Overcoming PD-1 Inhibitor Resistance with a Monoclonal Antibody to Secreted Frizzled-Related Protein 2 in Metastatic Osteosarcoma.

Secreted frizzled-related protein 2 (SFRP2) promotes the migration/invasion of metastatic osteosarcoma (OS) cells and tube formation by endothelial cells. However, its function on T-cells is unknown. We hypothesized that blocking SFRP2 with a humanized monoclonal antibody (hSFRP2 mAb) can restore immunity by reducing CD38 and PD-1 levels, ultimately overcoming resistance to PD-1 inhibitors. Treating two metastatic murine OS cell lines in vivo, RF420 and RF577, with hSFRP2 mAb alone led to a significant reduction in the number of lung metastases, compared to IgG1 control treatment. While PD-1 mAb alone had minimal effect, hSFRP2 mAb combination with PD-1 mAb had an additive antimetastatic effect. This effect was accompanied by lower SFRP2 levels in serum, lower CD38 levels in tumor-infiltrating lymphocytes and T-cells, and lower PD-1 levels in T-cells. In vitro data confirmed that SFRP2 promotes NFATc3, CD38 and PD-1 expression in T-cells, while hSFRP2 mAb treatment counteracts these effects and increases NAD+ levels. hSFRP2 mAb treatment further rescued the suppression of T-cell proliferation by tumor cells in a co-culture model. Finally, hSFRP2 mAb induced apoptosis in RF420 and RF577 OS cells but not in T-cells. Thus, hSFRP2 mAb therapy could potentially overcome PD-1 inhibitor resistance in metastatic osteosarcoma.

Development of a Novel Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein-2 That Inhibits Triple-Negative Breast Cancer and Angiosarcoma Growth In Vivo.

BACKGROUND: We previously reported that secreted frizzled-related protein-2 (SFRP2) is expressed in a variety of tumors, including sarcoma and breast carcinoma, and stimulates angiogenesis and inhibits tumor apoptosis. Therefore, we hypothesized that a humanized SFRP2 monoclonal antibody (hSFRP2 mAb) would inhibit tumor growth. METHODS: The lead hSFRP2 antibody was tested against a cohort of 22 healthy donors using a time course T-cell assay to determine the relative risk of immunogenicity. To determine hSFRP2 mAb efficacy, nude mice were subcutaneously injected with SVR angiosarcoma cells and treated with hSFRP2 mAb 4 mg/kg intravenously every 3 days for 3 weeks. We then injected Hs578T triple-negative breast cells into the mammary fat pad of nude mice and treated for 40 days. Control mice received an immunoglobulin (Ig) G1 control. The SVR and Hs578T tumors were then stained using a TUNEL assay to detect apoptosis. RESULTS: Immunogenicity testing of hSFRP2 mAb did not induce proliferative responses using a simulation index (SI) ≥ 2.0 (p < 0.05) threshold in any of the healthy donors. SVR angiosarcoma tumor growth was inhibited in vivo, evidenced by significant tumor volume reduction in the hSFRP2 mAb-treated group, compared with controls (n = 10, p < 0.001). Likewise, Hs578T triple-negative breast tumors were smaller in the hSFRP2 mAb-treated group compared with controls (n = 10, p < 0.001). The hSFRP2 mAb treatment correlated with an increase in tumor cell apoptosis (n = 11, p < 0.05). Importantly, hSFRP2 mAb treatment was not associated with any weight loss or lethargy. CONCLUSION: We present a novel hSFRP2 mAb with therapeutic potential in breast cancer and sarcoma that has no effect on immunogenicity.

TRAF3-interacting protein 3, a new oncotarget, promotes tumor growth in melanoma.

