Basal Cell Carcinoma Formation Research Articles

Abstract 7310: Dietary feeding of silibinin effectively protects against UVB-induced basal cell carcinoma

Abstract Exposure to UVB radiation causes various malignancies, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). BCC is the most prevalent skin cancer type worldwide; ~3.6 million cases of BCC are diagnosed annually in the USA. Silibinin (SB) is a naturally occurring flavonolignan that is present in milk thistle seeds. It has been shown to effectively reduce BCC occurrence upon UVB radiation exposure, in both pre- and post-treatment studies, when applied topically. However, SB efficacy, when fed in diet, against UVB-induced BCC formation and growth has not been reported. We addressed this question employing Ptch+/- mice, where animals were divided into three groups. Group 1: control group, mice were fed adjusted vitamin diet (AIN-76A; TD94096) with no UVB exposure. Group 2: UVB group, mice were fed pellet control diet (semi-purified, modified AIN-76A) and exposed to UVB (240 mJ/cm2) 3 times/week (Mon, Wed, Fri). Group 3: SB group, mice were fed 1% SB (w/w)-supplemented diet and exposed to UVB similar to that in Group 2. This protocol was followed for 26 weeks; thereafter, animals were sacrificed at 14 weeks and 26 weeks post-UVB initiation, and skin samples were harvested and analyzed for BCC formation. β-galactosidase staining showed the development of BCC lesions at 26 weeks in the UVB group; however, SB-fed group had both lesser number of BCC lesions and decreased area covered by BCCs as compared to the UVB group. Non-UVB exposed control group had no BCC lesions. To address if SB feeding starts exerting its efficacy earlier than 26 weeks, we selected the early efficacy timepoint for transcriptomics analysis to determine the mechanism of SB efficacy. Skin RNA samples from the 14-week time point (n=4/group; RIN≥7; DV200>85%) were subjected to library preparation (550 ng of total RNA; Zymo-Seq RiboFree Total RNA Library Kit), ribosomal RNA-depletion RNA sequencing (80 million paired-end reads/sample), and data processing. About 16,000 unique genes were identified, of which ~65% were protein-coding genes. The samples within each group were highly correlated (mean>0.99; Pearson’s correlation). The UVB cluster was sequestered away from the other groups, indicating that the SB diet effectively mitigated the effects of UVB exposure, even at the transcriptome level. Additionally, gene ontology and pathway enrichment analysis (Ingenuity Pathway Analysis, Qiagen) of differentially expressed genes (p<0.05; fold change≤2) showed UVB-induced aberration and SB-associated enrichment in mitotic prometaphase, metaphase, and anaphase pathways, RHO GTPase signaling, and cell cycle regulation; the top functional network found to be dysregulated was lipid metabolism. Taken together, these results highlight the potential of dietary SB intake (apart from topical SB) to be an effective modality against UVB-induced BCC formation and growth and identify molecular targets that are plausibly involved in SB efficacy. Citation Format: Sandeep Paudel, Neha Mishra, Komal Raina, Chapla Agarwal, Rajesh Agarwal. Dietary feeding of silibinin effectively protects against UVB-induced basal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7310.

Skin Cancer

ABSTRACT Basal cell nevus syndrome is a genetically linked multisystem disorder with a hallmark tendency for development of multiple basal cell carcinomas at a young age. It is associated with a multitude of other anomalies including keratocytes of the jaw, palmar or plantar pits, and ectopic intracranial calcifications. This disease is most commonly caused by loss of function in tumor suppressor gene PTCH1 resulting in overactivation of the Hedgehog pathway and basal cell carcinoma formation. Diagnosis is largely clinical; patients must meet criteria of both major and minor categories. Genetic testing is warranted in specific situations where clinical diagnosis is unclear or for genetic counseling purposes. Given that basal cell carcinoma is a very common dermatologic disorder, it is important to recognize when it arises in the setting of genetically associated diseases. Early detection allows for proper management and surveillance of not only basal cell carcinomas but also the other multisystem effects of basal cell nevus syndrome. This review gives an in-depth overview of the etiology, pathogenesis, diagnosis, and management of basal cell nevus syndrome.

