Abstract
Stem-like cancer cells, with characteristic self-renewal abilities, remain highly refractory to various clinical interventions. As such, stemness-inhibiting entities, such as tumor suppressor p53, are therapeutically pursued for their anticancer activities. Interestingly, similar implications for tumor suppressor TAp73 in regulating stemness features within stem-like cancer cells remain unknown.Experimental Design: This study utilizes various in vitro molecular biology techniques, including immunoblotting, qRT-PCR, and mass spectrometry-based proteomics, and metabolomics approaches to study the role of TAp73 in human and murine embryonal carcinoma stem-like cells (ECSLC) as well as human breast cancer stem-like cells (BCSLC). These findings were confirmed using patient-derived brain tumor-initiating cells (BTIC) and in vivo xenograft models. TAp73 inhibition decreases the expression of stem cell transcription factors Oct4, Nanog, and Sox-2, as well as tumorsphere formation capacity in ECSLCs. In vivo, TAp73-deficient ECSLCs and BCSLCs demonstrate decreased tumorigenic potential when xenografted in mice. Mechanistically, TAp73 modifies the proline regulatory axis through regulation of enzymes GLS, OAT, and PYCR1 involved in the interconversion of proline-glutamine-ornithine. Further, TAp73 deficiency exacerbates glutamine dependency, enhances accumulation of reactive oxygen species through reduced superoxide dismutase 1 (SOD1) expression, and promotes differentiation by arresting cell cycle and elevating autophagy. Most importantly, the knockdown of TAp73 in CD133HI BTICs, separated from three different glioblastoma patients, strongly decreases the expression of prosurvival factors Sox-2, BMI-1, and SOD1, and profoundly decreases their self-renewal capacity as evidenced through their reduced tumorsphere formation ability. Collectively, we reveal a clinically relevant aspect of cancer cell growth and stemness regulation through TAp73-mediated redox-sensitive metabolic reprogramming.
Highlights
Cancer stem–like cells (CSLC) constitute a distinct population of cells within a heterogeneous cancer mass
TAp73 inhibition decreases the expression of stem cell transcription factors Oct4, Nanog, and Sox-2, as well as tumorsphere formation capacity in embryonal carcinoma stem-like cells (ECSLC)
TAp73 modifies the proline regulatory axis through regulation of enzymes GLS, ornithine aminotransferase (OAT), and pyrroline-5carboxylate reductase 1 (PYCR1) involved in the interconversion of proline–glutamine–ornithine
Summary
TAp73 is required for maintaining the stemness of embryonal carcinoma stem-like cells. Replenishment of glutamine in TAp73 KD ECSLCs from two distinct shRNA clones restored the expression of pluripotency factor Oct and reversed the upregulation of differentiation marker b3-tubulin and autophagy protein LC3B-II (Fig. 5B and C; Supplementary Fig. S7B and S7C). Together, these results highlight that glutamine deprivation, as opposed to proline, is more critical for TAp73 KD–mediated effects and indicate that TAp73 KD cells may require supply of external glutamine to maintain their growth rate, pluripotency, and undifferentiated state. This comparative analysis of patient-derived CD133HI and CD133LO GBM cells directly demonstrates the differential role of TAp73 in stem-like cancer cells versus non–stem-like differentiated cancer cells, and confirmed the clinical relevance of TAp73 inhibition-mediated effects on the growth and stemness of stem-like cancer cells
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