Running Head: SONIC HEDGEHOG PATHWAY MODULATION IN BASAL CELL CARCINOMA

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Sonic Hedgehog Pathway Modulation In Basal Cell Carcinoma Justin M. Janoska The University of Bridgeport

Of all the types of skin malignancies known in the scientific literature, basal cell carcinoma (BCC) is the most prevalent non-melanoma skin cancer in the United States. With nearly 2 million diagnoses per year, it is very susceptible to rapid metastasis [11, 12]. It is most frequently seen in people over 45 years of age and those exposed to extreme UV radiation and sun exposure [12]. Basal cell carcinoma arises from mutations in genes (Patch) PTCH1, PTCH2, (Smoothened) SMO or (Suppressor of Fused) SUFU that code for proteins responsible for stimulating or taking part in the sonic hedgehog pathway (Shh), which ultimately induces unwarranted cell proliferation and differentiation. These mutations are found to be associated with disruptions in the hedgehog signaling; specifically, mutations in PTCH1 and SMO that have been found in patients with basal cell carcinoma [12]. Through manipulation of the sonic hedgehog pathway, there are a number natural substances, including a first and only FDA approved drug, Vismodegib, that can serve as a pathway intervention and inhibit cell proliferation and differentiation, thus subduing basal cell carcinoma metastasis.

The hedgehog pathway regulates cell differentiation, cellular proliferation, tissue polarity and of most importance, carcinogenesis; specifically, basal call carcinoma [9]. The hedgehog pathway is activated during embryonic development for the growth of tissues, limbs and organs through cell differentiation. It is inactive in adults for a majority of the time except for hair follicle, skim, stem cells and taste bud development [4, 11]. There are 3 Hedgehog (Hh) homologues: Desert Hedgehog (DHH), responsible for the development of germline and Schwann cells of the peripheral nervous system; Indian Hedgehog (IHH) responsible for development of long bones and Sonic Hedgehog (SHH), the protein most

prominently expressed and responsible for the brain, spinal cord and axial skeleton development [1].

The mechanism commonly originates from the release of hedgehog ligands from epithelial tumor cells that bind to a receptor protein PTCH at the cell membrane where it undergoes endocytosis. Normally, the PTCH1 gene functions as a tumor suppressor by inhibiting SMO. If no hog ligand is present (Figure 1), PTCH remains located at the primary cilia and prevents SMO from reaching the membrane; hence, SMO remains inactive [12].

Figure 1 [12]

However, when SMO is activated by the binding effect of the PTCH-Hedgehog complex (Figure 2), SMO translocates to the membrane of the cilium where a number of proteins such as SUFU are stimulated. When SUFU receives the signal from SMO, SUFU no longer prevents the release of transcription factors (GLI family zinc finger 1) GLI1 and GLI2; consequently, allowing for GLI to transition to the nucleus and regulate DNA transcription [12]. The target genes include GLI1, PTCH1, PTCH2, MYCN and specifically, upregulation of Cyclin D, Cyclin E and FOXM1. These genes then induce cellular proliferation as well as angiogenesis [7, 13]. The target gene expression results from direct binding at the gene promoter site (5TGGGTGGTC-3). Moreover, activation of the PDGFRa gene as seen in BCC yields cellular proliferation. SMO has the potential to function as an oncogene and induce unrepressed cell proliferation if it undergoes a mutation by becoming resistant to the PTCH protein [9].

Figure 2 [12]

Basal cell carcinoma is the most common type of skin cancer that accounts for 1/3 of all cancers in the United States [1]. As mentioned earlier, the pathogenesis of BCC arises from hedgehog ligand independent mechanisms stemming from mutations in hedgehog genes. This abnormal pathway activation is believed to transform stem cells into cancer stem cells [4]. Hedgehog pathway activity is present in at least 90% of BCC with at least one mutated allele of PTCH1 [5]. The PTCH1 gene that expresses the PTCH1 receptor protein is the most common mutated gene, which encourages unrelenting SMO activation and downstream signaling of GLI. PTCH1 mutations are associated with random and hereditary BBC due to UV radiation and is found in nearly 70% of people with BCC without Gorlin syndrome. SMO gene mutations have been discovered in almost 20% of people along with a small percentage who have procured SUFU or PTCH2 mutations. [13].

