Objective: Pancreatic cancer (PC) is a type of highly malignant tumor associated with poor prognosis, whose progression is driven by hypoxia in the tumor microenvironment. This study aims to explore the effects of hypoxia-induced upregulation of acetyl-CoA synthetase 2 (ACSS2) on the proliferation and stemness of PC cells and its potential molecular mechanism, so as to provide new targets and therapy strategies for the PC.

Materials and Methods: PC cells (PANC-1) were cultured under separate conditions: hypoxic and normoxic. Cell models of ACSS2 overexpression, ACSS2 knockdown and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) knockdown were constructed using transfection technique. Cell counting kit 8 (CCK-8) and clonal formation assay were used to assess cell viability, and cell stemness was analyzed by means of sphere-formation assay and detection of stem-related markers. A mouse tumor model was established by axilla injection of tumor cells, and tumor growth was evaluated by measuring the volume and weight of the isolated tumors. Relative mRNA and protein levels were analyzed by quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry.

Results: Hypoxic condition upregulated the expression of ACSS2 in PC cells. CCK-8 and clonal formation assays showed that upregulation of ACSS2 promoted cell proliferation (p < 0.001), while knockdown of ACSS2 inhibited cell proliferation (p < 0.001). Sphere formation assay and stemness marker detection showed that ACSS2 upregulation could maintain cell stemness (p < 0.001), while knockdown could inhibit it (p < 0.01). Through mechanistic studies, we found that ACSS2 activated phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway through HMGCS1. Interference with HMGCS1 inhibited pathway activation caused by ACSS2 upregulation and hindered cell proliferation and stemness. In vivo experiments further demonstrated that ACSS2 accelerated PC xenograft tumor growth and promoted tumor stemness.

Conclusion: Hypoxia induces upregulation of ACSS2 and activates PI3K/AKT/mTOR pathway through HMGCS1, thereby enhancing the proliferation and stemness of PC cells. This finding offers a novel perspective for understanding the development mechanism of PC and highlights a potential molecular target for developing targeted therapeutic strategies.