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Background: Castrate-resistant prostate cancer (CRPC) is the second leading cause of cancer deaths among US men. Currently, a large number of small and low grade prostate cancers (PCa) are being diagnosed. Only a few of them will metastasize and become lethal. A clinical method to distinguish aggressive from the indolent tumors is warranted. An enhanced glucose metabolism (Warbürg effect) and invasion of cells from their organs of origin and metastasis to distant organs are two characteristics that are ubiquitous in most solid tumors. In the last few years, the non-glycolytic activities (“moonlighting functions”) of glycolytic enzymes have been investigated in relation to cancer cell invasion. We propose that oxidatively stressed PCa cells, as a countermeasure, redirect some of the glycolytic enzymes to their moonlighting functions. Methods: We used a novel microfluidic device that separates the invading from the non-invading PCa cells based on their migratory characteristics in a 3D collagen matrix. We performed ICC analysis of the separated cells for oxidative stress generating enzyme spermidine/spermine N1 acetyl transferase (SSAT) and glycolytic enzymes aldolase, GAPDH and F-actin levels. We also carried out proteomic analysis of aldolase and GAPDH levels and oxidation state and metabolomic analysis for enzymatic activities of those two enzymes. Results: Under identical condition, LNCaP cells show minimum invasion, whereas between 30-40% of C4-2 cells invade more than 0.8 mm in 16 hours. ICC assay of C4-2 cells shows that most migratory C4-2 cells have SSAT overexpression, but less than 5% stationary cells show this effect. Proteomic analysis shows that androgen treatment that increases overall oxidative stress in both LNCaP and C4-2 cells actually decreases oxidation status of GAPDH in LNCaP cells and increases that in C4-2 cells. Anti-androgen enzalutamide reverses the androgen effect in both cell lines. The glycolytic activity of GAPDH decreases with an increase in protein oxidation. Our metabolomic data further confirmed an increase in GAPDH activity in LNCaP and a decrease in C4-2 cells after androgen treatment and the reversal with enzalutamide as expected from its oxidation status. Discussion: SSAT expression and a consequent increase in oxidative stress are related to invasion of CRPC. Oxidation of certain glycolytic enzymes in CRPC cells may divert them to their moonlighting function related to cellular invasion and metastasis. These functions may be monitored in patient biopsies and/or prostatectomy tissues for PCa prognosis. Application of the microfluidic method for the separation of invading from the non-invading cells and proteogenomic and metabolomic analysis of these cells isolated from patient prostatectomy tissues are currently being standardized. Citation Format: Jiaquan Yu, Ashley M. Weichmann, Alexandria Craig, Wei Huang, Dawn R. Church, Farideh Mehraein, Laurie L. Parker, David J. Beebe, George Wilding, Hirak S. Basu. “Moonlighting Functions” of glycolytic enzymes relate to human prostate cancer invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4968.