Magdalena Martinka

Prognostic Significance of Activated Akt Expression in Melanoma: A Clinicopathologic Study of 292 Cases

PURPOSE Akt is a serine/threonine kinase that leads to stimulation of cell cycle progression, cell proliferation, and inhibition of apoptosis. To investigate the role of Akt in melanoma pathogenesis, we examined the expression of phospho-Akt (p-Akt; Ser-473) in melanocytic lesions at different stages and analyzed the correlations between the p-Akt expression level and clinicopathologic factors and patient survival. PATIENTS AND METHODS We evaluated the p-Akt expression in 12 cases of normal nevi, 58 cases of dysplastic nevi, 170 cases of primary melanomas, and 52 cases of melanoma metastases using tissue microarray and immunohistochemistry. RESULTS Strong p-Akt expression was observed in 17%, 43%, 49%, and 77% of the biopsies in normal nevi, dysplastic nevi, primary melanoma, and melanoma metastases, respectively. Significant differences for p-Akt staining pattern were observed between normal nevi and primary melanomas (P < .05), and between primary melanomas and melanoma metastases (P < .001). Furthermore, our Kaplan-Meier survival curves showed that strong p-Akt expression is inversely correlated with both overall and disease-specific 5-year survival of patients with primary melanoma (P < .05 for both). Strikingly, our multivariate Cox regression analysis revealed that p-Akt is an independent prognostic factor in low-risk melanomas (thickness < or = 1.5 mm; relative risk, 6.44; 95% CI, 1.28 to 32.55; P = .018). CONCLUSION The expression of p-Akt increases dramatically with melanoma invasion and progression and is inversely correlated with patient survival. In addition, p-Akt may serve as an independent prognostic marker and help to identify those patients with low-risk melanomas who are at increased risk of death.

Aberrant Expression of Collagen Triple Helix Repeat Containing 1 in Human Solid Cancers

Purpose: The collagen triple helix repeat containing 1 (CTHRC1) is a promigratory protein first found to be expressed during rat tissue repair process. Recent preliminary results revealed CTHRC1 mRNA in melanoma and breast cancer. However, the full significance of CTHRC1 to human carcinogenesis remains unclear. This study is to further characterize the clinical and functional relevance of CTHRC1 in melanoma and other human solid cancers. Experimental Design: First, semiquantitative immunohistochemistry analysis was done on 304 clinically annotated, paraffin-embedded biopsies representing different stages of melanoma progression. Then, short interfering RNA was used to inhibit expression of CTHRC1 protein for migration analysis on cultured melanoma cells. Finally, the CTHRC1 expression was surveyed in 310 samples representing 19 types of human solid cancers. Results: In benign nevi and noninvasive melanoma biopsies, there was little CTHRC1 protein expression. In contrast, in invasive primary melanomas, there was a significant increase of CTHRC1 protein (P < 0.01, χ2 test). There was a further increase of CTHRC1 protein in metastatic melanoma specimens compared with nonmetastatic lesions (P < 0.01, χ2 test). In addition, inhibition of CTHRC1 expression resulted in decreased cell migration in vitro. Finally, transcription survey in 19 types of human solid cancers revealed aberrant CTHRC1 expression in 16 cancer types, especially cancers of the gastrointestinal tract, lung, breast, thyroid, ovarian, cervix, liver, and the pancreas. Conclusions: Aberrant expression of CTHRC1 is widely present in human solid cancers and seems to be associated with cancer tissue invasion and metastasis. It potentially plays important functional roles in cancer progression, perhaps by increasing cancer cell migration.

The histologic spectrum of cutaneous sarcoidosis: a study of twenty‐eight cases

Background:  Naked sarcoidal granulomas (NSGs) are the characteristic histologic finding in sarcoidosis. This descriptive study was designed to identify the frequency of other histologic changes in cutaneous sarcoidosis.

Role of ING4 in human melanoma cell migration, invasion and patient survival

Inhibitor of growth (ING) 4 has been reported as a tumor suppressor and shown to diminish colony-forming efficiency, induce p53-dependent apoptosis and arrest cell cycle at G(2)-M phase. In this study, we investigated the role of ING4 in human melanoma pathogenesis. Using the tissue microarray technology, we found that ING4 expression is significantly decreased in malignant melanoma compared with dysplastic nevi (P < 0.0001, chi(2) test) and reduced ING4 staining is associated with melanoma thickness, ulceration (P = 0.034 and 0.002, respectively, chi(2) test) as well as poor overall and disease-specific 5-year survival of primary melanoma patients (P = 0.0002 and 0.001, respectively, chi(2) test). Cox regression analysis revealed that reduced ING4 staining is an independent factor for the poor prognosis of patients with primary melanomas. Furthermore, we found that overexpression of ING4 suppressed cell migration by 63% and inhibited the activity of Ras homolog gene family member A (RhoA) small GTPase protein and Rho-associated kinase (ROCK)-mediated formation of stress fiber in melanoma cells. Moreover, our data showed that overexpression of ING4 inhibited melanoma cell invasion by 43% compared with the control (P = 0.006, t-test) and ING4-overexpressing melanoma cells showed significantly reduced activity of matrix metalloproteinase (MMP)-2 and MMP-9. Taken together, this study highlights the importance of ING4 in melanoma pathogenesis and ING4 may serve as a promising prognostic marker and a potential therapeutic target for human melanoma.

