferation. Using both unbiased and targeted approaches, we found that low dose progestins facilitate cell cycle entry by enhanced expression of CCND1 (cyclin D1) and SGK1 (serum and glucocorticoid related kinase 1), which are required for initiation of the downstream molecular cascade including phosphorylation of retinoblastoma protein (Rb) and expression E2F1. Expression of CCND1 and SGK1 mRNA are proximal responses to low dose progestin treatment, but transcriptional activation is not mediated by canonical progesterone receptor (PR) activity. Future work is needed to identify previously unexplored mechanisms of PR action in the context of low dose progestin treatments. In summary, these results challenge the assumption of dose response linearity to progestins and show unique functional and molecular effects of low dose progestin treatment. Of potential concern, our findings suggest that certain breast cancers, especially those expressing high levels of PR, may be accelerated by normal post-menopausal circulating concentrations of progestins (0.1-0.3nM). However, these findings also offer a sound rationale for the clinical therapeutic use of high dose progestins for patients with PR+ breast cancer.

Prostate and breast cancers are major health concerns, being amongst the most common forms of cancers in both men and women. The majority of prostate and breast cancers are driven by the hormone receptors androgen receptor (AR) and estrogen receptor (ESR1), respectively, and as such, endocrine therapies targeting the actions of these receptors has been a cornerstone of treatment for these patients. While these endocrine therapies are generally initially efficacious, resistance inevitably emerges. Resistance can emerge through various mechanisms, such as amplification of the receptor, generation of activating point mutations, alternative splicing of the receptor resulting in constitutively active forms of the receptor, and activating cross-talk from growth factor signaling pathways. A salient feature of these diseases is that the nuclear receptor (AR or ER) often remains engaged upon the emergence of resistance, and thus targeting of the receptor still provides therapeutic benefit. Therefore, much work in these fields has been performed to design better forms of endocrine therapy to help patients upon tumor progression. As cells are altering their signaling to deal with these pressures, this thesis work investigated the global genomic changes which arise in prostate and breast cancer cells after endocrine therapy to understand the effects of utilizing different forms of endocrine therapy, and whether these alterations in the cells induce novel vulnerabilities which can be therapeutically exploited. In the first set of studies, the differences between utilizing a competitive antagonist (enzalutamide-Enz) vs an AR degrader (AR-targeting proteolysis targeting chimera-PROTAC) were evaluated in prostate cancer. PROTACs are a new form of therapy for prostate cancer which have encouraging results in early clinical trials, so we wanted to better understand the genomic architecture and gene expression landscape after this new treatment modality compared to the current standard of care with an aim to use this knowledge to understand endocrine therapy resistance and identify therapeutically targetable pathways emerging from treatment. A factor agnostic approach was taken utilizing ATACseq and RNAseq to compare the genomic landscape after Enz or PROTAC treatment. It was found that the different AR inhibitors create distinct genomic landscapes which appear to be driven by unique sets of transcription factors. Further, it was discovered that AR inhibition, especially through degradation creates a novel liability which can be therapeutically exploited. AR was found to mediate these effects through regulating expression of a key transcription factor, and we propose a model in which the two proteins interact to regulate this axis. As AR is expressed in many other malignancies, it is feasible this strategy of degrading AR to induce this therapeutic vulnerability could have efficacy beyond prostate cancer. In the second set of studies, we investigated the genomic changes which are manifest after the emergence of endocrine therapy resistance in breast cancer and identified a novel signaling pathway that, when targeted, impairs tumor progression. Utilizing, DNAse hypersensitivity analysis, ChIP-seq, and RNAseq, it was found that GRHL2 cooperates with FOXA1 to drive a novel cistrome in endocrine therapy resistant breast cancer cells. The protein LYPD3 was found to be a downstream effector of GRHL2 and targeting LYPD3, or its ligand AGR2, with monoclonal antibodies significantly impaired primary tumor growth. Further studies into the functional role of LYPD3 were then undertaken, and it was discovered that LYPD3 knockdown significantly alters metastatic outgrowth of breast cancer cells in the lung. Investigation into the signaling of LYPD3 revealed a novel function of this protein. This work and future mechanistic studies will elucidate the signaling of LYPD3, and as LYPD3 is expressed in numerous subtypes of advanced cancers, understanding its signaling could provide a new biomarker for cancers which would be amenable to the targeted therapies identified in these studies in combination with LYPD3 targeted therapies.