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Proper control of Hedgehog (Hh) signaling is critical for hair follicle morphogenesis and ectopic Hh pathway activity is a hallmark of basal cell carcinoma (BCC), the most common type of skin cancer. Mutations in Hh pathway components such as the Hh-binding receptor PATCHED1 (PTCH1) are frequently found in BCC. However, how Hh pathway activation disrupts normal skin homeostasis to promote BCC formation remains poorly understood. Gli2, the major mediator of Hh signaling is essential for hair follicle development and its overexpression in the epidermis induces BCC formation. Despite the importance of Gli2 in the skin, how it is regulated during skin development and tumorigenesis is unclear. Using a genetic approach with loss-of-function mouse mutants and primary keratinocyte cultures, I have uncovered the distinct and overlapping functions of Sufu and Kif7, two evolutionarily conserved regulators of the Hh pathway, during skin development and tumorigenesis. Sufu and Kif7 play opposing roles in Hh signaling through the regulation of Gli2 subcellular distribution, and Kif7 performs distinct Sufu-dependent and –independent functions. In addition, deletion of both Sufu and Kif7 in embryonic skin leads to complete loss of follicular fate and compromised epidermal differentiation. In the adult skin, inactivation of Sufu does not drive BCC formation and requires additional genetic alterations such as the loss of Kif7. Using a Ptc1 mouse model for BCC, I have identified previously unrecognized molecular pathways and cellular events involved in BCC pathogenesis. This includes, aberrant cell cycle progression, loss of cell cycle checkpoint regulation, and suppression of the p53 response. Overall my work provides critical insight into the molecular control of Hh signaling and the downstream events driving BCC formation.

Proper control of Hedgehog (Hh) signaling is critical for hair follicle morphogenesis and ectopic Hh pathway activity is a hallmark of basal cell carcinoma (BCC), the most common type of skin cancer. Mutations in Hh pathway components such as the Hh-binding receptor PATCHED1 (PTCH1) are frequently found in BCC. However, how Hh pathway activation disrupts normal skin homeostasis to promote BCC formation remains poorly understood. Gli2, the major mediator of Hh signaling is essential for hair follicle development and its overexpression in the epidermis induces BCC formation. Despite the importance of Gli2 in the skin, how it is regulated during skin development and tumorigenesis is unclear. Using a genetic approach with loss-of-function mouse mutants and primary keratinocyte cultures, I have uncovered the distinct and overlapping functions of Sufu and Kif7, two evolutionarily conserved regulators of the Hh pathway, during skin development and tumorigenesis. Sufu and Kif7 play opposing roles in Hh signaling through the regulation of Gli2 subcellular distribution, and Kif7 performs distinct Sufu-dependent and –independent functions. In addition, deletion of both Sufu and Kif7 in embryonic skin leads to complete loss of follicular fate and compromised epidermal differentiation. In the adult skin, inactivation of Sufu does not drive BCC formation and requires additional genetic alterations such as the loss of Kif7. Using a Ptc1 mouse model for BCC, I have identified previously unrecognized molecular pathways and cellular events involved in BCC pathogenesis. This includes, aberrant cell cycle progression, loss of cell cycle checkpoint regulation, and suppression of the p53 response. Overall my work provides critical insight into the molecular control of Hh signaling and the downstream events driving BCC formation.

Thesis (M.S.)--Missouri University of Science and Technology, 2009. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 13, 2009) Includes bibliographical references (p. 46).

