A Novel Role for the NLRC4 Inflammasome in Mucosal Defenses against the Fungal Pathogen Candida albicans

Candida sp. are opportunistic fungal pathogens that colonize the skin and oral cavity and, when overgrown under permissive conditions, cause inflammation and disease. Previously, we identified a central role for the NLRP3 inflammasome in regulating IL-1β production and resistance to dissemination from oral infection with Candida albicans. Here we show that mucosal expression of NLRP3 and NLRC4 is induced by Candida infection, and up-regulation of these molecules is impaired in NLRP3 and NLRC4 deficient mice. Additionally, we reveal a role for the NLRC4 inflammasome in anti-fungal defenses. NLRC4 is important for control of mucosal Candida infection and impacts inflammatory cell recruitment to infected tissues, as well as protects against systemic dissemination of infection. Deficiency in either NLRC4 or NLRP3 results in severely attenuated pro-inflammatory and antimicrobial peptide responses in the oral cavity. Using bone marrow chimeric mouse models, we show that, in contrast to NLRP3 which limits the severity of infection when present in either the hematopoietic or stromal compartments, NLRC4 plays an important role in limiting mucosal candidiasis when functioning at the level of the mucosal stroma. Collectively, these studies reveal the tissue specific roles of the NLRP3 and NLRC4 inflammasome in innate immune responses against mucosal Candida infection

Abstract A58: Expression and function of endothelin converting enzyme 1 and endothelin receptors in breast cancer invasion

Abstract The endothelin axis, comprised of the active peptides endothelin 1-3 (ET1-3), the endothelin A receptor (ETAR) and endothelin B receptor (ETBR), and endothelin converting enzyme 1 (ECE1) has been studied in numerous cancers. In clinical samples of breast cancer, increased levels of ET1, ECE1, and the endothelin receptors have been reported, and several studies correlate expression of these proteins with poor prognosis. The endothelin pathway is known to induce mitogen activated protein kinase activation, leading to increased survival, proliferation, and migration, but the precise role of ECE1 and the endothelin receptors in breast cancer invasion is not fully understood. We hypothesized that the endothelin axis is instrumental in modulating breast cancer cell invasion. To initially confirm that the endothelin axis regulates breast cancer cell invasion, we measured MDA-MB-231 breast cancer cell invasion using an in vitro assay in the presence of an ECE1 inhibitor, CGS 35066, and found that abrogating ECE1 decreases breast cancer invasion. Next, we measured expression of ECE1 mRNA and protein using RT-PCR and Western blots in normal human mammary epithelial cells (HMECs), low-invading MCF-7 breast cancer cells, and highly invasive MDA-MB-231 breast cancer cells. We found that the major ECE1 isoform, ECE-1c, was most highly expressed on MCF-7 cells and that HMECs expressed more of the intracellular ECE1 isoform, ECE-1b, than either cancer cell line. Western blot analysis confirmed these results, as total ECE1 protein expression was highest in the MCF-7 cells. While multiple studies have been published on the expression of the endothelin receptors ETAR and ETBR in clinical breast cancer samples, less is known about the specific expression and function of these receptors in these breast cancer cell lines. Western blot analysis demonstrated notable up-regulation of ETAR expression in the MCF-7 and MDA-MB-231 cells compared to the HMECs, which is consistent with clinical findings. Interestingly, ETBR expression was expressed in HMECs but its expression was low or negative in both breast cancer cell lines. Previous studies have demonstrated that knockdown of ETAR inhibits breast cancer cell invasion, but whether ETBR functions similarly in breast cancer is unknown. To determine if ETBR might be regulating invasion in mammary cells, we transfected MCF7 cells with ETBR-specific siRNA and found that knocking down the gene encoding for ETBR (EDNRB) resulted in significantly increased cell invasion. This result suggests that while ETAR positively regulates breast cancer cell invasion, as reported in other studies, ETBR may be negatively regulating invasion through the endothelin axis. Together, our findings support an important role for the endothelin axis in breast cancer invasion, and specifically implicate ETAR and ETBR as performing complimentary and opposing roles in the regulation of breast cancer cell invasion. Citation Format: Mary Elizabeth Belles, Meena Halaka, Jessica M. Do, Parth Majmudar, Reem Sidani, Hannah Stephen, Molly Watson, Rebecca E. Conway. Expression and function of endothelin converting enzyme 1 and endothelin receptors in breast cancer invasion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A58.</jats:p

KLF4 defines the efficacy of the epidermal growth factor receptor inhibitor, erlotinib, in triple-negative breast cancer cells by repressing the EGFR gene

Abstract Background Triple-negative breast cancer (TNBC) is characterized by high rates of recurrence and poor overall survival. This is due, in part, to a deficiency of targeted therapies, making it essential to identify therapeutically targetable driver pathways of this disease. While epidermal growth factor receptor (EGFR) is expressed in 60% of TNBCs and drives disease progression, attempts to inhibit EGFR in unselected TNBC patients have had a marginal impact on outcomes. Hence, we sought to identify the mechanisms that dictate EGFR expression and inhibitor response to provide a path for improving the utility of these drugs. In this regard, the majority of TNBCs express low levels of the transcription factor, Krüppel-like factor 4 (KLF4), while a small subset is associated with high expression. KLF4 and EGFR have also been reported to have opposing actions in TNBC. Thus, we tested whether KLF4 controls the expression of EGFR and cellular response to its pharmacological inhibition. Methods KLF4 was transiently overexpressed in MDA-MB-231 and MDA-MB-468 cells or silenced in MCF10A cells. Migration and invasion were assessed using modified Boyden chamber assays, and proliferation was measured by EdU incorporation. Candidate downstream targets of KLF4, including EGFR, were identified using reverse phase protein arrays of MDA-MB-231 cells following enforced KLF4 expression. The ability of KLF4 to suppress EGFR gene and protein expression and downstream signaling was assessed by RT-PCR and western blot, respectively. ChIP-PCR confirmed KLF4 binding to the EGFR promoter. Response to erlotinib in the context of KLF4 overexpression or silencing was assessed using cell number and dose-response curves. Results We report that KLF4 is a major determinant of EGFR expression and activity in TNBC cells. KLF4 represses transcription of the EGFR gene, leading to reduced levels of total EGFR, its activated/phosphorylated form (pEGFR), and its downstream signaling intermediates. Moreover, KLF4 suppression of EGFR is a necessary intermediary step for KLF4 to inhibit aggressive TNBC phenotypes. Most importantly, KLF4 dictates the sensitivity of TNBC cells to erlotinib, an FDA-approved inhibitor of EGFR. Conclusions KLF4 is a major regulator of the efficacy of EGFR inhibitors in TNBC cells that may underlie the variable effectiveness of such drugs in patients. </jats:sec

S3 from TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis

&lt;p&gt;Supplemental Figure 3&lt;/p&gt;</jats:p

S3 from TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis

&lt;p&gt;Supplemental Figure 3&lt;/p&gt;</jats:p

S8 Legend from TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis

&lt;p&gt;Supplemental Figure 8 Legend&lt;/p&gt;</jats:p

S7 Legend from TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis

&lt;p&gt;Supplemental Figure 7 Legend&lt;/p&gt;</jats:p

S8 from TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis

&lt;p&gt;Supplemental Figure 8&lt;/p&gt;</jats:p

S6 from TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis

&lt;p&gt;Supplemental Figure 6&lt;/p&gt;</jats:p

S2 from TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis

&lt;p&gt;Supplemental Figure 2&lt;/p&gt;</jats:p