Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as ‘vasculogenic mimicry’ (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody–endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear β-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases.
Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of, and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). VM is more frequently seen in TNBC than other breast cancer subtypes, and is associated with aggressive metastatic behavior. We previously developed αEGFR-E-P125A, an antibody-endostatin fusion protein, linking an anti-EGFR antibody targeting domain to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and markedly decreases metastasis in both the MDA-MB-231-4175 and MDA-MB-468 TNBC xenograft models. To determine the mechanism of αEGFR-E-P125A action on inhibition of VM, we studied the effects of αEGFR-E-P125A on both the transcriptome and proteome. RNA-seq was conducted on MDA-MB-231-4175 TNBC cells plated on matrigel and undergoing VM, and on TNBC cells plated in matrigel and treated with αEGFR-E-P125A. Gene set enrichment analysis demonstrated that αEGFR-E-P125A treatment downregulated genes on the KRAS, JAK-STAT, and angiogenesis signaling pathways, including EGF, VEGFA, STAT3, PTK2, ITGB1, ITGB3, ITGAV, and ITGA5. Phospho-array analysis was used to interrogate the proteome and demonstrated downregulation of phosphorylation on multiple sites downstream of the EGFR and the α5β1 integrin receptors, such as the EGFR Y1069 site, the FAK Y397 site, implicated in the regulation of endothelial and TNBC motility, and the STAT3 Y705 and S727 sites, known for their role in promoting the transcription of angiogenic genes. Since inhibition of EGFR signaling alone does not inhibit VM, we interrogated the mechanistic relationship between αEGFR-E-P125A and VM inhibition through the α5β1 integrin/FAK signaling pathway. Specific inhibition of FAK at the Y397 site using the small molecule inhibitor, PF-573228 inhibited TNBC VM in vitro. Transient siRNA knockdown of FAK in MDA-MB-231-4175 cells and shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM in vitro. To understand the effect of αEGFR-E-P125A on the EGFR and α5β1 integrin receptors, competition assays were conducted. αEGFR-E-P125A competed with EGF for the EGFR receptor and with the binding of fibronectin to α5β1 integrin. Treatment of TNBC cells with αEGFR-E-P125A reduced total α5 integrin protein levels and decreased co-localization of EGFR and α5β1 integrin receptors. These results indicate that αEGFR-E-P125A is bound to both EGFR and α5β1 integrin, simultaneously suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and α5β1integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases. Citation Format: Ankita P. Sankar, Hava Gil Henn, Hyun Mi Cho, Dania Nassar, Sundaram Ramakrishnan, Rathin Das, Yu Zhang, Christian Elledge, Seung-Uon Shin, Joseph Rosenblatt. αEGFR-E-P125A ANTIBODY-ENDOSTATIN FUSION PROTEIN REDUCES VASCULOGENIC MIMICRY AND TUMOR CELL MOTILITY BY INHIBITION OF EGFR, INTEGRIN AND FAK SIGNALING [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-06.
Mantle Cell Lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma that frequently metastasizes. Lymphoma dissemination into distant sites is the primary cause of cancer mortality. Lymphoma metastasis requires development of supporting vasculature in which the interaction between malignant B cells and the vasculature or lymphatics is critical to tumor cell growth and dissemination. Human umbilical vein endothelial cells (HUVEC) or human lymphatic endothelial cells (HLEC) cultured on Matrigel, in vitro, assemble into networks of capillary like structures (CLS) or lymphatic like structures (LLS). When Mino or Jeko-1 MCL cells are co-cultured with vascular or lymphatic cells they migrate toward and anneal to nascent CLS or LLS. We constructed a novel antibody fusion protein, αCD20-IgG1-huEndoP125A, to inhibit such interactions. αCD20-IgG1-huEndoP125A links an αCD20 human IgG1 targeting domain and a mutant version of human endostatin, huEndoP125A, with enhanced anti-angiogenic properties. αCD20-IgG1-huEndoP125A completely inhibits CLS or LLS formation and prevents the alignment and annealing of MCL to CLS or LLS. αCD20-IgG1-huEndoP125A also significantly reduces both endothelial and MCL migration and invasion in vitro. Cell migration and trafficking is tightly regulated by chemokine interactions with corresponding chemokine receptors. We assayed for the presence of chemokines using a dot blot chemokine array. Jeko-1 and HUVEC cells were cultured on Matrigel, alone or together in co-culture, and either treated with αCD20-IgG1-huEndoP125A or left untreated. CXCL12 was expressed at high levels in HUVEC cultures undergoing CLS formation and expression was further increased in HUVEC and MCL co-cultures. HUVEC or HUVEC/MCL co-cultures treated with αCD20-IgG1-huEndoP125A showed markedly decreased CXCL12 expression. Flow cytometry showed that corresponding chemokine receptor CXCR4 was upregulated on MCL cells compared to normal B cells. CXCR4 was also upregulated on HUVEC undergoing CLS formation, and further increased on HUVEC in co-culture with MCL cells. αCD20-IgG1-huEndoP125A inhibited HUVEC CLS formation, reduced MCL migration to and alignment with nascent CLS or LLS, downregulated CXCL12 levels in HUVEC or coculture supernatants, and reduced CXCR4 expression on both HUVEC and MCL cells. Murine 38c13-huCD20 lymphoma cells were implanted s.c. into C3H mice and mice were treated i.p. with either PBS, Rituximab, or equimolar αCD20-IgG1-huEndoP125A. Mice treated with αCD20-IgG1-huEndoP125A showed significantly reduced tumor growth compared to mice treated with PBS or rituximab. αCD20-IgG1-huEndoP125A ability to inhibit angiogenesis, lymphangiogenesis, and lymphoma association with angiogenic or lymphatic vasculature may be a potent means of preventing lymphoma growth and metastasis. Citation Format: Christian Elledge, Seung-Uon Shin, Rathin Das, Yu Zhang, Hyun-Mi Cho, Sundaram Ramakrishnan, Ankita Sankar, Joseph Rosenblatt. Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2654.
Dating abuse (DA) victimization, alcohol use, and marijuana use are some of the most prevalent public health concerns among adolescents, particularly among Latinx adolescents. DA victimization was a robust predictor of subsequent alcohol and marijuana use among white and Black/African American adolescents, but existing longitudinal studies have not examined these relations among Latinx adolescents. The present study examined whether Latinx adolescents’ psychological and physical DA victimization predicted their alcohol and marijuana use one year later. We also explored sex differences in these relationships across time. A school-based sample of 315 Latinx adolescents (M age = 15.06 years; 52.6% girls) completed baseline, self-report assessments (Time 1 [T1]) and 89.6% of the sample completed follow-up assessments one year later (Time 2 [T2]). Results of a path analysis revealed that paths did not vary by sex. T1 psychological DA victimization predicted T2 alcohol use, but not T2 marijuana use. T1 physical DA victimization did not predict T2 alcohol or marijuana use; however, T1 marijuana use predicted T2 physical DA victimization. Latinx adolescents’ DA victimization may have important implications for subsequent substance use. Targeting alcohol use, marijuana use, and DA in culturally-grounded intervention programs may reduce Latinx adolescents’ continued victimization and substance misuse.
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