VHL Deletion Impairs Mammary Alveologenesis but Is Not Sufficient for Mammary Tumorigenesis

Abstract

Overexpression of hypoxia inducible factor-1 (HIF-1)␣, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1␣ protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1␣ expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1␣ overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl ؊/؊ mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl The tumor suppressor protein von Hippel-Lindau (VHL) provides substrate specificity to a large Elongin B/C, RING-box 1 (RBX1), Cullin2 (CUL2) complex that functions as an E3 ubiquitin ligase to target proteins for polyubiquitinylation and degradation by the 26S proteasome. Under normoxic conditions, VHL mediates the rapid, oxygen-dependent destruction of the hypoxia-inducible factor (HIF)␣ subunits, HIF-1␣ and HIF-2␣.1 In contrast, in response to decreasing oxygen, or as a result of inactivating VHL mutations, HIF-1␣ and HIF-2␣ are stabilized, facilitating interaction with the constitutively expressed aryl hydrocarbon nuclear receptor translocator (ARNT; HIF-1ß) to form the HIF-1 or HIF-2 transcription factor complexes. Microarray profiling has demonstrated that the HIFs stimulate or repress transcription of over 100 genes in breast, renal, endothelial, or embryonic fibroblast cells as part of the hypoxic response.2-4 These targets are implicated in control of a variety of cellular processes, including glucose transport, glycolysis, angiogenesis, cell cycle, stress response, and apoptosis.

Novel Insight into KLF4 Proteolytic Regulation in Estrogen Receptor Signaling and Breast Carcinogenesis

“…Studies from us and other groups (40) have shown that VHL was down-regulated in breast cancer in comparison with normal tissues, although the underlying mechanisms remain unknown. It is noteworthy that overexpression of HIF␣ resulting from VHL knockout was not sufficient for breast carcinogenesis (62), further corroborating the role of HIF-independent function of VHL in tumorigenesis. The VHL-KLF4 circuit, as revealed by our current studies, could at least represent one important mechanism for breast cancer development.…”

“…It would be of great worth to test whether the E2-KLF4 cascade, as revealed by our current study, plays a role in mammary stem cell biology. Surprisingly, VHL deletion has been shown to impair the differentiation of progenitor cells into alveolar epithelium, implicating that VHL has a role in stem cell renewal (62). Importantly, co-deletion of HIF1␣ could not rescue the vhl Ϫ/Ϫ -dependent phenotype, suggesting that additional VHL-regulated genes besides HIF1A function to regulate the regenerative potential of the breast epithelium.…”

HIF targets in bone remodeling and metastatic disease

“…Interestingly, VHL knockout mice have significant impairment of alveologenesis but deletion of VHL is not sufficient to induce mammary tumorigenesis (Seagroves et al, 2010). Normal ductal branching occurs in mammary-specific VHL −/− mice but alveoli in pregnant VHL −/− mice are undifferentiated, altering their secretory function and resulting in failure to lactate.…”

p63, Sharp1, and HIFs: Master Regulators of Metastasis in Triple-Negative Breast Cancer

“…However, despite the overarching relevance of O 2 levels in HIF regulation, this should not be considered the whole story. First, HIF levels remain high in a number of cancer types even if overtly hypoxic areas are missing (15,16); second, although HIFs are aberrantly stabilized in breast cancers, genetic VHL deletion in mouse mammary epithelium does not lead to breast cancer formation, VHL-mutant patients are not prone to develop mammary tumors, and no VHL mutations have been detected in sporadic breast cancers (17). At the same time, HIFs do remain unstable proteins, degraded by the proteasome, even under hypoxic conditions (18).…”