468 Preferentially Expressed Antigen in Melanoma Modulates Sensitivity to Retinoic Acid and Cell Cycle Control in Immortalized and Malignant Keratinocytes

Retinoids can be used to treat and prevent the formation of basal cell carcinoma (BCC) and squamous cell carcinomas (SCCs; notably cutaneous and oral SCC). Preferentially Expressed Antigen in Melanoma (PRAME), a gamete-specific repressor of retinoid-induced differentiation, is ectopically expressed in BCC and SCC tumors. However, the effects of PRAME on the development of these cancers and their sensitivity to retinoid therapies are unknown. The purpose of this study was to investigate PRAME’s influence on retinoid response and proliferation in benign human keratinocytes, BCC and SCC cells.

Abstract 716: Silibinin: A novel potential therapeutic agent against UVB-induced basal cell carcinoma

Abstract Herein, we report silibinin (a natural flavonolignan from milk thistle seeds) efficacy against basal cell carcinoma (BCC), the major non-melanoma skin cancer. To determine the preventive/therapeutic activity of silibinin on the progression of UVB-induced microscopic BCC lesions to more advanced BCC, both male and female 8 weeks old Ptch1+/- mice were irradiated with 240 mJ/cm2 UVB dose 3 times/week (M, W, F) for 26 weeks, and thereafter, UVB irradiation was stopped. At this point (after initial UVB exposure for 26 weeks was stopped), these Ptch1+/- mice were randomized into 3 groups: Baseline group (mice euthanized for baseline data); Vehicle group: mice treated topically with acetone for 20 more weeks, and Silibinin group: mice treated topically with silibinin (9 mg in 200 µL acetone), once a day for five days/week for 20 more weeks i.e., till 54 weeks of mice age. Assessment of BCC and non-BCC lesion pathology was performed following β-galactosidase and H&E based histopathological analysis. Results indicated that compared to BCC-associated pathologies observed at baseline, acetone exposure for another 20 weeks resulted in a significant increase in the number (~2 folds, P<0.001) and area (~3.4 folds, P<0.001) covered by BCC lesions. There was also a significant increase in epidermal dysplasia, fibrosarcoma, and squamous cell carcinoma incidence. Notably, topical application of silibinin during this phase significantly decreased BCC numbers as well as area covered by BCC lesions by ~76% (P<0.001) and ~88% (P<0.001), respectively. This protective effect of silibinin was associated with decreased proliferation of basal cells and decreased expression of Hh signaling molecules (Smo and Gli1). To further delineate the changes associated with silibinin’ s protective effect at the transcriptomic level, RNA sequencing studies were performed in skin samples from all three groups as well as non-UVB exposed control mice. Clustering of mRNA profile by sparse Partial Least Squares - Discriminant Analysis (sPLS-DA) showed that UVB exposed tissues (not treated with silibinin) were significantly different from non-UVB controls; most notably, silibinin treatment (after UVB exposure) reversed this phenomenon and the transcriptomic profile of silibinin group was almost similar to non-UVB controls. This indicated that topical silibinin was not only able to protect against progression to advanced BCC but had the potential to even normalize the aberrant gene expression driving BCC formation. In addition, Gene ontology enrichment analysis as well as Pathway enrichment analysis of differentially expressed genes showed silibinin-associated enrichment in the calcium and CX3CR1-mediated signaling pathway, and TGF-β-mediated regulation of the extracellular matrix. Taken together, these results highlight the potential of silibinin to be an effective preventive and/or therapeutic modality against BCC growth and progression. Citation Format: Komal Raina, Sandeep Paudel, Neha Mishra, Sushil Kumar, David J. Orlicky, Zhiying You, Rama Kant, Chapla Agarwal, Rajesh Agarwal. Silibinin: A novel potential therapeutic agent against UVB-induced basal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 716.

Biochemical pathways and targeted therapies in basal cell carcinoma: A systematic review

<p>Basal cell carcinoma (BCC) is the most common type of human malignancy. It is a slow-growing skin cancer with little ability to metastasize, but it is aggressive and can cause local tissue destruction. Descriptions of Basal Cell Nevus Syndrome (BCNS), characterized by a predisposition to the formation of BCC and other neoplasms, and identification of the genetic defect in this syndrome, has led to significant advancement in our understanding of the pathogenesis of BCC. Unregulated expression of target genes in the sonic Hedgehog (SHH) signaling pathway plays a prominent role in the pathogenesis of BCC. An understanding of the signaling components has allowed for the development of pharmacologic agents that inhibit the SHH pathway. The first inhibitor of the SHH pathway approved by the Food and Drug Administration (FDA) for the treatment of BCC is vismodegib. In this review, we will discuss the biochemical pathways involved in BCC as targets of novel pharmacologic therapies.</p>

Spreading of Isolated Ptch Mutant Basal Cell Carcinoma Precursors Is Physiologically Suppressed and Counteracts Tumor Formation in Mice.

