Kathylynn Saboda

Inhibition of phosphatidylinositol-3-kinase and mitogen-activated protein kinase kinase 1/2 prevents melanoma development and promotes melanoma regression in the transgenic TPRas mouse model

A number of human melanomas show hyperactivation of the Ras pathway due to mutations of the molecule or alteration of upstream or downstream effectors. In this study, we evaluated the effect of blocking the two Ras downstream pathways phosphatidylinositol-3-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal–regulated kinase on melanoma development and regression in the TPRas mouse model. The inhibition of these two signaling cascades by topically applied Ly294002 and U0126 significantly delayed melanoma development and significantly decreased the tumor incidence, particularly when the drugs were applied in combination. Treatment with the inhibitors of established melanomas resulted in complete remission in 33% of mice and partial regression in 46% of mice when drugs were delivered in combination. These responses correlated with increased apoptosis and decreased proliferation both in vitro and in vivo and reduced tumor angiogenesis. In conclusion, this study strongly supports the role of the phosphatidylinositol-3-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal–regulated kinase pathways in the development and maintenance of Ras-dependent melanomas and supports the notion that specific inhibition of these effectors may represent a very promising avenue for the treatment and prevention of the disease. [Mol Cancer Ther 2006;5(12):3071–7]

Investigating Sun-damaged Skin and Actinic Keratosis with Optical Coherence Tomography: A Pilot Study

Actinic Keratosis (AK) arises from sun-damaged skin and is the first clinical manifestation in the multistep process of skin carcinogenesis to invasive squamous cell carcinoma. Thus, it is an ideal target for chemopreventive efforts. Noninvasive measures of AK severity are needed to assess the efficacy of chemoprevention agents. We performed a pilot study on 20 participants to investigate the OCT appearance of sun-protected skin of the upper inner arm as well as sun-damaged skin and early AKs of the dorsal forearms, and to determine if features or quantitative measures in Optical Coherence Tomography (OCT) images could be used to reliably differentiate between these categories. OCT images of upper inner arm (normal appearing skin) showed skin layers and features (stratum corneum, epidermis, dermis, blood vessels) seen in previous studies; additionally in this participant group the subcutaneous fat layer was usually identified. Sun-damaged skin was characterized by increased signal in the epidermis and rapid attenuation of light. AKs were diverse in appearance but frequently characterized by high surface reflection, the presence of a low-signal band in the stratum corneum, and heterogeneous appearance in the epidermis/dermis. Significant differences were found between skin categories using measures of stratum corneum and epidermal/dermal depths and intensities. The presence of a dark band in the stratum corneum was 79% sensitive and 100% specific for AK. This study indicates that OCT holds promise as a useful technique for identifying and characterizing AKs and monitoring their response to chemoprevention agents.

Inhibition of Activator Protein-1 by Sulforaphane Involves Interaction with Cysteine in the cFos DNA-Binding Domain: Implications for Chemoprevention of UVB-Induced Skin Cancer

Sulforaphane is an isothiocyanate derived from cruciferous vegetables that has been linked to decreased risk of certain cancers. Although the role of sulforaphane in the induction of the transcription factor Nrf2 has been studied extensively, there is also evidence that inhibition of the transcription factor activator protein-1 (AP-1) may contribute to the chemopreventive properties of this compound. In this study, we show for the first time that sulforaphane is effective at reducing the multiplicity and tumor burden of UVB-induced squamous cell carcinoma in a mouse model using cotreatment with the compound and the carcinogen. We also show that sulforaphane pretreatment is able to reduce the activity of AP-1 luciferase in the skin of transgenic mice after UVB. Chromatin immunoprecipitation analysis verified that a main constituent of the AP-1 dimer, cFos, is inhibited from binding to the AP-1 DNA binding site by sulforaphane. Electrophoretic mobility shift assay analysis of nuclear proteins also shows that sulforaphane and diamide, both known to react with cysteine amino acids, are effective at inhibiting AP-1 from binding to its response element. Using truncated recombinant cFos and cJun, we show that mutation of critical cysteines in the DNA-binding domain of these proteins (Cys(154) in cFos and Cys(272) in cJun) results in loss of sensitivity to both sulforaphane and diamide in electrophoretic mobility shift assay analysis. Together, these data indicate that inhibition of AP-1 activity may be an important molecular mechanism in chemoprevention of squamous cell carcinoma by sulforaphane.