Prognostic Significance of Nuclear ING3 Expression in Human Cutaneous Melanoma

Purpose: The novel tumor-suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. Our previous study showed that ING3 promotes UV-induced apoptosis via the Fas/caspase-8–dependent pathway in melanoma cells. To investigate the putative role of ING3 in the development of melanoma, we examined the expression of ING3 in melanocytic lesions at different stages and analyzed the correlation between ING3 expression and clinicopathologic variables and patient survival. Experimental Design: Using tissue microarray and immunohistochemistry, we evaluated nuclear and cytoplasmic ING3 staining in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas. Results: Nuclear ING3 expression was remarkably reduced in malignant melanomas compared with dysplastic nevi (P < 0.001), which was significantly correlated with the increased ING3 level in cytoplasm (P < 0.05). Furthermore, the reduced nuclear ING3 expression was significantly correlated with a poorer disease-specific 5-year survival of patients with primary melanoma, especially for the high-risk melanomas (thickness ≥2.0 mm) with the survival rate reducing from 93% for patients with strong nuclear ING3 staining in their tumor biopsies to 44% for those with negative-to-moderate nuclear ING3 staining (P = 0.004). Strikingly, our multivariate Cox regression analysis revealed that reduced nuclear ING3 expression is an independent prognostic factor to predict patient outcome in primary melanomas (P = 0.038). Conclusions: Our data indicate that ING3 may be an important marker for human melanoma progression and prognosis as well as a potential therapeutic target.

Cutaneous GVHD is associated with the expansion of tissue-localized Th1 and not Th17 cells

Studies in mice have shown that proinflammatory Th17 cells can cause acute graft-versus-host disease (aGVHD) related tissue damage; however, whether they play a role in human aGVHD remains unclear. In a prospective study, we measured the proportion of Th17 cells in the blood and skin of patients at the onset of aGVHD. We found no difference in the proportion or amount of IL-17 produced by T cells in the blood of patients with aGVHD (n = 20) compared with time-matched patients without GVHD (n = 14). Moreover, Th17 cells were not increased in the skin of patients with cutaneous aGVHD (n = 7) compared with healthy controls (n = 10). In contrast, we found significantly more interferon-γ-producing T cells in the skin of patients with aGVHD compared with controls. These data support the long-standing paradigm that tissue localized interferon-γ-producing cells are the perpetrators of aGVHD.

Reduced Apaf-1 expression in human cutaneous melanomas

Malignant melanoma is a life-threatening skin cancer due to its highly metastatic character and resistance to radio- and chemotherapy. It is believed that the ability to evade apoptosis is the key mechanism for the rapid growth of cancer cells. However, the exact mechanism for failure in the apoptotic pathway in melanoma cells is unclear. p53, the most frequently mutated tumour suppressor gene in human cancers, is a key apoptosis inducer. However, p53 mutation is only found in 15–20% of melanoma biopsies. Recently, it was found that Apaf-1, a downstream target of p53, is inactivated in metastatic melanoma. Specifically, loss of heterozygosity (LOH) of the Apaf-1 gene was found in 40% of metastatic melanoma. To determine if loss of Apaf-1 expression is indeed involved in melanoma progression, we employed the tissue microarray technology and examined Apaf-1 expression in 70 human primary malignant melanoma biopsies by immunohistochemistry. Our data showed that Apaf-1 expression is significantly reduced in melanoma cells compared with normal nevi (χ2=6.02, P=0.014). Our results also revealed that loss of Apaf-1 was not associated with the tumour thickness, ulceration or subtype, patients gender, age and 5-year survival. In addition, our in vitro apoptosis assay revealed that overexpression of Apaf-1 can sensitise melanoma cells to anticancer drug treatment. Taken together, our data indicate that Apaf-1 expression is significantly reduced in human melanoma and that Apaf-1 may serve as a therapeutic target in melanoma.