Basal cell carcinoma (BCC) originate from Hedgehog/Patched signaling-activated epidermal stem cells. However, the chemically induced tumorigenesis of mice with a CD4Cre-mediated biallelic loss of the Hedgehog signaling repressor Patched also induces BCC formation. Here, we identified the cellular origin of CD4Cre-targeted BCC progenitors as rare Keratin 5+ epidermal cells and show that wildtype Patched offspring of these cells spread over the hair follicle/skin complex with increasing mouse age. Intriguingly, Patched mutant counterparts are undetectable in age-matched untreated skin but are getting traceable upon applying the chemical tumorigenesis protocol. Together, our data show that biallelic Patched depletion in rare Keratin 5+ epidermal cells is not sufficient to drive BCC development, because the spread of these cells is physiologically suppressed. However, bypassing the repression of Patched mutant cells, e.g., by exogenous stimuli, leads to an accumulation of BCC precursor cells and, finally, to tumor development.

Mechano‐vibrational spectroscopy of skin: Are changes in collagen and vascular tissue components early signs of basal cell carcinoma formation?

The modulus and resonant frequency of cancer cells and extracellular matrix are increased in both basal cell and squamous cell carcinomas, and in addition, the collagen stiffness is increased. The organization of the extracellular matrix surrounding cancer cells is clearly different than the extracellular matrix that is seen in normal skin. We have used vibrational optical coherence tomography (VOCT) to measure the resonant frequency and stiffness of collagen, vascular, and reorganized fibrous extracellular matrix components. Measurements of vessels and fibrotic collagen content made on basal cell carcinomas (BCCs) are compared to similar measurements made on normal skin from different anatomical locations. Lesions with basal cell carcinomas exhibit characteristic resonant frequencies and moduli of reorganized extracellular matrix characteristic of fibrotic tissue. Cancerous lesions are characterized by dermal collagen (100Hz), vascular (150Hz), and fibrotic peaks (200-230Hz). The fibrotic peak is not found in areas of normal skin. Based on this pilot study, it is hypothesized inflammation may promote precancerous lesion formation in the surrounding extracellular matrix. Measurement of the increases in vaculature and fibrotic content may be useful in early detection of BCCs.

Update in the Management of Basal Cell Carcinoma.

Basal cell carcinomas are the most frequent skin cancers in the fair-skinned adult population over 50 years of age. Their incidence is increasing throughout the world. Ultraviolet (UV) exposure is the major carcinogenic factor. Some genodermatosis can predispose to formation of basal cell carcinomas at an earlier age. Basal cell carcinomas are heterogeneous, from superficial or nodular lesions of good prognosis to very extensive difficult-to-treat lesions that must be discussed in multidisciplinary committees. Recent guidelines have updated the management of basal cell carcinoma. The prognosis is linked to the risk of recurrence of basal cell carcinoma or its local destructive capacity. Characteristic molecular events in these tumours are: (i) activation of the hedgehog pathway, which has allowed the development of hedgehog inhibitors for difficult-to-treat lesions that are not accessible to surgery or radiotherapy; (ii) high mutational burden, which suggests that hedgehog inhibitor refractory tumours could be offered immunotherapy; some trials are ongoing. The standard treatment for most basal cell carcinomas is surgery, as it allows excision margin control and shows a low risk of recurrence. Superficial lesions can be treated by non-surgical methods with significant efficacy.

Evaluation of Serum Protein Carbonyl Levels and Total Antioxidant Capacity in Patients With Basal Cell Carcinoma

Abstract- Basal cell carcinoma (BCC) is one of the most common types of human cancer. Since oxidative stress is believed to be one of the pathogenic mechanisms involved in BCC formation, understanding the probable relation of inflammatory, oxidative factors, such as serum protein carbonyl and total antioxidant capacity, to BCC can help identify other pathogenic aspects of this tumor. Forty cases of BCC and forty cases of healthy controls without BCC or any other systemic disease were included in this study. After 5-ml venous blood samples were obtained from each patient, the separated serum fractions were stored at -70° C until analysis. Serum protein carbonyl level in the BCC group was significantly higher than that in the healthy controls ((5.86±3.25 µM vs. 0.86±0.20 µM, P<0.001). The average serum total antioxidant capacity was 3.41±0.05 mM in the BCC group and 1.15±0.03 mM in the control group (statistically significant at P<001). Oxidative stress might play a role in the pathogenesis of BCC.

