Derek L. Dai

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.

IL-1 Blockade Attenuates Islet Amyloid Polypeptide-Induced Proinflammatory Cytokine Release and Pancreatic Islet Graft Dysfunction

Islets from patients with type 2 diabetes exhibit β cell dysfunction, amyloid deposition, macrophage infiltration, and increased expression of proinflammatory cytokines and chemokines. We sought to determine whether human islet amyloid polypeptide (hIAPP), the main component of islet amyloid, might contribute to islet inflammation by recruiting and activating macrophages. Early aggregates of hIAPP, but not nonamyloidogenic rodent islet amyloid polypeptide, caused release of CCL2 and CXCL1 by islets and induced secretion of TNF-α, IL-1α, IL-1β, CCL2, CCL3, CXCL1, CXCL2, and CXCL10 by C57BL/6 bone marrow-derived macrophages. hIAPP-induced TNF-α secretion was markedly diminished in MyD88-, but not TLR2- or TLR4-deficient macrophages, and in cells treated with the IL-1R antagonist (IL-1Ra) anakinra. To determine the significance of IL-1 signaling in hIAPP-induced pancreatic islet dysfunction, islets from wild-type or hIAPP-expressing transgenic mice were transplanted into diabetic NOD/SCID recipients implanted with mini-osmotic pumps containing IL-1Ra (50 mg/kg/d) or saline. IL-1Ra significantly improved the impairment in glucose tolerance observed in recipients of transgenic grafts 8 wk following transplantation. Islet grafts expressing hIAPP contained amyloid deposits in close association with F4/80-expressing macrophages. Transgenic grafts contained 50% more macrophages than wild-type grafts, an effect that was inhibited by IL-1Ra. Our results suggest that hIAPP-induced islet chemokine secretion promotes macrophage recruitment and that IL-1R/MyD88, but not TLR2 or TLR4 signaling is required for maximal macrophage responsiveness to prefibrillar hIAPP. These data raise the possibility that islet amyloid-induced inflammation contributes to β cell dysfunction in type 2 diabetes and islet transplantation.

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.

Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

The liver and pancreas share a common origin and coexpress several transcription factors. To gain insight into the transcriptional networks regulating the function of these tissues, we globally identify binding sites for FOXA2 in adult mouse islets and liver, PDX1 in islets, and HNF4A in liver. Because most eukaryotic transcription factors bind thousands of loci, many of which are thought to be inactive, methods that can discriminate functionally active binding events are essential for the interpretation of genome-wide transcription factor binding data. To develop such a method, we also generated genome-wide H3K4me1 and H3K4me3 localization data in these tissues. By analyzing our binding and histone methylation data in combination with comprehensive gene expression data, we show that H3K4me1 enrichment profiles discriminate transcription factor occupied loci into three classes: those that are functionally active, those that are poised for activation, and those that reflect pioneer-like transcription factor activity. Furthermore, we demonstrate that the regulated presence of H3K4me1-marked nucleosomes at transcription factor occupied promoters and enhancers controls their activity, implicating both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment in determining tissue-specific gene expression. Finally, we apply these approaches to generate novel insights into how FOXA2, PDX1, and HNF4A cooperate to drive islet- and liver-specific gene expression.

PUMA expression is significantly reduced in human cutaneous melanomas

Cutaneous malignant melanoma is an aggressive form of skin cancer, characterized by strong chemoresistance and poor patient prognosis. The molecular mechanisms underlying its resistance to chemotherapy remain unclear but are speculated to involve the dysregulation of apoptotic pathways. In this study, we sought to determine whether PUMA (p53 upregulated modulator of apoptosis) contributes to human melanoma formation, tumor progression, and survival. We used tissue microarray and immunohistochemistry to examine PUMA expression in 107 primary melanomas, 51 metastatic melanomas, and 64 dysplastic nevi. Here we report that PUMA expression is significantly weaker in primary melanomas compared to dysplastic nevi (P<0.0001), and is further reduced in metastatic melanomas compared to primary tumors (P=0.001). We show that weak PUMA expression in melanoma correlates with poorer overall and disease-specific 5-year survival (P<0.005 and P<0.001, respectively) of melanoma patients and that PUMA expression in tumor tissue is an independent predictor of both overall and disease-specific 5-year survival (P=0.05). Additionally, we show that exogenous PUMA expression in human melanoma cell lines (both wild type and mutant p53) results in significant apoptotic cell death. Our results suggest that PUMA expression may be an important prognostic marker for human melanoma and that adenoviral delivery of PUMA sensitizes melanoma cells to apoptosis.

