Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia

The hedgehog signaling network regulates pattern formation, proliferation, cell fate and stem/progenitor cell self-renewal in many organs. Altered hedgehog signaling is implicated in 20-25% of all cancers, including breast cancer. We demonstrated previously that heterozygous disruption of the gene encoding the patched-1 (PTCH1) hedgehog receptor, a negative regulator of smoothened (Smo) in the absence of ligand, led to mammary ductal dysplasia in virgin mice. We now show that expression of activated human SMO (SmoM2) under the mouse mammary tumor virus (MMTV) promoter in transgenic mice leads to increased proliferation, altered differentiation, and ductal dysplasias distinct from those caused by Ptch1 heterozygosity. SMO activation also increased the mammosphere-forming efficiency of primary mammary epithelial cells. However, limiting-dilution transplantation showed a decrease in the frequency of regenerative stem cells in MMTV-SmoM2 epithelium relative to wild type, suggesting enhanced mammosphere-forming efficiency was due to increased survival or activity of division-competent cell types under anchorage-independent growth conditions, rather than an increase in the proportion of regenerative stem cells per se. In human clinical samples, altered hedgehog signaling occurs early in breast cancer development, with PTCH1 expression reduced in approximately 50% of ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC). Conversely, SMO is ectopically expressed in 70% of DCIS and 30% of IBC. Surprisingly, in both human tumors and MMTV-SmoM2 mice, SMO rarely colocalized with the Ki67 proliferation marker. Our data suggest that altered hedgehog signaling may contribute to breast cancer development by stimulating proliferation, and by increasing the pool of division-competent cells capable of anchorage-independent growth.     

Hedgehog signaling in skin cancers

An increasing progress on the role of Hedgehog (Hh) signaling for carcinogenesis has been achieved since the link of Hh pathway to human cancer was firstly established. In particular, the critical role of Hh signaling in the development of Basal cell carcinoma (BCC) has been convincingly demonstrated by genetic mutation analyses, mouse models of BCCs, and successful clinical trials of BCCs using Hh signaling inhibitors. In addition, the Hh pathway activity is also reported to be involved in the pathogenesis of Squamous Cell Carcinoma (SCC), melanoma and Merkel Cell Carcinoma. These findings have significant new paradigm on Hh signaling transduction, its mechanisms in skin cancer and even therapeutic approaches for BCC. In this review, we will summarize the major advances in the understanding of Hh signaling transduction, the roles of Hh signaling in skin cancer development, and the current implications of “mechanism-based” therapeutic strategies.     

Liver kinase B1 (LKB1) in the pathogenesis of UVB-induced murine basal cell carcinoma

LKB1, a known tumor suppressor, is mutated in Peutz–Jeghers Syndrome (PJS). It is responsible for the enhanced cancer risk in patients with PJS. Dysregulation of LKB1-dependent signaling also occurs in various epithelial cancers. UVB alters the expression of LKB1, though its role in the pathogenesis of skin cancer is unknown. Here we describe upregulation of LKB1 expression in UVB-induced murine basal cell carcinoma (BCC) and in human skin tumor keratinocytes. AMP-kinase and acetyl Co-A carboxylase, the downstream LKB1 targets, are also enhanced in this neoplasm. In addition, p-Akt, a kinase which inactivates GSK3β by its phosphorylation, is enhanced in BCCs. Consistently, an accumulation of p-GSK3β and an increase in activated nuclear β-catenin are found. mTOR signaling, which is also inhibited by LKB1, remains upregulated in BCCs. However, a marked decrease in the expression of sestrins, which function as potent negative regulators of mTOR is observed. Metformin, a known chemical inducer of this pathway, was found effective in immortalized HaCaT keratinocytes, but failed to activate the LKB1-dependent signaling in human carcinoma A431 cells. Thus, our data show that the LKB1/AMPK axis fails to regulate mTOR pathway, and a complex regulatory mechanism exists for the persistent mTOR activation in murine BCCs.     

A population-based study of hedgehog pathway gene variants in relation to the dual risk of basal cell carcinoma plus another cancer

