RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells

Abstract

Rationale: PI3K/mTOR signaling is frequently upregulated in breast cancer making inhibitors of this pathway highly promising anticancer drugs. However, PI3K-inhibitors have a low therapeutic index. Therefore, finding novel combinatory treatment options represents an important step towards clinical implementation of PI3K pathway inhibition in breast cancer therapy. Here, we propose proteases as potential synergistic partners with simultaneous PI3K inhibition in breast cancer cells. Methods: We performed mRNA expression studies and unbiased functional genetic synthetic lethality screens by a miR-E based knockdown system targeting all genome-encoded proteases, i.e. the degradome of breast cancer cells. Importantly theses RNA interference screens were done in combination with two PI3K pathway inhibitors. Protease hits were validated in human and murine breast cancer cell lines as well as in non-cancerous cells by viability and growth assays. Results: The degradome-wide genetic screens identified 181 proteases that influenced susceptibility of murine breast cancer cells to low dose PI3K inhibition. Employing independently generated inducible knockdown cell lines we validated 12 protease hits in breast cancer cells. In line with the known tumor promoting function of these proteases we demonstrated Usp7 and Metap2 to be important for murine and human breast cancer cell growth and discovered a role for Metap1 in this context. Most importantly, we demonstrated that Usp7, Metap1 or Metap2 knockdown combined with simultaneous PI3K inhibition resulted in synergistic impairment of murine and human breast cancer cell growth Conclusion: We successfully established proteases as combinatory targets with PI3K inhibition in human and murine breast cancer cells. Usp7, Metap1 and Metap2 are synthetic lethal partners of simultaneous protease/PI3K inhibition, which may refine future breast cancer therapy.

Degradome-focused RNA interference screens to identify proteases important for breast cancer cell growth

“…In addition, many other depletion hits were supported by literature, e.g., Metap1 and Metap2. Metap2 is known to act as tumor-promoting in different cancers ( 54 , 55 ) including breast cancer ( 19 , 56 ). Metap1 is known to act as tumor-promoting in cervical cancer, fibrosarcoma, and lung cancer ( 57 , 58 ), and we recently discovered a role for Metap1 in promoting the sensitivity of breast cancer cells to phosphoinositide 3-kinase (PI3K) inhibition ( 19 ).…”

“…Metap2 is known to act as tumor-promoting in different cancers ( 54 , 55 ) including breast cancer ( 19 , 56 ). Metap1 is known to act as tumor-promoting in cervical cancer, fibrosarcoma, and lung cancer ( 57 , 58 ), and we recently discovered a role for Metap1 in promoting the sensitivity of breast cancer cells to phosphoinositide 3-kinase (PI3K) inhibition ( 19 ). Also, MMP17 was already likened to breast cancer, promoting tumor growth ( 59 ).…”

“…Based on internal controls and validation of many screen hits by experiment and literature, we prove degradome-focused RNAi-based pooled competitive growth screens as a suitable discovery pipeline to analyze the role of proteases in breast cancer cell proliferation/survival. The usability of our screening approach has previously been validated in context of synthetic lethality screens ( 19 ). Applying multistep selection criteria, our screens yielded 100 proteases that were identified to be important for breast cancer growth.…”

“…In 2022, 290,560 new cases and 43,780 deaths are estimated to occur in the United States alone ( 18 ). For our degradome-wide RNAi screens, we employed a customized shRNA library targeting the entire murine degradome ( 19 ). Specifically, we made use of a third-generation shRNA backbone, the enhanced microRNA (miR-E), generated by optimization of the native human miR-30a scaffold ( 20 ).…”

“…Moreover, we employed an advanced RNAi vector system with inducible promoters for coexpression of the miR-E and a fluorescent reporter combined with a constitutive fluorescent marker to monitor vector integration. We have recently successfully applied this degradome-wide screening approach in a synthetic lethality setting ( 19 ). Now, we use it to define sets of proteases enabling or promoting breast cancer cell growth.…”