FOXC1 Activates Smoothened-Independent Hedgehog Signaling in Basal-like Breast Cancer

Abstract

Summary The mesoderm- and epithelial-mesenchymal transition-associated transcription factor FOXC1 is specifically overexpressed in basal-like breast cancer (BLBC), but its biochemical function is not understood. Here we demonstrate that FOXC1 controls cancer stem cell (CSC) properties enriched in BLBC cells via activation of Smoothened (SMO)-independent Hedgehog (Hh) signaling. This non-canonical activation of Hh is specifically mediated by Gli2. We further show that the N-terminal domain of FOXC1 (aa 1–68) binds directly to an internal region (aa 898–1168) of Gli2, enhancing the DNA-binding and transcription-activating capacity of Gli2. FOXC1 expression correlates with that of Gli2 and its targets in human breast cancers. Moreover, FOXC1 overexpression reduces sensitivity to anti-Hedgehog (Hh) inhibitors in BLBC cells and xenograft tumors. Together, these findings reveal FOXC1-mediated non-canonical Hh signaling that determines the BLBC stem-like phenotype and anti-Hh sensitivity, supporting inhibition of FOXC1 pathways as potential approaches for improving BLBC treatment.

FOXC1-induced Gli2 activation: A non-canonical pathway contributing to stemness and anti-Hedgehog resistance in basal-like breast cancer

“…FOXC1 can elicit Hh signaling in 2 BLBC cell lines MDA-MB-231 and HCC1500, neither of which expresses SMO. 6 Furthermore, the FOXC1 effect was not affected by SMO knockdown in SMO-expressing BLBC cells. 6 Consistent with our results, it has been shown that Hh signaling is highly active in breast CSCs that are characterized by the cell-surface glycoprotein CD44 C /CD24 ¡ phenotype.…”

“…6 Furthermore, the FOXC1 effect was not affected by SMO knockdown in SMO-expressing BLBC cells. 6 Consistent with our results, it has been shown that Hh signaling is highly active in breast CSCs that are characterized by the cell-surface glycoprotein CD44 C /CD24 ¡ phenotype. 9 Because FOXC1 stimulates Hh signaling independently of SMO, we postulated that elevated expression of FOXC1 may render cancer cells refractory to SMO targeting inhibitors.…”

“…6 Previous studies have demonstrated that Hh signaling is essential for maintenance of CSC in multiple cancers. In support of this finding in a clinical setting, a positive correlation between FOXC1 levels and the activation of Hh signaling was detected in gene expression analysis of breast tumor samples, and the presence of FOXC1/Hh signaling predicted worse prognosis.…”

“…We recently reported that Forkhead box C1 (FOXC1), a key diagnostic biomarker specific for BLBC, 4,5 increases CSC properties in BLBC cells through a Glioma-Associated Oncogene Family Zinc Finger 2 (GLI2)-mediated, Smoothened (SMO)-independent Hedgehog (Hh) pathway. 6 We initially discovered in our in vitro studies that FOXC1 increases aldehyde dehydrogenase activity and mammosphere formation rate, both of which are indicators of breast CSC. We also found that FOXC1 is essential for tumorigenicity in human breast cancer xenograft models.…”

Tissue-inappropriate derepression of FOXC1 is frequent and functional in human acute myeloid leukemia

“…These cellular effects are dependent on downstream effectors such as MMP7 (matrix metalloproteinase 7) and SNAI1 (Snail family zinc finger 1), or activation of Hedgehog signaling. [7][8][9] Given the expression pattern and essential functional roles of FOXC1 in mesenchymal tissues, and the absence of any functional role for the gene in normal hematopoiesis, 2,5 we were surprised to discover though bioinformatics analyses that FOXC1 was among the most highly upregulated transcription factor genes in primary human acute myeloid leukemia (AML) stem and progenitor cells when compared with normal hematopoietic stem and progenitor cells (HSPCs). Indeed, in an extended analysis of 461 cases of human AML, FOXC1 was expressed at high level in approximately 20% of patient samples, 10 almost invariably in association with concomitant highlevel HOXA/B (Homeobox gene clusters A and B) expression (Fig.…”