GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples.

Although not completely understood, the role of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and epithelial skin tumors has been reported before. In this study, we confirmed in various melanoma cell line models that keratin 16 (KRT16) and S100 Calcium-Binding Protein A7 (S100A7) are transcriptional targets of GLI Family Zinc Finger (GLI) proteins. Besides their important role in protecting and maintaining the epidermal barrier, keratins are somehow tightly connected with the S100 family of proteins. We found that stronger expression of KRT16 indeed corresponds to stronger expression of S100A7 in our clinical melanoma samples. We also report a trend regarding staining of GLI1, which corresponds to stronger staining of GLI3, KRT16, and S100A7 proteins. The most interesting of our findings is that all the proteins are detected specifically in the epidermis overlying the tumor, but rarely in the tumor itself. The examined proteins were also not detected in the healthy epidermis at the edges of the sample, suggesting that the staining is specific to the epidermis overlaying the tumor mass. Of all proteins, only S100A7 demonstrated a statistically significant trend regarding tumor staging and staining intensity. Results from our clinical samples prove that immune infiltration is an important feature of melanoma. Pigmentophages and tumor-infiltrating lymphocytes (TIL) demonstrate a significant association with tumor stage, while mononuclear cells are equally present in all stages. For S100A7, we found an association between the number of TILs and staining intensity. Considering these new findings presented in our study, we suggest a more detailed examination of the possible role of the S100A7 protein as a biomarker in melanoma.

The Role of Estrogen and Estrogen Receptors in Head and Neck Tumors.

Head and neck squamous cell carcinoma (HNSCC) is the most common histological form of head and neck tumors (HNTs), which originate from the epithelium of the lips and oral cavity, pharynx, larynx, salivary glands, nasal cavity, and sinuses. The main risk factors include consumption of tobacco in all forms and alcohol, as well as infections with high-risk human papillomaviruses or the Epstein-Barr virus. Regardless of the etiological agent, the risk of developing different types of HNTs is from two to more than six times higher in males than in females. The reason for such disparities probably lies in a combination of both biological and psychosocial factors. Therefore, it is hypothesized that exposure to female sex hormones, primarily estrogen, provides women with protection against the formation and metastasis of HNTs. In this review, we synthesized available knowledge on the role of estrogen and estrogen receptors (ERs) in the development and progression of HNTs, with special emphasis on membrane ERs, which are much less studied. We can summarize that in addition to epidemiologic studies unequivocally pointing to the protective effect of estrogen in women, an increased expression of both nuclear ERs, ERα, and ERß, and membrane ERs, ERα36, GPER1, and NaV1.2, was present in different types of HNSCC, for which anti-estrogens could be used as an effective therapeutic approach.

Signaling Switching from Hedgehog-GLI to MAPK Signaling Potentially Serves as a Compensatory Mechanism in Melanoma Cell Lines Resistant to GANT-61.

BACKGROUND: Melanoma represents the deadliest skin cancer due to its cell plasticity which results in high metastatic potential and chemoresistance. Melanomas frequently develop resistance to targeted therapy; therefore, new combination therapy strategies are required. Non-canonical signaling interactions between HH-GLI and RAS/RAF/ERK signaling were identified as one of the drivers of melanoma pathogenesis. Therefore, we decided to investigate the importance of these non-canonical interactions in chemoresistance, and examine the potential for HH-GLI and RAS/RAF/ERK combined therapy. METHODS: We established two melanoma cell lines resistant to the GLI inhibitor, GANT-61, and characterized their response to other HH-GLI and RAS/RAF/ERK inhibitors. RESULTS: We successfully established two melanoma cell lines resistant to GANT-61. Both cell lines showed HH-GLI signaling downregulation and increased invasive cell properties like migration potential, colony forming capacity, and EMT. However, they differed in MAPK signaling activity, cell cycle regulation, and primary cilia formation, suggesting different potential mechanisms responsible for resistance occurrence. CONCLUSIONS: Our study provides the first ever insights into cell lines resistant to GANT-61 and shows potential mechanisms connected to HH-GLI and MAPK signaling which may represent new hot spots for noncanonical signaling interactions.