A Cancer Screening Intervention for Underserved Latina Women by Lay Educators

OBJECTIVES Inadequate screening adherence for breast, cervical, and colorectal cancer among Latinas places them at greater risk for poor survival rates, once diagnosed. The purpose of this study was to examine two delivery methods of lay health educators (promotoras de salud) to increase screening behavior and evaluate costs. METHODS This community-based group randomized trial assigned Latinas due for breast, cervical, or colorectal cancer screening (n=1006) to promotora-taught cancer screening/prevention classes delivered individually (IND) or in social support groups (SSG) over 8 weeks. Screening behaviors were assessed immediately after and 3 and 15 months after intervention. Intervention costs per study arm were compared. RESULTS Screening and maintenance behaviors were not significantly different between SSG and IND for any one type of cancer screening, but with a study entry requirement that participants were either never screened or due for screening, postintervention screening rates (that is, completing a screening that was due) were notable (39.4% and 45.5%, respectively). The cost of achieving any one screening was much higher for IND participants. CONCLUSIONS SSG vs. IND delivery did not significantly affect cancer screening behaviors, but both interventions produced robust achievement of screenings for previously nonadherent participants. Group-based promotora-led interventions supporting social involvement are recommended as a more cost-effective approach to achieving cancer screening among Latina women.

p38 MAP kinase plays a functional role in UVB-induced mouse skin carcinogenesis.

UVB irradiation of epidermal keratinocytes results in the activation of the p38 mitogen‐activated protein kinase (MAPK) pathway and subsequently activator protein‐1 (AP‐1) transcription factor activation and cyclooxygenase‐2 (COX‐2) expression. AP‐1 and COX‐2 have been shown to play functional roles in UVB‐induced mouse skin carcinogenesis. In this study, the experimental approach was to express a dominant negative p38α MAPK (p38DN) in the epidermis of SKH‐1 hairless mice and assess UVB‐induced AP‐1 activation, COX‐2 expression, and the skin carcinogenesis response in these mice compared to wild‐type littermates. We observed a significant inhibition of UVB‐induced AP‐1 activation and COX‐2 expression in p38DN transgenic mice, leading to a significant reduction of UVB‐induced tumor number and growth compared to wild‐type littermates in a chronic UVB skin carcinogenesis model. A potential mechanism for this reduction in tumor number and growth rate is an inhibition of chronic epidermal proliferation, observed as reduced Ki‐67 staining in p38DN mice compared to wild‐type. Although we detected no difference in chronic apoptotic rates between transgenic and nontransgenic mice, analysis of acutely irradiated mice demonstrated that expression of the p38DN transgene significantly inhibited UVB‐induced apoptosis of keratinocytes. These results counter the concerns that inhibition of p38 MAPK in a chronic situation could compromise the ability of the skin to eliminate potentially tumorigenic cells. Our data indicate that p38 MAPK is a good target for pharmacological intervention for UV‐induced skin cancer in patients with sun damaged skin, and suggest that inhibition of p38 signaling reduces skin carcinogenesis by inhibiting COX‐2 expression and proliferation of UVB‐irradiated cells.

Primary Cilia Are Lost in Preinvasive and Invasive Prostate Cancer

Prostate cancer is the second most commonly diagnosed cancer in men worldwide. Little is known about the role of primary cilia in preinvasive and invasive prostate cancer. However, reduced cilia expression has been observed in human cancers including pancreatic cancer, renal cell carcinoma, breast cancer, cholangiocarcinoma, and melanoma. The aim of this study was to characterize primary cilia expression in preinvasive and invasive human prostate cancer, and to investigate the correlation between primary cilia and the Wnt signaling pathway. Human prostate tissues representative of stages of prostate cancer formation (normal prostate, prostatic intraepithelial neoplasia (PIN), and invasive prostate cancer (including perineural invasion)) were stained for ciliary proteins. The frequency of primary cilia was determined. A decrease in the percentage of ciliated cells in PIN, invasive cancer and perineural invasion lesions was observed when compared to normal. Cilia lengths were also measured to indirectly test functionality. Cilia were shorter in PIN, cancer, and perineural invasion lesions, suggesting dysfunction. Primary cilia have been shown to suppress the Wnt pathway. Increased Wnt signaling has been implicated in prostate cancer. Therefore, we investigated a correlation between loss of primary cilia and increased Wnt signaling in normal prostate and in preinvasive and invasive prostate cancer. To investigate Wnt signaling in our cohort, serial tissue sections were stained for β-catenin as a measure of Wnt signaling. Nuclear β-catenin was analyzed and Wnt signaling was found to be higher in un-ciliated cells in the normal prostate, PIN, a subset of invasive cancers, and perineural invasion. Our results suggest that cilia normally function to suppress the Wnt signaling pathway in epithelial cells and that cilia loss may play a role in increased Wnt signaling in some prostate cancers. These results suggest that cilia are dysfunctional in human prostate cancer, and increase Wnt signaling occurs in a subset of cancers.

Expression of vascular endothelial growth factor in early cutaneous melanocytic lesion progression.

A considerable body of evidence supports the concept that a significant number of cutaneous malignant melanomas progress through a precursor lesion or dysplastic melanocytic nevi (DN). Tumor angiogenesis likely plays a critical role in early development of melanoma, and intermediate biomarkers of angiogenesis could be useful as chemoprevention and prognostic markers.

