Abstract
Simple SummaryCancer cell survival in hypoxia areas, with low oxygen and food supply as well as abundant waste material, is critical to their aggressiveness and associated with disease relapse and mortality. Therefore, it is vital to understand the molecular regulators of cancer cell survival under these harsh physiological conditions. In the present study, we assessed the role of a mitochondrial protein carnitine palmitoyltransferase (CPT1A) in regulating prostate cancer (PCa) cell survival and proliferation under hypoxic conditions in both cell culture and animal models. The results showed that CPT1A expression in PCa cells is key to their survival and proliferation in the hypoxic tumor microenvironment. These results have high translational significance in improving cancer prognosis and therapy.Hypoxia and hypoxia-related biomarkers are the major determinants of prostate cancer (PCa) aggressiveness. Therefore, a better understanding of molecular players involved in PCa cell survival under hypoxia could offer novel therapeutic targets. We previously reported a central role of mitochondrial protein carnitine palmitoyltransferase (CPT1A) in PCa progression, but its role in regulating PCa survival under hypoxia remains unknown. Here, we employed PCa cells (22Rv1 and MDA-PCa-2b) with knockdown or overexpression of CPT1A and assessed their survival under hypoxia, both in cell culture and in vivo models. The results showed that CPT1A knockdown in PCa cells significantly reduced their viability, clonogenicity, and sphere formation under hypoxia, while its overexpression increased their proliferation, clonogenicity, and sphere formation. In nude mice, 22Rv1 xenografts with CPT1A knockdown grew significantly slower compared to vector control cells (~59% reduction in tumor volume at day 29). On the contrary, CPT1A-overexpressing 22Rv1 xenografts showed higher tumor growth compared to vector control cells (~58% higher tumor volume at day 40). Pathological analyses revealed lesser necrotic areas in CPT1A knockdown tumors and higher necrotic areas in CPT1A overexpressing tumors. Immunofluorescence analysis of tumors showed that CPT1A knockdown strongly compromised the hypoxic areas (pimonidazole+), while CPT1A overexpression resulted in more hypoxia areas with strong expression of proliferation biomarkers (Ki67 and cyclin D1). Finally, IHC analysis of tumors revealed a significant decrease in VEGF or VEGF-D expression but without significant changes in biomarkers associated with microvessel density. These results suggest that CPT1A regulates PCa survival in hypoxic conditions and might contribute to their aggressiveness.
Highlights
Prostate cancer (PCa) is the most common non-cutaneous cancer in men—accounting for more than 1 in 5 new diagnoses in men—with 248,538 new cases and more than 34,130 deaths estimated in the United States in 2021 [1]
To assess the effects of CPT1A KD or OE on prostate cancer (PCa) cell proliferation and colony formation, we first confirmed the knockdown and overexpression of stably transfected 22Rv1 cells and knockdown of stably transfected MDA-PCa-2b cells compared to their respective vector controls (Figure 1A, full blot shown in Figures S1 and S2)
The MTT assay results showed that 22Rv1 PCa cells with CPT1A KD had a statistically significant decrease in viability under both normoxia and hypoxia compared to vector control cells (Figure 1C)
Summary
Prostate cancer (PCa) is the most common non-cutaneous cancer in men—accounting for more than 1 in 5 new diagnoses in men—with 248,538 new cases and more than 34,130 deaths estimated in the United States in 2021 [1]. A chronic condition of low oxygen in the tumor microenvironment, known as hypoxia, is an early event occurring in solid neoplasms that contributes to tumor aggressiveness. Cancer cells within this stressed microenvironment adopt many tumor-promoting characteristics, such as genomic instability, invasive behavior, and altered cellular bioenergetics [6]. Due to the important role of hypoxia in the tumor microenvironment, several approaches have been developed to target hypoxia-induced signaling and hypoxia inducible factor 1α (HIF-1α) inhibitors to circumvent therapeutic resistance [11,12]. Targeting hypoxia-induced signaling in tumors, such as inhibitors of HIF-1α and HIF-2α, may improve the efficacy of therapeutic regimens. Since hypoxic areas of the tumor have decreased blood supply, further studies are needed to efficiently deliver these pharmacological agents to target hypoxic areas
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