Abstract 5088: Silibinin inhibits UVB-induced promotion and progression of microscopic basal cell carcinoma formation via targeting bone morphogenic protein 2

Abstract Non-melanoma skin cancers (NMSCs) account for about half of all malignancies diagnosed annually in the United States. Around 80% of NMSCs are basal cell carcinoma (BCC) and 20% are squamous cell carcinoma (SCC). Whereas the efficacy of several chemopreventive agents has been examined and reported against both BCC and SCC, a majority of these studies have focused on the test agent’s activity in a long-term setting to determine the amount of tumors formed. Notably, the studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well-established patched (Ptch)+/- mouse model of UVB-induced BCC formation, we excised skin samples from UVB exposed mice prior to tumor formation to study the promotion/progression of BCC and to determine the target/s of silibinin, a well-known skin cancer (SCC) chemopreventive agent, in tumor growth inhibition. Ptch+/- and Ptch+/+ mice were irradiated with UVB 240mJ/cm2 3 times per week with or without topically applied silibinin 5 times per week. As early as one month, we found that UVB exposure significantly increased the number of mast cells in Ptch+/- mice by about 48% (P<0.05), which was completely inhibited (to control levels) by silibinin topical treatments. In Ptch+/+ mice, which do not develop BCC tumors, we did not observe any increase in mast cells following UVB exposure, suggesting this could be a specific pathway in the development of BCC. To decipher the molecular mechanism of these findings, we performed a PCR profiler array analysis of several genes involved in signal transduction pathways which showed strong differences between Ptch+/+ and Ptch+/- mice that were unexposed, UVB irradiated, and silibinin treated. Most notably, following UVB exposure for 1 month, in Ptch+/- mice the expression of Bone morphogenetic protein 2 (BMP-2), Hairy/enhancer-of-split related with YRPW motif 1 (Hey1), and Inhibitor of DNA binding 1 (Id1) was significantly upregulated when compared to Ptch+/+ mice. Additional studies focusing on BMP-2 found that silibinin strongly inhibits UVB-induced expression of BMP-2 in Ptch+/- mouse skin. Consistent with these results, we also found that silibinin strongly attenuates UVB-induced BMP-2 expression and DNA damage in Ptch+/- mouse skin ex vivo. Regarding BCC formation, silibinin treatment inhibited UVB-induced microscopic BCC formation in Ptch+/- mice; microscopic tumor number and size were reduced by 73% and 84%, respectively. Together, our results suggest a possible role of BMP-2 in early stages of BCC development and that silibinin plausibly acts through BMP-2 to inhibit microscopic BCC formation. In conclusion, our previous studies in SCC models and current findings in BCC model suggest that silibinin could be a multi-target agent capable of being a chemopreventive agent for both types of NMSCs. Citation Format: Cynthia M. Rigby, Gagan Deep, Anil K. Jain, David J. Orlicky, Chapla Agarwal, Komal Raina, Rajesh Agarwal. Silibinin inhibits UVB-induced promotion and progression of microscopic basal cell carcinoma formation via targeting bone morphogenic protein 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5088.

349 Regulation of epithelial stem cell proliferation and differentiation by the Gαi heterotrimeric G protein

Regulation of cAMP signaling in the skin and other organs plays a central role in the regulation of cell differentiation and tumor formation. Decrease in cAMP signaling as a result of Gαs or protein kinase A (PKA) inactivation causes an expansion of basal progenitor keratinocytes in the mouse skin, resulting in basal cell carcinoma formation (Iglesias-Bartolome et al, Nature Cell Biology, 17:p793, 2015). These observations suggest that the regulation of cAMP levels is potentially involved in epithelial stem cell fate decisions. We hypothesized that activation of Gαi subunit, which opposes Gαs signaling by reducing cAMP production, might participate in this regulatory network. To study Gαi signaling we took advantage of a synthetic biology approach by using a modified human muscarinic receptor coupled to Gαi (hM4Di) which is exclusively activated by the synthetic ligand clozapine N-oxide (CNO). By expressing and activating hM4Di in primary keratinocytes we demonstrated that the activation of Gαi induces cell proliferation and decreases differentiation. Moreover, treatment with CNO in organotypic cultures led to an increase in skin thickness and cell proliferation. This effect was rescued when the cells were treated with the Gαi inhibitor pertussis toxin (PTX). We next validated these results using a tetracycline inducible mouse model. We targeted the expression of hM4Di to the basal/stem cell compartment of the skin and induced its activation by treating with CNO. Analysis of skin histology reveals that Gαi activation results in skin hyperplasia, with expansion of the K5+ basal compartment and increase of the proliferation marker PCNA. Mechanistically, activation of Gαi in keratinocytes leads to increase in ERK and AKT phosphorylation. Our findings indicate that signaling through Gαi and its yet to be identified coupled GPCRs could play a central role in the regulation of keratinocytes differentiation and proliferation.