Prognostic Significance of Sox4 Expression in Human Cutaneous Melanoma and Its Role in Cell Migration and Invasion

The Sox4 transcription factor is involved in various cellular processes, such as embryonic development and differentiation. Deregulated expression of Sox4 in several human cancers has been reported to date, but its role in melanoma is unknown. We explored the role of Sox4 in melanoma pathogenesis in vivo and in vitro. Using tissue microarray, we evaluated Sox4 expression in 180 melanocytic lesions and investigated its role in melanoma cell migration and invasion. Sox4 expression was remarkably reduced in metastatic melanoma compared with dysplastic nevi (P < 0.05) and primary melanoma (P < 0.01). This reduction was correlated with a poorer disease-specific survival of melanoma patients (P = 0.039). Multivariate Cox regression analysis revealed that reduced Sox4 expression is an independent prognostic factor (P = 0.049). Knockdown of Sox4 enhanced melanoma cell invasion, migration, and stress fiber formation. The increased migration and invasion on Sox4 knockdown depends on the presence of nuclear factor (NF)-κB p50 and is abrogated when p50 is knocked down. We further observed inhibition of NF-κB p50 transcription by Sox4, in addition to a reverse pattern of expression of Sox4 and NF-κB p50 in different stages of melanocytic lesions. Our results suggest that Sox4 regulates melanoma cell migration and invasion in an NF-κB p50-dependent manner and may serve as a prognostic marker and potential therapeutic target for human melanoma.

Loss of SNF5 Expression Correlates with Poor Patient Survival in Melanoma

Purpose: Aberrant expression of SWI/SNF chromatin remodeling complex is involved in cancer development. The tumor suppressor SNF5, the core subunit of SWI/SNF complex, has been shown to regulate cell differentiation, cell cycle control, and apoptosis. To investigate the role of SNF5 in the development of melanoma, we examined the expression of SNF5 in melanocytic lesions at different stages and analyzed the correlation between SNF5 expression and clinicopathologic variables and patient survival. Experimental Design: Using tissue microarry and immunohistochemistry, we evaluated SNF5 staining in 51 dysplastic nevi, 88 primary melanomas, and 48 metastatic melanomas. We studied chemosensitivity of melanoma cells with reduced SNF5 expression by siRNA using cell survival and apoptosis assays. Results: SNF5 expression was reduced in metastatic melanoma compared with dysplastic nevi (P = 0.005), in advanced primary melanoma (Clarks level V) compared with low risk Clarks level II melanoma (P = 0.019), and in melanoma at sun-exposed sites compared with sun-protected sites (P = 0.044). Furthermore, we showed a strong correlation between negative SNF5 expression and a worse 5-year survival in melanoma patients (P = 0.016). Multivariate Cox regression analysis revealed that negative SNF5 expression is an independent prognostic factor to predict patient outcome in primary melanomas (P = 0.031). Finally, we showed that knockdown of SNF5 in melanoma cell lines resulted in significant chemoresistance. Conclusions: Our data indicate that SNF5 may be an important marker for human melanoma progression and prognosis as well as a potential therapeutic target. (Clin Cancer Res 2009;15(20):6404–11)

Sox4-mediated Dicer expression is critical for suppression of melanoma cell invasion

We previously reported reduced expression of Sox4 in metastatic melanoma and its role in suppression of cell migration and invasion through inhibition of nuclear factor (NF)-κB p50. Sox4 can also bind to the promoter sequence of Dicer, a microRNA (miRNA) biogenesis factor. Interestingly, altered expression of Dicer was also observed in cancers. However, the potential mechanisms that regulate Dicer expression and its potential significance in melanoma progression are unknown. Here, we studied the regulation of Dicer expression by Sox4 and its role in suppression of melanoma invasion. Our data showed that Sox4 positively regulates Dicer expression by binding to its promoter sequences and enhancing its activity. We found that knockdown of Dicer enhances the matrigel invasion of melanoma cells by at least twofold. In addition, we revealed that overexpression of exogenous Dicer reverts the enhanced melanoma cell invasion upon Sox4 knockdown. Furthermore, we examined the expression of Dicer protein in a large set of melanocytic lesions (n=514) at different stages by tissue microarray and found that Dicer expression is inversely correlated with melanoma progression (P<0.0001). Consistently, reduced Dicer expression was correlated with a poorer overall and disease-specific 5-year survival of patients (P=0.015 and 0.0029, respectively). In addition, we found a significant correlation between expression of Sox4 and Dicer proteins in melanoma biopsies (P=0.009), further indicating the regulation of Dicer expression by Sox4. Finally, we revealed that knockdown of Sox4 induces a major change in the expression pattern of miRNAs in melanoma cells, mainly due to reduced expression of Dicer. Our results pinpoint the regulation of Dicer expression by Sox4 in melanoma and the critical role of Dicer in suppression of melanoma invasion. Our findings on Sox4-regulated miRNA biogenesis pathway may aid toward the development of novel targeted therapeutic approaches for melanoma.