'Toxic Masculinity': What Is Known about the Role of Androgen Receptors in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC), the most prevalent cancer in the head and neck region, develops from the mucosal epithelium of the upper aerodigestive tract. Its development directly correlates with alcohol and/or tobacco consumption and infection with human papillomavirus. Interestingly, the relative risk for HNSCC is up to five times higher in males, so it is considered that the endocrine microenvironment is another risk factor. A gender-specific risk for HNSCC suggests either the existence of specific risk factors that affect only males or that females have defensive hormonal and metabolic features. In this review, we summarized the current knowledge about the role of both nuclear and membrane androgen receptors (nAR and mARs, respectively) in HNSCC. As expected, the significance of nAR is much better known; it was shown that increased nAR expression was observed in HNSCC, while treatment with dihydrotestosterone increased proliferation, migration, and invasion of HNSCC cells. For only three out of five currently known mARs-TRPM8, CaV1.2, and OXER1-it was shown either their increased expression in various types of HNSCC or that their increased activity enhanced the migration and invasion of HNSCC cells. The primary treatments for HNSCC are surgery and radiotherapy, but targeted immunotherapies are on the rise. On the other hand, given the evidence of elevated nAR expression in HNSCC, this receptor represents a potential target for antiandrogen therapy. Moreover, there is still plenty of room for further examination of mARs' role in HNSCC diagnosis, prognosis, and treatment.

Higher Expressions of SHH and AR Are Associated with a Positive Receptor Status and Have Impact on Survival in a Cohort of Croatian Breast Cancer Patients.

Breast cancers (BC) are usually classified into four molecular subtypes according to the expression of estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors and proliferation marker Ki-67. Despite available anti-hormonal therapies and due to the inherent propensity of some subtypes to develop metastasis, there is a permanent need to discover new prognostic and predictive biomarkers, as well as therapeutic targets for BC. In this study, we used immunohistochemical staining to determine the expression of androgen receptor (AR) and sonic hedgehog protein (SHH), the main ligand of the Hedgehog-GLI (HH-GLI) signaling pathway, in 185 archival primary BC tissue samples and correlated it with clinicopathological characteristics, molecular subtypes, receptors statuses, and survival in a cohort of Croatian BC patients. Results showed that higher SHH and AR expressions were associated with positive receptor status, but increased SHH expression had a negative impact on survival in receptor-negative BCs. On the contrary, higher AR expression was mostly protective. However, multivariate analysis showed that only higher AR expression could be considered as an independent prognostic biomarker for poorer overall survival in triple-negative breast cancer patients (TNBC) (HR 10.9, 95% CI 1.43-83.67; p = 0.021), what could be Croatian population-related. SHH could be a potential target for treating TNBCs and HER2-enriched BCs, in cases where HH-GLI signaling is canonical (SHH-dependent).

RNA-seq and ChIP-seq Identification of Unique and Overlapping Targets of GLI Transcription Factors in Melanoma Cell Lines.

BACKGROUND: Despite significant progress in therapy, melanoma still has a rising incidence worldwide, and novel treatment strategies are needed. Recently, researchers have recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its consistent crosstalk with the MAPK pathway. In order to further investigate the link between the two pathways and to find new target genes that could be considered for combination therapy, we set out to find transcriptional targets of all three GLI proteins in melanoma. METHODS: We performed RNA sequencing on three melanoma cell lines (CHL-1, A375, and MEL224) with overexpressed GLI1, GLI2, and GLI3 and combined them with the results of ChIP-sequencing on endogenous GLI1, GLI2, and GLI3 proteins. After combining these results, 21 targets were selected for validation by qPCR. RESULTS: RNA-seq revealed a total of 808 differentially expressed genes (DEGs) for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. ChIP-seq identified 527 genes that contained GLI1 binding sites in their promoters, 1103 for GLI2 and 553 for GLI3. A total of 15 of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq. CONCLUSIONS: Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma. We suggest that our findings could provide new potential targets to consider while designing melanoma-targeted therapy.