WWOX Possesses N-Terminal Cell Surface-Exposed Epitopes WWOX7-21 and WWOX7-11 for Signaling Cancer Growth Suppression and Prevention In Vivo

Abstract

Membrane hyaluronidase Hyal-2 supports cancer cell growth. Inhibition of Hyal-2 by specific antibody against Hyal-2 or pY216-Hyal-2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal-2. Alternatively, WWOX undergoes self-polymerization and localizes in the cell membrane. Proapoptotic pY33-WWOX binds Hyal-2, and TGF-β induces internalization of the pY33-WWOX/Hyal-2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14-WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface-exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3-dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7-21 peptide, or truncation to 5-amino acid WWOX7-11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time-lapse microscopy revealed that WWOX7-21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33-WWOX, downregulation of prosurvival pERK, prompt increases in Ca2+ influx, and disruption of the IkBα/WWOX/ERK prosurvival signaling. In contrast, pS14-WWOX7-21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib-mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14-WWOX significantly enhanced the ceritinib-induced apoptosis. Together, the N-terminal epitopes WWOX7-21 and WWOX7-11 are potent in blocking cancer growth in vivo. WWOX7-21 and WWOX7-11 peptides and pS14-WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.

Therapeutic Zfra4-10 or WWOX7-21 Peptide Induces Complex Formation of WWOX with Selective Protein Targets in Organs that Leads to Cancer Suppression and Spleen Cytotoxic Memory Z Cell Activation In Vivo

“…Zfra binds to membrane Hyal-2 to activate spleen memory cytotoxic Z cells to kill cancer cells [ 2 , 6 , 10 , 11 , 12 , 13 ]. Additionally, when Hyal-2/pY33-WWOX/SMAD4 complex is overexpressed, hyaluronan binds to Hyal-2 and activates the signaling of Hyal-2/pY33-WWOX/SMAD4 to cause apoptosis in vivo [ 2 , 6 , 10 , 11 , 12 , 13 ]. To establish the role of Hyal-2 and WWOX in cancer suppression, nude mice received tail vein injections with sterile Milli-Q water or Zfra4-10 peptide (1 mM in 100 μL sterile Milli-Q water) in every two other days for three times.…”

“…In our recent study, we have demonstrated that ceritinib, an anaplastic lymphoma kinase (ALK)-positive inhibitor for lung cancer treatment [ 23 , 24 ], is able induce breast cancer stem cell sphere explosion and death [ 10 ]. Whether activated Z cells attacked and killed cancer cells and cancer stem cell spheres in vitro was investigated.…”

“…Naïve wild type Wwox B6 mice were treated with Zfra (1 mM in 100 μL PBS) once via tail vein injection, followed by resting for one week, and then were subjected to purification of activated Z cells in the spleen [ 6 , 7 ]. Activated Z cells (8 × 10 5 cells) were co-cultured with mouse breast 4T1 cell monolayers, in the presence of nuclear stains 4’,6-diamidino-2-phenylindole (DAPI; blue fluorescence) and propidium iodide (PI; red fluorescence), for imaging by time-lapse microscopy [ 9 , 10 , 13 , 16 , 25 ]. Activated spleen Z cells underwent clonal expansion and aggressively attacked and killed 4T1 cells ( Figure 7 A,B; Videos S1–S3 ).…”

“…Zfra4-10 peptide increases the binding of HIF-1α with C1qBP and COX2. Additionally, Zfra4-10 or WWOX7-21 peptide increases the complex formation of WWOX with C1qBP, CD133, p21, JNK1 [ 44 ], COX2, and p-ERK [ 10 ] in the spleen, which correlates with cancer suppression in vivo. Loss of the binding between WWOX and target proteins leads to increased cancer cell growth.…”

“…For example, ionophore/phorbol ester-mediated forced differentiation of leukemia T cells or normal T cells requires WWOX phosphorylation at Ser14, but dephosphorylation at pY33 and pY61 [ 9 ]. The matured T cells fail to block cancer growth [ 9 , 10 , 11 ]. By comparison, we showed here that Zfra suppresses WWOX phosphorylation at S14, Y33, and Y61 to allow spleen Z cell activation.…”