Silibinin inhibits ultraviolet B radiation-induced mast cells recruitment and bone morphogenetic protein 2 expression in the skin at early stages in Ptch(+/-) mouse model of basal cell carcinoma.

Around 80% of nonmelanoma skin cancers (NMSCs) are basal cell carcinoma (BCC), still studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well-established patched (Ptch)+/- mouse model of ultraviolet B (UVB) radiation-induced BCC formation, we excised skin samples from UVB exposed Ptch+/- and Ptch+/+ mice before tumor formation to study the promotion/progression of BCC and to determine the efficacy and target/s of silibinin, a well-known skin cancer chemopreventive agent. UVB exposure for 1 month increased the number of mast cells in Ptch+/- mice by ~48% (P < 0.05), which was completely inhibited by silibinin. Polymerase chain reaction profiler array analysis of skin samples showed strong molecular differences between Ptch+/+ and Ptch+/- mice which were either unexposed or UVB irradiated+/- silibinin treatment. Most notably, silibinin treatment significant decreased the expression of BMP-2, Bbc3, PUMA, and Ccnd1 in Ptch+/- mice irradiated with silibinin + UVB. Additional studies showed that silibinin targets UVB-induced expression of bone morphogenetic protein 2 (BMP-2) in Ptch+/- mouse skin. Last, our studies found that silibinin strongly attenuates UVB-induced BMP-2 expression and DNA damage in Ptch+/- mouse skin ex vivo only after single UVB exposure. Together, our results suggest a possible role of mast cell recruitment and BMP-2 activation in the early stages of BCC development; these are strongly inhibited by silibinin suggesting its possible chemopreventive efficacy against BCC formation in long-term UVB exposure regimen.

Risk Factors for Basal Cell Carcinoma in Men Younger Than 40 Years: A Case-Control Study.

Basal cell carcinoma (BCC) is the most common malignancy in the United States and is more prevalent in older populations. The aim of this study was to investigate BCC risk factors in male patients younger than 40 years. A consecutive series of male patients with pathology-proven BCC and younger than 40 years at time of diagnosis were retrospectively identified along with matched controls. Phone interviews were conducted using a structured questionnaire, and differences between patients with and without BCC were investigated. A total of 50 patients with BCC and 27 controls were included in this study. Compared with controls, patients with BCC worked outdoor jobs for longer lengths of time (43.2 vs 15.6 months; p = .04), were more likely to have a family history of skin cancer (66% vs 44%; p = .02), and were more likely to use sunscreen heavily after biopsy (p = .02). Patients with multiple BCCs (n = 20) were more likely to have a history of substantial recreational sun exposure (p = .01) than patients with solitary lesions (n = 30). The authors conclude that outdoor sun exposure in patients with underlying genetic susceptibility is the most likely mechanism of BCC formation in young male patients.

Overexpression of Desmoglein 2 in a Mouse Model of Gorlin Syndrome Enhances Spontaneous Basal Cell Carcinoma Formation through STAT3-Mediated Gli1 Expression