Infiltrative morphological mimicry at sites of biopsy-proven nodular basal cell carcinoma has been described. The immunoprofile of scar BCCs (scar BCCs,SBCCs) has not been documented. The aim of this study was to assess the histopathological spectrum, stromal (fibronectin, laminin, actin, desmin and vimentin) response and proliferation (bcl-2, MIB1 and p53) status of SBCCs. Twenty nine BCCs occurring in scars, unrelated to previous malignancy (de novo scar BCCS, DN-SBCCs), 27 BCCs that were incompletely excised and regrew at the same site (regrowth scar BCCs, RG-SBCCs) and 25 BCCs that were completely excised with tumour free margins, but recurred at the same site (recurrent scar BCCs, R-SBCCs) were accessed from the files of the Department of Pathology and Plastic and Reconstructive Surgery of the Faculty of Medicine, University of KwaZulu Natal, and formed the basis of this study. The morphological features of DN-SBCCs was pure (3%), predominantly nodular (79%), micronodular (7%) and infiltrative (11 %). RG-SBCCs were predominantly nodular (82%), micronodular (7%) and infiltrative (11%). RSBCCs were predominantly nodular (80%), micronodular (4%) and infiltrative (16%). The majority of DN-SBCCs, RG-SBCCs and R-SBCCs showed intact basement membrane laminin staining, while two (7%) DN-SBCCs showed 1 + and 2+ loss of basement membrane laminin staining. Three (11 %) and two (8%) RG-SBCCs and R-SBCCs,respectively, showed 2+ or 3+ basement membrane laminin discontinuity. The majority of DN-SBCCs (83%), RGSBCCs (75%) and R-SBCCs (88%) were actin negative. No desmin immunopositivity was demonstrated in the epithelial or stromal components of DN-SBCCs, RG-SBCCs and R-SBCCs. All BCC groups showed high 3+ or 4+ vimentin immunopositivity. The majority (>50%) of the SBCCs showed low (2+) bcl-2 immunopositivity. There was no significant difference in p53 immunopositivity in all SBCCs. SBCCs demonstrate phenotypic and immunophenotypic heterogeneity. That DN-SBCCs with the infiltrative and micronodular patterns have not recurred implies that the histomorphology is a pseudo-aggressive pattern. A similar view could pertain to RG-SBCCs, but because the scar did not cicatrise the incompletely excised BCC implies that the histomorphology of RG-BCC may be a potentially more aggressive phenotype. The recurrence of a completely excised basal cell carcinoma may be viewed as a feature of an aggressive tumour, especially when the recurrent BCC contains micronodular and infiltrative components. However, as most R-SBCCs occurred at head and neck sites that are exposed to ultraviolet light, it is also possible that these are simply new BCCs occurring within scars in head and neck sites prone to BCCs. Furthermore, these R-SBCCs were not destructive tumours. CONCLUSION: None of the infiltrative foci of DN-SBCCs demonstrated laminin loss. Three of 5 with intra-epithelial actin immunopositivity also demonstrated low bcl-2 and high p53 staining, immunoprofiling these with an aggressive infiltrative component. Of 11 RG-SBCCs with high p53 staining, 4 had high p53 staining in the infiltrative component, but only one had a low bcl-2 composite score and low bcl-2 score in the infiltrative focus. In addition, these infiltrative foci demonstrated intraepithelial MSA positivity and a "VA" immunophenotype of the stromal cells, indicating one RG-SBCC with an established, aggressive immunophenotype. Those positive with one or more, but not all, aggressive immunostains, are hypothesised to be RG-SBCCs evolving/developing an aggressive immunophenotype. Only one R-SBCC, with a predominantly infiltrative pattern, had a "full-house" of aggressive immunostaining in the infiltrative foci: low bcl-2, high p53, 2+ laminin discontinuity and intra-epithelial and stromal MSA positivity. Of significance is that 7 with a predominant nodular pattern had a high p53 score. Of these, 5 had high bcl-2 scores. Hence, while high p53 may be a feature of aggressive growth, it is important that this staining be complemented with that of bcl-2, laminin and MSA. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2006.

Periocular malignancy represents an increasing burden and currently requires disfiguring surgery in an attempt to cure patients. Basal cell carcinoma (BCC) is the commonest cancer worldwide and morphoeic BCC (mBCC) is an aggressive subtype. Sebaceous gland carcinoma (SGC) is a rare, but life-threatening condition that often requires blinding surgery to prevent mortality, especially in the pagetoid subtype. MBCC has a high risk of local recurrence compared to the more indolent nodular subtype reflected by a different set of driver genes including FLNB and HECTD4. Surrounding mBCC stroma is abnormal, containing mutations in EPHA3 and GLI3. Four common dysregulated pathways detected using both whole exome and RNA sequencing for mBCC were; 'hedgehog (Hh) signalling pathway', 'BCC', 'Natural killer cell mediated cytotoxicity' and 'Fc Epsilon RI signalling pathway'. Hh mutational profile for nodular BCC was not reflected in the RNA and protein expression. In contrast, Hh overexpression is seen in the tumour and stroma of morphoeic tissue with the latter potentially being partly responsible for its aggressive nature and risk of recurrence that may warrant removal to prevent recurrence. SGC has a low overall mutational burden, no UV signature and defective mismatch repair signature. Driver genes included TP53, RB1 and the dynein family is a novel driver possibly involved in chromatid segregation as marked chromosomal instability was demonstrated on copy number analysis. Correlation of whole exome and RNA sequencing data demonstrated upregulated 'cell cycle', 'ubiquitin mediated proteolysis' and 'wnt signalling'. Subtype analysis of pagetoid and nodular SGC revealed the histone gene cluster family as important to both. Oncomir hsa-miR-21 was overexpressed in both and loss of hsa-miR-199a occurs in pagetoid. Increased protein expression of HIST1H2BD was seen in both subtypes as was Hh expression. These novel SGC findings support a chromosomally unstable cancer with the ability to invade extracellular matrix.

