LKB1 is a tumor susceptibility gene for the Peutz-Jeghers cancer syndrome and is a target for mutational inactivation in sporadic human malignancies. LKB1 encodes a serine/threonine kinase that has critical roles in cell growth, polarity and metabolism. A novel and important function of LKB1 is its ability to regulate the phosphorylation of CREB-regulated transcription co-activators (CRTCs) whose aberrant activation is linked with oncogenic activities. However, the roles and mechanisms of LKB1 and CRTC in the pathogenesis of esophageal cancer have not been previously investigated. In this study, we observed altered LKB1-CRTC signaling in a subset of human esophageal cancer cell lines and patient samples. LKB1 negatively regulates esophageal cancer cell migration and invasion in vitro. Mechanistically, we determined that CRTC signaling becomes activated because of LKB1 loss, which results in the transcriptional activation of specific downstream targets including LYPD3, a critical mediator for LKB1 loss-of-function. Our data indicate that de-regulated LKB1-CRTC signaling might represent a crucial mechanism for esophageal cancer progression.

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive breast cancers. TNBC patients have a high risk of recurrence and metastasis, and current treatment options remain limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Emerging evidence suggests that cancer stem-like cells (CSCs) that escape chemo or radiation therapy in TNBC are often Notch-dependent. At the same time, there is evidence that active tumor immunity predicts good response to neo-adjuvant chemotherapy in TNBC. Notch inhibitors, including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC, where they eliminate CSCs resistant to chemotherapy. However, the success of GSIs in clinical trials is limited by their intestinal toxicity and adverse immunological effects. CD4 and CD8 T-cells, necessary to adaptive tumor immunity, require Notch1 for activation. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSI. SS has Gamma Secretase Modifier (GSM) activity. We confirmed that SS inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In contrast, SS did not inhibit Notch expression or cleavage in murine T cells. In C0321 tumors, which recapitulate human mesenchymal TNBC, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors. SS caused an increase in intra-tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD-1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the number of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. We are currently investigating the mechanisms of this anti-tumor activity. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy agents. Repurposing an FDA-approved, safe agent for the treatment of TNBC would be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Hossain F, Ucar D, Majumder S, Xu K, Ran Y, Minter L, Xi Y, Burow M, Golde T, Osborne B, Miele L. Repurposing sulindac sulfide as a notch inhibitor to target cancer stem-like cells in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-22-01.

Triple negative breast cancer (TNBC) is a molecularly heterogeneous, clinically aggressive disease group that is highly prevalent among African-Americans and younger patients. Standard chemo/radio therapy often produces clinical responses, but recurrence and metastasis are unfortunately common. Metastatic disease is generally incurable. Chemo/radiotherapy has been shown to induce EMT and enrich a chemo-resistant cancer stem-like cell (CSC) population in TNBC. CSCs are thought to drive disease recurrence. Notch signaling is critical for maintenance of TNBC CSC. Expression of Notch1 and its ligand Jagged1 are correlated with poor prognosis. Efforts to pharmacologically target Notch with Gamma Secretase Inhibitors (GSIs) have been impaired by the systemic toxicity of the GSIs, and by the fact that Notch1 also plays a key role in anti-tumor adaptive immunity. Therapeutic agents that indirectly and selectively target Notch signaling in breast cancer cells would be a potentially attractive strategy. However, no such agents have been identified to date. We have found that the MAPK5-ERK5 kinase pathway, which contains at least two druggable targets, functions as a master regulator of Notch signaling in TNBC cells. ERK5 knockout TNBC cells have dramatically decreased expression of Notch receptors, ligands and transcriptional targets. In vivo, these cells form barely detectable tumors that do not metastasize and express lower levels of Notch1 and its ligand Jagged1. Using in silico screening, we identified a class of compounds that selectively target MAP2K5 (MEK5) and decrease the phosphorylation of MAPK7 (ERK5). We selected compound SC-181 for further study. Consistent with ERK5KO cells, pharmacological suppression of ERK5 phosphorylation with SC-181 decreased Notch1 and Jagged1 mRNAs and proteins. SC-181 reversed EMT and reduced the CD44hi/CD24lo CSC population in TNBC cells, but had no effect on T-cell proliferation. SC-181 decreased the number and size of mammospheres in a concentration-dependent manner. Overexpression of the Notch1 intracellular domain (N1IC) in ERK5KO cells rescues their phenotype, dramatically increasing the CSC fraction and promoting EMT. Our results suggest that targeting the MEK5-ERK5 pathway is a promising new strategy to selectively modulate Notch signaling in TNBC CSC without compromising tumor immunity. Citation Format: Ucar DA, Matossian MD, Hoang-Barnes VT, Hossain FM, Gupta M, Burks HE, Wright TD, Cavanaugh J, Flaherty P, Burow ME, Miele L. A novel druggable target upstream of Notch: MEK5/ERK5 signaling regulates Jagged-1 and Notch1 expression in triple negative breast cancer stem cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-03-04.