Mitochondrial fission promotes self-renewal and tumorigenic potential in prostate cancer

ABSTRACT

The emergence of therapy-resistant cancer stem cells (CSCs) limit the efficacy of prostate cancer treatment. Using genetic knockdown and chemical inhibitors, we demonstrate the critical role of Bromodomain Containing 4 (BRD4) in promoting mitochondrial fission and sustaining CSC expansion. These findings provide a new paradigm for developing novel treatment strategies for prostate cancer.

Intratumoral heterogeneity due to the complex interplay of genetic and epigenetic variation and the almost inevitable emergence of therapy-resistant cells limit severely the efficacy of cancer treatment.^1,2 To date, there is compelling evidence of a hierarchical organization within tumors with stem-like tumor cells that sustain this biological heterogeneity.³ Cancer stem cells (CSCs) retain the ability of self-reproducing through symmetric cell division and generating the entire progeny of tumor cells at various stages of differentiation and proliferative capacity through progressive asymmetric cell division.³ The balance between these processes is essential for preserving the CSC pool and ensuring the continuous expansion of the tumor mass. Moreover, despite even massive reduction of bulk tumor cells (BTCs) consequent to an effective therapy, treatment-resistant CSCs can survive, progressively expand and reconstitute the original population of tumor cells leading to relapse.¹ The presence of CSCs also influence metastatic spread and disease recurrence in prostate cancer. Expansion of CSCs, which are independent of androgen receptor (AR) signaling, contributes to the emergence of castration-resistant prostate cancer (CRPC).⁴ Indeed, current therapies for locally advanced and metastatic prostate cancer target preferentially AR-dependent proliferating tumor cells that constitute the bulk of the tumor mass.⁵ However, prostate CSCs, insensitive to these therapies,⁴ can repopulate the tumors at primary and metastatic sites.

Epigenetic and transcriptional reprogramming in prostate cancer stem cells

Epigenetic modifiers are essential for instructing specific transcriptional programs and promoting phenotypic adaptation of CSCs and treatment resistance.^1,2 Bromodomain and extra-terminal domain (BET) proteins, like Bromodomain Containing 4 (BRD4), are chromatin readers that bind to acetylated histones and enable cell type-specific transcriptional programs.⁶ BRD4 cooperates with transcription factors, like MYC and Signal Transducer and Activator of Transcription 3 (STAT3), which are important in many types of cancer. BET inhibitors (BETi) block these interactions and interfere with the transcription of multiple genes. BETi are active in preclinical models and are under evaluation in clinical trials for hematological and solid tumors, including prostate cancer.⁶ Specifically, BETi disrupt BRD4 interaction with the androgen receptor (AR) and block the expression of AR-regulated genes in AR-positive prostate cancer cells.⁷ However, whether BETi affect prostate CSCs, which have attenuated AR signaling but increased activity of other oncogenic transcription factors, was an open question.

In our study, we compared the transcriptome of CSC-enriched tumor-sphere cells and bulk tumor cells (BTCs). This analysis revealed significantly divergent transcriptional programs in CSCs and BTCs.⁸ Genes associated with proliferation, mitosis, and DNA replication were predominantly active in BTCs. Conversely, these pathways were under-represented in CSCs, which exhibited preferential activation of metabolic and mitochondrial pathways. Interestingly, we found that BRD4 contributed to the activation of the distinct transcriptional programs in CSCs and BTCs. Notably, transcription of several genes in metabolic and mitochondrial pathways in CSCs depended on BRD4 activity. Blocking BRD4 by genetic tools or with BETi affected transcription of these genes selectively in CSCs and impaired key processes essential for CSC survival and expansion.

Cancer stem cells and mitochondria dynamics

The presence of many genes involved in mitochondria biogenesis, dynamics, and function among those preferentially activated in prostate CSCs was a revealing finding. Mitochondrial reprogramming has an emerging role in cancer pathogenesis, beyond the classical metabolic shift described as Warburg effect.⁹ Enhanced mitochondrial biogenesis and dynamics are critical for stem cell maintenance determining the outcome of symmetric and asymmetric cell division (Figure 1(a)).¹⁰ In prostate CSCs, we uncovered an important link between BRD4 function and regulation of mitochondria dynamics through the activation of Mitochondrial Fission Factor (MFF) transcription.⁸ Blocking BRD4 by genetic knockdown or the BETi OTX015/MK-8628 impaired fission and segregation of mitochondria during asymmetric cell division (Figure 1(b)). This resulted in senescence selectively in CSCs and progressive loss of self-renewal capability. Importantly, BRD4/BET inhibition reduced tumor growth in vivo with persistent depletion of tumor-propagating CSCs. Genetic knockdown of MFF further demonstrated that mitochondria fission is essential for prostate CSCs. Abrogation of mitochondrial fission, through failed during asymmetric cell division, promoted senescence, loss of proliferative potential and loss of tumorigenic capability in prostate CSCs. Thus, proteins involved in mitochondrial dynamics might be relevant targets for developing CSC-directed therapies.

Figure 1.

Mitochondrial fission and cancer stem cell propagation. (a) Mitochondrial fission is required for segregation of functional and dysfunctional mitochondria in the progeny of stem and non-stem tumor cells, respectively, during consecutive asymmetric cell division. (b) Impairing mitochondrial fission and the correct execution of asymmetric cell division, BET inhibitors and knockdown of BRD4 (BRD4 kd) or MFF (MFF kd) promote the progressive exhaustion of self-renewal capability and the elimination of prostate cancer stem cells.

BET inhibitors and combinatorial strategies for prostate cancer treatment

Our study clarifies relevant aspects of the antitumor activity of BETi that will likely guide their future clinical development. We demonstrated that BETi elicited different responses in CSCs and BTCs.⁸ BRD4 inhibition by both BETi and genetic knockdown caused reversible arrest of proliferation (cytostatic effect) in BTCs. Conversely, BRD4 inhibition in CSCs resulted in persistent loss of self-renewal and tumorigenic potential. Furthermore, the effects in CSCs were independent of AR status and AR signaling, whereas the cytostatic effect on BTCs was predominant in AR-proficient prostate cancer cells. The progressive exhaustion of prostate CSCs was due to the failed mitochondria segregation during asymmetric cell division and consequent selective impairment of their self-renewal.⁸ The efficacy of BETi towards prostate CSCs provides the opportunity to test the potential benefits of combining CSC-directed therapies with anticancer drugs affecting BTCs. This combinatorial strategy could reduce treatment failures by preventing the emergence of resistant CSCs and provide effective therapeutic options for patients with CRPC for which current treatment offers limited and not durable benefits.

Funding Statement

This work was supported by the Krebsliga Schweiz [KLS-4569-08-2018]; Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung [310030L-170182]; Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung [IZLSZ3-170898]; Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung [310030-169942].

Acknowledgments

Funding for this work was granted by the Swiss National Science Foundation, Swiss Cancer league, Ticino Foundation for Cancer Research and Oncology Therapeutic Development (OTD, Clichy, France).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest to disclose.