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.

Synthetic Zfra4-10 and WWOX7-21 peptides strongly suppress cancer growth in vivo. Hypothetically, Zfra4-10 binds to the membrane Hyal-2 of spleen Z cells and activates the Hyal-2/WWOX/SMAD4 signaling for cytotoxic Z cell activation to kill cancer cells. Stimulation of membrane WWOX in the signaling complex by a WWOX epitope peptide, WWOX7-21, is likely to activate the signaling. Here, mice receiving Zfra4-10 or WWOX7-21 peptide alone exhibited an increased binding of endogenous tumor suppressor WWOX with ERK, C1qBP, NF-κB, Iba1, p21, CD133, JNK1, COX2, Oct4, and GFAP in the spleen, brain, and/or lung which led to cancer suppression. However, when in combination, Zfra4-10 and WWOX7-21 reduced the binding of WWOX with target proteins and allowed tumor growth in vivo. In addition to Zfra4-10 and WWOX7-21 peptides, stimulating the membrane Hyal-2/WWOX complex with Hyal-2 antibody and sonicated hyaluronan (HAson) induced Z cell activation for killing cancer cells in vivo and in vitro. Mechanistically, Zfra4-10 binds to membrane Hyal-2, induces dephosphorylation of WWOX at pY33 and pY61, and drives Z cell activation for the anticancer response. Thus, Zfra4-10 and WWOX7-21 peptides, HAson, and the Hyal-2 antibody are of therapeutic potential for cancer suppression.

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

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-ß 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.

Zfra activates memory Hyal-2+ CD3- CD19- spleen cells to block cancer growth, stemness, and metastasis in vivo.

Zfra is a 31-amino-acid zinc finger-like protein, which participates in the tumor necrosis factor signaling. Here, we determined that when nude mice and BALB/c mice were pre-injected with nanogram levels of a synthetic Zfra1-31 or truncated Zfra4-10 peptide via tail veins, these mice became resistant to the growth, metastasis and stemness of melanoma cells, and many malignant cancer cells. The synthetic peptides underwent self-polymerization in phosphate-buffered saline. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra aggregation and its-mediated cancer suppression in vivo. Injected Zfra peptide autofluoresced due to polymerization and was trapped mainly in the spleen. Transfer of Zfra-stimulated spleen cells to naïve mice conferred resistance to cancer growth. Zfra-binding cells, designated Hyal-2+ CD3- CD19- Z cells, are approximately 25-30% in the normal spleen, but are significantly downregulated (near 0-3%) in tumor-growing mice. Zfra prevented the loss of Z cells caused by tumors. In vitro stimulation or education of naïve spleen cells with Zfra allowed generation of activated Z cells to confer a memory anticancer response in naïve or cancer-growing mice. In particular, Z cells are abundant in nude and NOD-SCID mice, and can be readily activated by Zfra to mount against cancer growth.

Tumor Suppressor WWOX and p53 Alterations and Drug Resistance in Glioblastomas.

Tumor suppressor p53 are frequently mutated in glioblastomas (GBMs) and appears to contribute, in part, to resistance to temozolomide (TMZ) and therapeutic drugs. WW domain-containing oxidoreductase WWOX (FOR or WOX1) is a proapoptotic protein and is considered as a tumor suppressor. Loss of WWOX gene expression is frequently seen in malignant cancer cells due to promoter hypermethylation, genetic alterations, and translational blockade. Intriguingly, ectopic expression of wild type WWOX preferentially induces apoptosis in human glioblastoma cells harboring mutant p53. WWOX is known to physically bind and stabilize wild type p53. Here, we provide an overview for the updated knowledge in p53 and WWOX, and postulate potential scenarios that wild type and mutant p53, or isoforms, modulate the apoptotic function of WWOX. We propose that triggering WWOX activation by therapeutic drugs under p53 functional deficiency is needed to overcome TMZ resistance and induce GBM cell death.