Phosphorylated forms of WWOX at Y33 and S14 counteract each other in controlling cell stemness in vitro and in vivo

Background We have recently classified cells into two categories: WWOXf cells expressing functional WW domain-containing oxidoreductase (WWOX), and WWOXd cells having WWOX deficiency or overexpressed gain-of-function mutants. When WWOXf cells encounter WWOXd cells, WWOXd cells, in many cases, undergo retrograde migration to avoid physical contact. Both cells may initiate remote, targeted mutual killing at a distance of 500 μm. pY33-WWOX is known to inhibit cancer growth and stemness, as well as the progression of neurodegeneration. In contrast, pS14-WWOX supports the progression of cancer and neurodegeneration, such as Alzheimer’s disease. Hypothetically, WWOXd cells exhibit stronger stemness than WWOXf cells, and pS14-WWOX is upregulated to increase stemness in WWOXd cells. Methods To test the differences of WWOXf and WWOXd cells in stemness, monolayers and spheres of normal and cancer cells were exposed to UV or treated with Zfra4-10, WWOX7-21, and modified peptides, followed by time-lapse bright-field and fluorescent microscopy to measure cell stemness, bubbling cell death (BCD), calcium influx, Fe 2+ uptake, calcein retention, mitochondrial potential, and stress fiber formation. We co-cultured non-sphere-forming WWOXf with sphere-forming WWOXd cells to determine sphere formation in the presence or absence of antibodies against WWOX and its phosphorylation forms. Nude and B6 mice received an aliquot of pY33-WWOX antiserum, non-immune serum, or Zfra4-10 peptide via tail-vein injections to assess organ stemness by immunohistochemistry and co-immunoprecipitation. We used Western blotting and immunofluorescence staining to assess cancer cell stemness. Results Compared to WWOXf, WWOXd cells were shown to have (1) poor efficacy in calcein retention (indicating reduced survival), (2) significantly reduced intracellular nitric oxide (NO), (3) failure in UV-induced calcium influx, yet (4) increased efficiency in Fe 2+ uptake and in ERK-dependent stress fiber formation under stress conditions. pS14-WWOX supports WWOXf cells in calcein retention and blocks stress fiber formation. Functional WWOX suppresses cancer cell stemness by directly binding stem cell markers, including OCT-4, SSEA-4, and Nanog. UV upregulates pY33 and simultaneously suppresses pS14 in WWOX, leading to cell death. Inhibition of pY33-WWOX by antibody induces normal stem cell generation in mice. Upregulation of pS14-WWOX, or exposure to a cell-repellent WWOX286-299 peptide, leads to enhanced stem cell-like sphere formation in breast cancer 4T1 cells. As a potent WWOX inhibitor, the Zfra4-10 peptide significantly upregulates many stem cell markers by 2- to 5-fold and, meanwhile, downregulates WWOX in the liver. Conclusion pS14-WWOX is essential in maintaining stemness in WWOXd cells. Zfra4-10 strongly suppresses cancer cell stemness by downregulating pS14-WWOX and stem cell markers. Mechanistically, pY33- and pS14-WWOX counteract each other in controlling cell stemness. Suppression of pY33-WWOX by antibody upregulates cell stemness in vitro and in vivo. Our study suggests that Zfra4-10 targets cancer cell stemness and growth, and that the pY33-WWOX antibody could promote the expansion of essential stem cells for repairing abnormal organs.