Activation of the hedgehog pathway is causative of virtually all sporadic and Gorlin syndrome-related basal cell carcinomas (BCCs), with loss of function of Ptc1 being the most common genomic lesion. Sporadic BCCs also overexpress Dsg2, a desmosomal cadherin normally found in the basal layer. Using a mouse model of Gorlin syndrome (Ptc1+/lacZ mice), we found that overexpressing Dsg2 in the basal layer (K14-Dsg2/Ptc1+/lacZ mice) or the superficial epidermis (Inv-Dsg2/Ptc1+/lacZ mice) resulted in increased spontaneous BCC formation at 3 and 6 months, respectively. The tumors did not show loss of heterozygosity of Ptc1, despite high levels of Gli1 and phosphorylated Stat3. A panel of sporadic human BCCs showed increased staining of both Dsg2 and phosphorylated Stat3 in all nine samples. Overexpression of Dsg2 in ASZ001 cells, a Ptc1-/- BCC cell line, induced Stat3 phosphorylation and further increased Gli1 levels, in both an autocrine and paracrine manner. Three different Stat3 inhibitors reduced viability and Gli1 expression in ASZ001 cells but not in HaCaT cells. Conversely, stimulation of Stat3 in ASZ001 cells with IL-6 increased Gli1 expression. Our results indicate that Dsg2 enhances canonical hedgehog signaling downstream of Ptc1 to promote BCC development through the activation of phosphorylated Stat3 and regulation of Gli1 expression.

Roles of the Hedgehog Signaling Pathway in Epidermal and Hair Follicle Development, Homeostasis, and Cancer

The epidermis is the outermost layer of the skin and provides a protective barrier against environmental insults. It is a rapidly-renewing tissue undergoing constant regeneration, maintained by several types of stem cells. The Hedgehog (HH) signaling pathway is one of the fundamental signaling pathways that contributes to epidermal development, homeostasis, and repair, as well as to hair follicle development and follicle bulge stem cell maintenance. The HH pathway interacts with other signal transduction pathways, including those activated by Wnt, bone morphogenetic protein, platelet-derived growth factor, Notch, and ectodysplasin. Furthermore, aberrant activation of HH signaling is associated with various tumors, including basal cell carcinoma. Therefore, an understanding of the regulatory mechanisms of the HH signaling pathway is important for elucidating fundamental mechanisms underlying both organogenesis and carcinogenesis. In this review, we discuss the role of the HH signaling pathway in the development and homeostasis epidermis and hair follicles, and in basal cell carcinoma formation, providing an update of current knowledge in this field.

Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to hedgehog-mediated osteogenic induction.

Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Causative mutations in several genes associated with the sonic hedgehog (SHH) signaling pathway, including PTCH1, have been identified in Gorlin syndrome patients. However, no definitive genotype—phenotype correlations are evident in these patients, and their clinical presentation varies greatly, often leading to delayed diagnosis and treatment. We generated iPSCs from four unrelated Gorlin syndrome patients with loss-of-function mutations in PTCH1 using the Sendai virus vector (SeVdp(KOSM)302). The patient-derived iPSCs exhibited basic iPSC features, including stem cell marker expression, totipotency, and the ability to form teratomas. GLI1 expression levels were greater in fibroblasts and patient-derived iPSCs than in the corresponding control cells. Patient-derived iPSCs expressed lower basal levels than control iPSCs of the genes encoding the Hh ligands Indian Hedgehog (IHH) and SHH, the Hh acetyltransferase HHAT, Wnt proteins, BMP4, and BMP6. Most of these genes were upregulated in patient-derived iPSCs grown in osteoblast differentiation medium (OBM) and downregulated in control iPSCs cultured in OBM. The expression of GLI1 and GLI2 substantially decreased in both control and patient-derived iPSCs cultured in OBM, whereas GLI3, SHH, and IHH were upregulated in patient-derived iPSCs and downregulated in control iPSCs grown in OBM. Activation of Smoothened by SAG in cells grown in OBM significantly enhanced alkaline phosphatase activity in patient-derived iPSCs compared with control iPSC lines. In summary, patient-derived iPSCs expressed lower basal levels than the control iPSCs of the genes encoding Hh, Wnt, and bone morphogenetic proteins, but their expression of these genes strongly increased under osteogenic conditions. These findings indicate that patient-derived iPSCs are hypersensitive to osteogenic induction. We propose that Hh signaling is constituently active in iPSCs from Gorlin syndrome patients, enhancing their response to osteogenic induction and contributing to disease-associated abnormalities.

From basal cell carcinoma morphogenesis to the alopecia induced by hedgehog inhibitors: connecting the dots.