Introduction: A personal history of basal cell carcinoma (BCC) is associated with increased risk of other malignancies, but the reason is unknown. The hedgehog pathway is critical to the etiology of BCC, and is also believed to contribute to susceptibility to other cancers. This study tested the hypothesis that hedgehog pathway and pathway-related gene variants contribute to the increased risk of subsequent cancers among those with a history of BCC. Methods: The study was nested within the ongoing CLUE II cohort study, established in 1989 in Washington County, Maryland, USA. The study consisted of a cancer-free control group (n=2296) compared to three different groups of cancer cases ascertained through 2007, those diagnosed with: (1) Other (non-BCC) cancer only (n=2349); (2) BCC only (n=534); and (3) BCC plus other cancer (n=446). The frequencies of variant alleles were compared among these four groups for 20 single nucleotide polymorphisms (SNPs) in 6 hedgehog pathway genes (SHH, IHH, PTCH2, SMO, GLI1, SUFU), and also 22 SNPs in VDR and 8 SNPs in FAS, which have cross-talk with the hedgehog pathway. Results: Comparing those with both BCC and other cancer versus those with no cancer, no significant associations were observed for any of the hedgehog pathway SNPs, or for the FAS SNPs. One VDR SNP was nominally significantly associated with the BCC cancer-prone phenotype, rs11574085 [per minor allele odds ratio (OR) 1.38, 95% confidence interval (CI) 1.05-1.82; p-value=0.02]. Conclusion: The hedgehog pathway gene SNPs studied, along with the VDR and FAS SNPs studied, are not strongly associated with the BCC cancer-prone phenotype.     

Selective expression of KrasG12D in granulosa cells of the mouse ovary causes defects in follicle development and ovulation

Activation of the RAS family of small G-proteins is essential for follicle stimulating hormone-induced signaling events and the regulation of target genes in cultured granulosa cells. To analyze the functions of RAS protein in granulosa cells during ovarian follicular development in vivo, we generated conditional knock-in mouse models in which the granulosa cells express a constitutively active KrasG12D. The KrasG12D mutant mice were subfertile and exhibited signs of premature ovarian failure. The mutant ovaries contained numerous abnormal follicle-like structures that were devoid of mitotic and apoptotic cells and cells expressing granulosa cell-specific marker genes. Follicles that proceeded to the antral stage failed to ovulate and expressed reduced levels of ovulation-related genes. The human chorionic gonadotropin-stimulated phosphorylation of ERK1/2 was markedly reduced in mutant cells. Reduced ERK1/2 phosphorylation was due, in part, to increased expression of MKP3, an ERK1/2-specific phosphatase. By contrast, elevated levels of phospho-AKT were evident in granulosa cells of immature KrasG12D mice, even in the absence of hormone treatments, and were associated with the progressive decline of FOXO1 in the abnormal follicle-like structures. Thus, inappropriate activation of KRAS in granulosa cells blocks the granulosa cell differentiation pathway, leading to the persistence of abnormal non-mitotic, non-apoptotic cells rather than tumorigenic cells. Moreover, those follicles that reach the antral stage exhibit impaired responses to hormones, leading to ovulation failure. Transient but not sustained activation of RAS in granulosa cells is therefore crucial for directing normal follicle development and initiating the ovulation process.     

Interference with SMO increases chemotherapy drug sensitivity of A2780/DDP cells by inhibiting the Hh/Gli signaling pathway

Aberrant activation of the Hedgehog (Hh)/Gli pathway contributes to the tumorigenesis of several human cancers, including ovarian cancers. We investigated the function of SMO on cell growth, drug resistance, and invasive ability in A2780/DDP cells. Moreover, we also tested the levels of the downstream target genes of the Hh/Gli pathway in SMO short hairpin RNA (shRNA) lentivirus-infected A2780/DDP cells. Western blot analysis results revealed that the Hh/Gli pathway was activated in cisplatin-resistant A2780/DDP cells. After infection by SMO shRNA lentivirus, the colony formation rate and invasive rate of cisplatin-resistant A2780/DDP cells were decreased. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that upon transfection with SMO shRNA, cell growth was decreased and drug sensitivity to cisplatin was upregulated. Moreover, interference with SMO decreased the expression of MMP-2, MMP-9, VEGF, and Snail in cisplatin-resistant cells. Thus, the Hh/Gli signaling pathway was aberrantly activated in A2780/DDP cells. The colony formation rate and invasive rate were decreased in SMO shRNA lentivirus–infected A2780/DDP cells. All results showed that inhibiting Hh/Gli signaling may negatively regulate the proliferation, invasion, and metastasis of cisplatin-resistant A2780/DDP cells, as well as increase the sensitivity of A2780/DDP to the chemotherapeutic drug of cisplatin.     

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.     

Pathways Sufficient to Induce Epidermal Carcinogenesis

Abnormal epidermal proliferation is characteristic of a number of disorders, including the two most common cancers in the United States, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Both cancers display a disruption in the normal homeostatic balance between cell division and programmed cell death. While abnormal activation of the sonic hedgehog/patched pathway has been established as sufficient to induce hallmark features of BCC in both human and murine epidermis,1-4 pathways sufficient to convert normal epidermis into SCC have been less well defined. Building on findings that indicate a potent role for Ras and NF-?B in normal epidermal growth regulation,5-9 recent work indicates that activation of Ras signaling in concert with inhibition of NF-?B function is entirely sufficient to transform normal human epidermis into tumor tissue with all the cardinal features of SCC.