TRAF3-interacting protein 3 (TRAF3IP3) is expressed in the immune system and participates in cell maturation, tissue development, and immune response. In a previous study, we reported that TRAF3IP3 levels were substantially increased in the vasculature of breast cancer tissues, suggesting a proangiogenic role. In this study, we investigated TRAF3IP3 tumorigenic function. TRAF3IP3 protein was present in several cancer cell lines, with highest levels in melanoma. In addition, tumor microarray analysis on 23 primary melanoma and nine positive lymph nodes revealed that 70% of human primary melanoma and 66% of lymph node metastases were positive for TRAF3IP3. Importantly, TRAF3IP3 downregulation correlated with an 83% reduction of tumor growth in a subcutaneous xenograft mouse model (n=10, P=0.005). Immunohistochemistry analysis of the tumors revealed that TRAF3IP3-shRNA tumors had increased apoptosis (n=4, P<0.01) and reduced microvascular density (n=4, P<0.002). In addition, TRAF3IP3 downregulation in malignant endothelial cells reduced tube formation in a Matrigel tube formation assay. In melanoma cells, decreased levels of TRAF3IP3 were also associated with reduced viability (n=4, P=0.03) and proliferation (n=3, P=0.03), together with increased sensitivity to ultraviolet-induced apoptosis (n=4, P=0.0004). Furthermore, TRAF3IP3 downregulation correlated with increased amounts of interferon-γ. Interferon-γ inhibits tumor growth and angiogenesis, thus suggesting a new pathway for TRAF3IP3 in cancer. Collectively, the association of TRAF3IP3 with malignant properties of melanoma suggest a clinical potential for targeted therapy.

Frizzled-5: a high affinity receptor for secreted frizzled-related protein-2 activation of nuclear factor of activated T-cells c3 signaling to promote angiogenesis.

Secreted frizzled-related protein 2 (SFRP2) is a pro-angiogenic factor expressed in the vasculature of a wide variety of human tumors, and modulates angiogenesis via the calcineurin-dependent nuclear factor of activated T-cells cytoplasmic 3 (NFATc3) pathway in endothelial cells. However, until now, SFRP2 receptor for this pathway was unknown. In the present study, we first used amino acid alignments and molecular modeling to demonstrate that SFRP2 interaction with frizzled-5 (FZD5) is typical of Wnt/FZD family members. To confirm this interaction, we performed co-immunofluorescence, co-immunoprecipitation, and ELISA binding assays, which demonstrated SFRP2/FZD5 binding. Functional knock-down studies further revealed that FZD5 is necessary for SFRP2-induced tube formation and intracellular calcium flux in endothelial cells. Using protein analysis on endothelial cell nuclear extracts, we also discovered that FZD5 is required for SFRP2-induced activation of NFATc3. Our novel findings reveal that FZD5 is a receptor for SFRP2 and mediates SFRP2-induced angiogenesis via calcineurin/NFATc3 pathway in endothelial cells.

Dysregulated connexin 43 in HER2-positive drug resistant breast cancer cells enhances proliferation and migration.

Connexin 43 (Cx43) is a gap junction protein whose function in the development of breast cancer and in breast cancer progression remains unclear. Evidence suggests that Cx43 (GJA1) mRNA and protein expression is altered in breast tumors. However, reports indicate both increased and decreased Cx43 levels in human breast cancer samples. Studies also suggest that loss of Cx43 regulated gap junction intercellular communication is a common feature of breast malignancies that potentially correlates with histological stage. Further evidence suggests that Cx43 (GJA1) mRNA expression is negatively correlated with HER2 positivity but a relationship between Cx43 and HER2 in breast cancer is not well defined. Therefore, in this study, we sought to evaluate the relationship between Cx43 activity, HER2, and drug resistance. Using HER2+ breast cancer cell lines that are sensitive or resistant to HER2 inhibitor, we evaluated Cx43 gap junction function. We found that Cx43 gap junction activity is completely lost in drug resistant HER2-positive (HER2+) breast cancer cells, whereas Cx43 gap junction activity can be restored by Cx43 overexpression in drug sensitive HER2+ cells. Moreover, the dysregulation of Cx43 resulted in increased tumorigenic and migratory capacity of the HER2+ drug resistant breast cancer cells.