Safety and Efficacy of Dose-Intensive Oral Vitamin A in Subjects with Sun-Damaged Skin

Purpose: Previously, we reported the results of a Phase III, placebo-controlled trial in 2,297 randomized participants with moderately severe actinic keratoses wherein 25,000 IU/day vitamin A caused a 32% risk reduction in squamous cell skin cancers. We hypothesized that dose escalation of vitamin A to 50,000 or 75,000 IU/day would be both safe and more efficacious in skin cancer chemoprevention. Experimental Design: One hundred and twenty-nine participants with severely sun-damaged skin on their lateral forearms were randomized to receive placebo or 25,000, 50,000, or 75,000 IU/day vitamin A for 12 months. The primary study end points were the clinical and laboratory safety of vitamin A, and the secondary end points included quantitative, karyometric image analysis and assessment of retinoid and rexinoid receptors in sun-damaged skin. Results: There were no significant differences in expected clinical and laboratory toxicities between the groups of participants randomized to placebo, 25,000 IU/day, 50,000 IU/day, and 75,000 IU/day. Karyometric features were computed from the basal cell layer of skin biopsies, and a total of 22,600 nuclei from 113 participants were examined, showing statistically significant, dose-response effects for vitamin A at the 25,000 and 50,000 IU/day doses. These karyometric changes correlated with increases in retinoic acid receptor α, retinoic acid receptor β, and retinoid X receptor α at the 50,000 IU/day vitamin A dose. Conclusions: The vitamin A doses of 50,000 and 75,000 IU/day for 1 year proved safe and equally more efficacious than the 25,000 IU/day dose and can be recommended for future skin cancer chemoprevention studies.

A Phase 2a Study of Topical Perillyl Alcohol Cream for Chemoprevention of Skin Cancer

The chemopreventive and antitumor properties of perillyl alcohol (POH) that were studied preclinically indicate that topical POH inhibits both UVB-induced murine skin carcinogenesis (squamous cell tumor models) and 7,12-dimethylbenz(a)anthracene–induced murine melanoma (transgenic models involving tyrosinase-driven Ras). A previous phase 1 clinical trial in participants with normal-appearing skin showed that topical POH cream was well tolerated at a dose of 0.76% (w/w). Here, we performed a 3-month, double-blind, randomized, placebo-controlled phase 2a trial of two different doses of topical POH in individuals with sun-damaged skin. Participants applied POH cream twice daily to each dorsal forearm. Baseline and end-of-study biopsies were taken from each participant to evaluate whether the topical application of POH was effective in reversing actinic damage as evidenced by normalization of quantitative skin histopathologic scores and change in nuclear chromatin pattern as measured by karyometric analysis. There was a borderline reduction in the histopathologic score of the lower-dose POH group compared with the placebo (P = 0.1), but this was not observed in the high-dose group. However, in the high-dose group, a statistically significant reduction in the proportion of nuclei deviating from normal was observed by the use of karyometric analysis (P < 0.01). There was no statistical significance shown in the lower-dose group. No changes were observed in p53 expression, cellular proliferation (by proliferating cell nuclear antigen expression), or apoptosis in either treatment group compared with the placebo group. These results suggest that whereas our karyometric analyses can detect a modest effect of POH in sun-damaged skin, improved delivery into the epidermis may be necessary. Cancer Prev Res; 3(2); 160–9

Cross-validation of Murine UV Signal Transduction Pathways in Human Skin

Acute UVB irradiation of mouse skin results in activation of phospatidyinositol‐3 (PI‐3) kinase and mitogen‐activated protein kinase (MAPK) pathways leading to altered protein phosphorylation and downstream transcription of genes. We determined whether activation of these pathways also occurs in human skin exposed to 4× minimal erythemic dose of UVB in 23 volunteers. Biopsies were taken prior to, at 30 min, 1 and 24 h post‐UVB. In agreement with mouse studies, the earliest UV‐induced changes in epidermis were seen in phospho‐CREB (two‐ and five‐fold at 30 min and 1 h) and in phospho‐MAPKAPK‐2 (three‐fold at both 30 min and 1 h). At 1 h, phospho‐c‐JUN and phospho‐p38 were increased five‐ and two‐fold, respectively. Moreover, phospho‐c‐JUN and phospho‐p38 were further increased at 24 h (12‐ and six‐fold, respectively). Phospho‐GSK‐3β was similarly increased at all time points. Increases in phospho‐p53 (12‐fold), COX‐2 (four‐fold), c‐FOS (14‐fold) and apoptosis were not seen until 24 h. Our data suggest that UVB acts through MAPK p38 and PI‐3 kinase with phosphorylation of MAPKAPK‐2, CREB, c‐JUN, p38, GSK‐3β and p53 leading to marked increases in c‐FOS, COX‐2 and apoptosis. Validation of murine models in human skin will aid in development of effective skin cancer chemoprevention and prevention strategies.