Prevention of murine autoimmune diabetes by CCL22-mediated Treg recruitment to the pancreatic islets

Type 1 diabetes is characterized by destruction of insulin-producing β cells in the pancreatic islets by effector T cells. Tregs, defined by the markers CD4 and FoxP3, regulate immune responses by suppressing effector T cells and are recruited to sites of action by the chemokine CCL22. Here, we demonstrate that production of CCL22 in islets after intrapancreatic duct injection of double-stranded adeno-associated virus encoding CCL22 recruits endogenous Tregs to the islets and confers long-term protection from autoimmune diabetes in NOD mice. In addition, adenoviral expression of CCL22 in syngeneic islet transplants in diabetic NOD recipients prevented β cell destruction by autoreactive T cells and thereby delayed recurrence of diabetes. CCL22 expression increased the frequency of Tregs, produced higher levels of TGF-β in the CD4+ T cell population near islets, and decreased the frequency of circulating autoreactive CD8+ T cells and CD8+ IFN-γ–producing T cells. The protective effect of CCL22 was abrogated by depletion of Tregs with a CD25-specific antibody. Our results indicate that islet expression of CCL22 recruits Tregs and attenuates autoimmune destruction of β cells. CCL22-mediated recruitment of Tregs to islets may be a novel therapeutic strategy for type 1 diabetes.

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.

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.

Role of p53 Up-regulated Modulator of Apoptosis and Phosphorylated Akt in Melanoma Cell Growth, Apoptosis, and Patient Survival

Malignant melanoma is an aggressive and chemoresistant form of skin cancer characterized by rapid metastasis and poor patient prognosis. The development of innovative therapies with improved efficacy is critical to treatment of this disease. Here, we show that aberrant expression of two proteins, p53 up-regulated modulator of apoptosis (PUMA) and phosphorylated Akt (p-Akt), is associated with poor patient survival. Using tissue microarray analysis, we found that patients exhibiting both weak PUMA expression and strong p-Akt expression in their melanoma tumor tissue had significantly worse 5-year survival than patients with either weak PUMA or strong p-Akt expression alone (P < 0.001). Strikingly, no patients exhibiting strong PUMA expression and weak p-Akt expression in primary tumor tissue died within 5 years of diagnosis. We propose a two-pronged therapeutic strategy of (a) boosting PUMA expression and (b) inhibiting Akt phosphorylation in melanoma tumor tissue. Here, we report that a recombinant adenovirus containing human PUMA cDNA (ad-PUMA) efficiently inhibits human melanoma cell survival in vitro, rapidly induces apoptosis, and dramatically suppresses human melanoma tumor growth in a severe combined immunodeficient mouse xenograft model. In melanoma cells strongly expressing p-Akt, we show that Akt/protein kinase B signaling inhibitor-2 (API-2; a small-molecule Akt inhibitor) reduces cell survival in a dose- and time-dependent manner and enhances ad-PUMA-mediated growth inhibition of melanoma cells. Finally, we show that, by combining ad-PUMA and API-2 treatments, human melanoma tumor growth can be inhibited by >80% in vivo compared with controls. Our results suggest that a strategy to correct dysregulated PUMA and p-Akt expression in malignant melanoma may be an effective therapeutic option.

Nuclear ING2 expression is reduced in human cutaneous melanomas

Cutaneous malignant melanoma is a severe and sometimes life-threatening cancer. The molecular mechanism of melanomagenesis is incompletely understood. Deregulation of apoptosis is probably one of the key factors contributing to the progression of melanoma. The inhibitor of growth (ING) family proteins are candidate tumour suppressors which play important roles in apoptosis. Downregulated expression of ING proteins have been reported in several tumour types, including the loss of nuclear expression of p33ING1b in melanoma. As ING2 exhibits 58.9% homology with p33ING1b, we hypothesized that the aberrant expression of ING2 may be involved in melanomagenesis. Here, we used tissue microarray technology and immunohistochemistry to examine ING2 expression in human nevi and melanoma biopsies. Our data showed that nuclear ING2 expression was significantly reduced in radial growth phase (RGP), vertical growth phase (VGP), and metastatic melanomas compared with dysplastic nevi (P<0.05). Our data also revealed that nuclear ING2 expression was not associated with patients gender, age or tumour thickness, ulceration, American Joint Committee on Cancer (AJCC) stage, tumour subtype, location and 5-year survival (P>0.05). Taken together, our results suggest that nuclear ING2 expression is significantly reduced in human melanomas and that reduced ING2 may be an important molecular event in the initiation of melanoma development.