The p53 gene is a powerful tumor suppressor gene that deters unbridled cell division of damaged cells and stimulates cellular death via apoptosis. P53 gene mutations have been found in 44100% of BCC patients as well as discovery of mutations in Cyclin-dependent kinase inhibitor 2A [11, 13]. Mouse models have demonstrated that these mutations conduct uncontrolled hedgehog pathway signaling leading to BCC. Mice with PTCH1 mutations develop BCC when exposed to carcinogens with at least 2 tumors per mouse. Studies have demonstrated BCC development in knock-in mouse models showing SMO activation and GLI2 expression [5]. There is also confirmation that PTCH1 induced tumors also are correlated with spina bifida occulta [9]. Arsenic has been indicated to prompt BCC via p53 methylation b y turning the gene off [13]. Mutations in the MC1R gene (production of the pigment melanin), BRM gene, basal

keratinocyte keratin K5 gene, P450 CYP gene (foreign substance detoxifier), glutathione-Stransferase gene (oxidative stress fighter) and the XRCC3 gene (responsible for DNA repair), have all been shown in studies to make one susceptible to spontaneous BCC [13].

There are 3 potential hedgehog models that can engender BCC tumor cells. In Figure 3, model A, the PTCH protein becomes incapable of restraining SMO in place; subsequently, SMO becomes active both due to mutations at their respective genes. In model B, mutated cells release ligands of which the same tumor cell releasing the ligand responds to and activates the hedgehog pathway. In model C, tumor cells still release ligands, but they interact with neighboring connective tissue stromal cells provoking them to secrete IGF-1 and VEGF hormones which ultimately energize tumor cell proliferation [6].

Figure 3 [6]

There are more than 50 substances ranging from natural products to drugs that modulate the hedgehog pathway by directly inhibiting Hh molecules, SMO or GLI proteins. Cyclopamine, a naturally found plant alkaloid, has been shown to be effective in interfering with SMO signaling by binding to the SMO membrane, but also as in resisting SMO and PTCH1 gene mutation expressions [13]. In vivo mouse models have demonstrated that Cyclopamine blocks 50% of BCC growth and even brain tumor growth in medulloblastoma through oral administration [9]. In smaller trials, 4 patients with BCC have experienced a suppression of BCC multiplication [13].

Perhaps the most documented and scientifically proven substance to combat BCC is a synthetic Hh antagonist drug called Vismodegib. It is the first (2012) FDA approved SMO inhibitor for basal cell carcinoma and has proven to be decently effective in obstructing SMO receptors and downstream proteins in the pathway, which ultimately prevent further cell proliferation [2]. A 150 mg daily dose of Vismodegib is particularly useful in patients who relapse or are not eligible for surgery. In phase I of clinical studies, Vismodegib was tested on 33 patients with BCC and revealed that 18 patients experienced a 50% reduction in tumors. 2 patients responded completely and 16 experienced a partial response. In phase II studies, the drug was reported to induce a 43% response rate in 63 patients with advanced BCC and a 30% response rate in 33 patients with metastatic BCC. 20-40% of the patients who were on the drug exhibited symptoms of dysgeusia, muscle spasms, fatigue, weight loss and hyponatremia, diarrhea, decreased appetite, nausea, vomiting and arthralgia [2, 11, 12].

Figure 4 outlines the sequence of events of the Hh pathway and where along the path Vismodegib intervenes.