The deciphering of the hedgehog (Hh) signalling pathway implicated in the tumorigenesis of basal cell carcinoma (BCC) led to the development of targeted drug therapies, the Hh pathway inhibitors (HPIs) vismodegib and sonidegib. In the skin, physiological Hh signalling is activated in growing hair follicles (HFs), where it is required for proliferation of the epithelium of HFs during morphogenesis and for their postnatal growth. The effects of HPI treatment leading to the regression of BCC and the development of alopecia underpin the central role of the Hh pathway in BCC formation, as well as hair cycling. Given the fact that BCC is a follicular-driven tumour, it is a fine tuning of events that regulate hair cycling that may drive towards the formation of benign follicular hamartomas or malignant BCC neoplasms. Wnt/β-catenin signalling interacts with the Hh signalling during HF morphogenesis, normal hair cycling and BCC development. The aim of this review is to present how key molecular events implicated in Hh pathway crosstalk in the HF are also involved in BCC pathogenesis and result in the alopecia developed by HPI treatment.

INTU is essential for oncogenic Hh signaling through regulating primary cilia formation in basal cell carcinoma.

Inturned (INTU), a cilia and planar polarity effector (CPLANE), performs prominent ciliogenic functions during morphogenesis, such as in the skin. INTU is expressed in adult tissues but its role in tissue maintenance is unknown. Here, we report that the expression of the INTU gene is aberrantly elevated in human basal cell carcinoma (BCC), coinciding with increased primary cilia formation and activated hedgehog (Hh) signaling. Disrupting Intu in an oncogenic mutant Smo (SmoM2)-driven BCC mouse model prevented the formation of BCC through suppressing primary cilia formation and Hh signaling, suggesting that Intu performs a permissive role during BCC formation. INTU is essential for IFT-A complex assembly during ciliogenesis. To further determine whether Intu is directly involved in the activation of Hh signaling downstream of ciliogenesis, we examined the Hh signaling pathway in mouse embryonic fibroblasts, which readily respond to Hh pathway activation. Depleting Intu blocked SAG-induced Hh pathway activation, whereas the expression of Gli2ΔN, a constitutively active Gli2, restored Hh pathway activation in Intu-deficient cells, suggesting that INTU functions upstream of Gli2 activation. In contrast, overexpressing Intu did not promote ciliogenesis or Hh signaling. Taken together, data obtained from this study suggest that INTU is indispensable during BCC tumorigenesis and that its aberrant upregulation is likely a prerequisite for primary cilia formation during Hh-dependent tumorigenesis.

Tumor-Derived Suppressor of Fused Mutations Reveal Hedgehog Pathway Interactions.

The Hedgehog pathway is a potent regulator of cellular growth and plays a central role in the development of many cancers including basal cell carcinoma (BCC). The majority of BCCs arise from mutations in the Patched receptor resulting in constitutive activation of the Hedgehog pathway. Secondary driver mutations promote BCC oncogenesis and occur frequently due to the high mutational burden resulting from sun exposure of the skin. Here, we uncover novel secondary mutations in Suppressor of Fused (SUFU), the major negative regulator of the Hedgehog pathway. SUFU normally binds to a Hedgehog transcriptional activator, GLI1, in order to prevent it from initiating transcription of Hedgehog target genes. We sequenced tumor-normal pairs from patients with early sporadic BCCs. This resulted in the discovery of nine mutations in SUFU, which were functionally investigated to determine whether they help drive BCC formation. Our results show that four of the SUFU mutations inappropriately activate the Hedgehog pathway, suggesting they may act as driver mutations for BCC development. Indeed, all four of the loss of function SUFU variants were found to disrupt its binding to GLI, leading to constitutive pathway activation. Our results from functional characterization of these mutations shed light on SUFU’s role in Hedgehog signaling, tumor progression, and highlight a way in which BCCs can arise.

Biochemical pathways and targeted therapies in basal cell carcinoma: A systematic review

&lt;p&gt;Basal cell carcinoma (BCC) is the most common type of human malignancy. It is a slow-growing skin cancer with little ability to metastasize, but it is aggressive and can cause local tissue destruction. Descriptions of Basal Cell Nevus Syndrome (BCNS), characterized by a predisposition to the formation of BCC and other neoplasms, and identification of the genetic defect in this syndrome, has led to significant advancement in our understanding of the pathogenesis of BCC. Unregulated expression of target genes in the sonic Hedgehog (SHH) signaling pathway plays a prominent role in the pathogenesis of BCC. An understanding of the signaling components has allowed for the development of pharmacologic agents that inhibit the SHH pathway. The first inhibitor of the SHH pathway approved by the Food and Drug Administration (FDA) for the treatment of BCC is vismodegib. In this review, we will discuss the biochemical pathways involved in BCC as targets of novel pharmacologic therapies.&lt;/p&gt;