Basal-like breast cancer represents a subgroup of mammary cancers associated with a particularly poor prognosis, as they are refractory to current targeted therapies employed for the treatment of breast cancer. In this work I aimed to explore the therapeutic potential of selective estrogen receptor modulators (SERMs), a targeted breast cancer treatment, in combination with epigallocatechin gallate (EGCG), for the treatment of basal-like breast cancer, using MDA-MB-231 cell as an in vitro model of the disease. A significant reduction in MDA-MB-231 cell number and a significant increase in cytotoxicity was observed following treatment with 25 [mu]M of EGCG in combination with 1 [mu]M of 4-hydroxytamoxifen (4-OHT) (EGCG+4-OHT) or 4 [mu]M of raloxifene (EGCG+Ral) over a 36 h time course. However, these effects were not resolved in time, with an increase in G₁-phase cell cycle arrest. Changes in the metabolism of EGCG were dismissed as a possible mechanism through which the combination treatments may be eliciting the cytotoxicity. Changes in the expression and phosphorylation of various signaling proteins, important for the proliferation and survival of basal-like breast cancer, were investigated through Western blotting. Interestingly, the two combination treatments produced very similar results; reductions in the phosphorylation of EGFR and AKT occurred after 6, 12, and 18 h with EGCG+4-OHT and 6, 12, 18 and 24 h with EGCG+Ral, while a reduction in S6K phosphorylation was observed following 6, 12, 18 and 24 h of both combination treatments. Interestingly, both SERMs contributed significantly to the net reduction in S6K phosphorylation, induced by the combination treatments. Both combination treatments were also associated with a significant increase in the phosphorylation and total expression of stress activated protein kinases, p38 and JNK1/2 following 12, 18 and 24 h of treatment. As changes were observed at an intracellular signaling level, the effect of the combination treatments were investigated at the transcriptomic level after 18 h of treatment, using human oligonucleotide microarrays. This transcriptomic analysis revealed that both combination treatments reduced the transcript expression of five enzymes involved with cholesterol synthesis, which was confirmed through qRT-PCR. Cholesterol is an important component of the plasma membrane and is critical for the transduction of extracellular signals. Furthermore, both combination treatments induced the transcriptomic expression of the zinc coordinating metallothionein (MT) proteins. This was associated with an increased nuclear localization of MTF-1, the transcription factor responsible for MT expression, after 6, 12 and 18 h of both combination treatments. Finally, nuclear Western blotting of the NF-[kappa]B subunit, p65, revealed that both combination treatments reduced the nuclear localization of NF-[kappa]B following 6, 12 and 18 h. In collating this data, it appears that the combination treatments of EGCG+4-OHT and EGCG+Ral are inducing cytotoxicity through various mechanisms, including reduced cellular signaling through EGFR, AKT and S6K, increased stress signaling through JNK1/2 and p38 and altered gene expression of MTs and enzymes involved with cholesterol synthesis. Therefore, the combination treatment of EGCG+SERMs exhibits therapeutic potential in MDA-MB-231 cells, a model of basal-like breast cancer.

<p>The human skin provides us with an excellent protective barrier and possesses a remarkable ability of constant renewal. Basal cell carcinoma is the most common type of skin cancer. The aim of this project was to verify results from an earlier study investigating the molecular differences between basal cell carcinoma (BCC) and basal cells of normal human epidermis. In that study microdissection of cell populations from BCC and basal cells of normal epidermis respectively was performed in five cases of confirmed BCC. Following RNA extraction and amplification, a gene expression analysis was performed using a 46 k human cDNA microarray. Comparison of expression profiles showed a differential expression of approximately 300 genes in BCC. An upregulation of signaling pathways previously known to be of importance in BCC development could be observed, as well as a downregulation of differentiation markers, MHC class II molecules, and proteins active in scavenging of oxygen radicals. We wanted to confirm these findings for a number of selected genes, using real time PCR. The focal point of this project was microdissection of cells from BCC and subsequent isolation of RNA. Microdissection based methods offer a possibility of selecting well defined cell populations for further analysis by using a focused laser beam. Initially tests in order to optimize the method were also performed, concerning the dehydration process and choice of slides used in microdissection. Isolation of RNA may, as we experienced, be associated with problems due to destruction of RNA by degrading enzymes.</p>

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Basal cell carcinoma is an indolent, slow-growing tumor that rarely metastasizes. Approximately 70% of tumors occur in the head and neck regions. If a basal cell tumor metastasizes, it usually spreads to the regional lymph nodes first, followed by the lungs. We describe a patient with basal cell carcinoma of the right lower extremity with skin metastases. Skin biopsy of one tumor revealed fibroepithelioma of Pinkus, a rare variant of basal cell carcinoma.