Figure 4 [11]

Based on these trials, a daily oral dose of 150 mg for a minimum of 10 months is the current recommendation until the cancer has reached to a severe level of metastasis. Nevertheless, the long-term effectiveness and safety is still equivocal and requires further trials. While Vismodegib looks promising in patients with BCC, it has been documented that some patients with medulloblastoma developed a resistance to the drug. A SMO mutation concomitant with a

PTCH mutation, binding of Vismodegib to SMO was hampered, denoting that more SMO inhibitors to bypass a SMO mutation may justify a reason for further investigation [12]. Additionally, there are other feasible drugs like Saridegib which function much like Vismodegib. GANT-58, GANT-61 and JK 184 function as downstream inhibitors, specifically targeted towards the GLI protein. In patients with sonic hedgehog mutations and SMO inhibitor resistance, GLI inhibitors offer another viable treatment option [11].

From a naturopathic standpoint, there is further evidence that more commonly found natural substances in foods provide therapeutic strategies in inhibiting Hh signaling. Among the list of substances that prevent tumorigenesis through the Hh pathway include vitamin D3, zerumbone, norcantharidin, arsenic trioxide, curcumin, EGCG, resveratrol and genistein [9, 10]. Figure 5 marks where these substances affect proteins within the Hh pathway.

Vitamin D3 can even influence the pathway by directly binding to SMO and inhibiting its activity. The root herb, Zerumbone has the potential to inhibit PTCH1, GLI1 and GLI2 gene transcription, thus stimulating cellular apoptosis. Norcantharidin, a molecule derived from cantharidin excreted by the blister beetle is quite effective in generating programmed cellular death via anoikis and apoptosis as well as precluding angiogenesis and metastasis. While the mechanism of how arsenic trioxide exactly inhibits GLI2 is somewhat vague, studies have indicated that the compound can effectively be used in carcinomas that integrate Hh pathway activation such as with Ewing sarcoma and medulloblastoma. Aside from curcumins strong anti-inflammatory properties, it has the additional advantage of inducing apoptosis and inhibiting sonic hedgehog signaling to the PTCH1 receptor. EGCG, a powerful antitumor polyphenol

found in commonly in green tea has remarkable benefits in inhibiting GLI1 transcription, inducing cellular apoptosis and overall reducing cellular multiplication. Studies also indicate that the synergistic effects of the flavonoid quercetin and EGCG hinders cancer stem cell regeneration [9].

Figure 5 [10]

Another polyphenol resveratrol, has shown to restrict cell proliferation in vitro cancer cells and in animal models. Much like other substances, resveratrol interferes with sonic hedgehog signaling and BCR-ABL expression in myeloid leukemia through IL-6 down-regulation [10].

Lastly, there is the isoflavone genistein, which is abundant in soybeans that has been shown to inhibit tumorigensis. In vitro studies have shown that genistein even prevents prostate cancer stem cell differentiation and proliferation through Gli signaling intervention [9, 10]. Figure 6 illustrates genisteins positive effect on shrinking tumorspheres after 14 days of administration [8]. While most studies have proven GLI1 signaling is frequent in prostate cancer, GLI1 activation still appears to be a common denominator in cancer stem cell tumorigenesis. In the context of prostate cancer, genistein has a unique ability to subdue cancer cell revival [8].

Figure 6 [8]

Irrespective of the fact that basal cell carcinoma is a benign skin cancer, there is an abundance of evidence that the cancer can be reversed, treated and prevented. Based on the evidence in clinical trials on modulating the sonic hedgehog pathway, there are copious amounts of options proven to act as mediators and disrupt cellular differentiation and proliferation; the foundation for malignant tumors. Despite the first FDA approved Shh inhibitor Vismodegib, naturally found substances prevalent in our diet can serve to inhibit tumorigensis such as with the synergistic effect of quercetin and EGCG. This is just a simple testament to the idea that there are extraordinary benefits to making phytonutrients an integral part of ones diet. Many of these compounds interfere with downstream signaling in various ways that indicate a promising outlook in treating and preventing not only basal cell carcinoma, but medulloblastoma and potentially other virulent diseases.

References

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