Loss of prohormone convertase 2 promotes beta cell dysfunction in a rodent transplant model expressing human pro-islet amyloid polypeptide.

AIMS/HYPOTHESIS: A contributor to beta cell failure in type 2 diabetes and islet transplants is amyloid formation by aggregation of the beta cell peptide, islet amyloid polypeptide (IAPP). Similar to the proinsulin processing pathway that generates insulin, IAPP is derived from a prohormone precursor, proIAPP, which requires cleavage by prohormone convertase (PC) 1/3 and PC2 in rodent pancreatic beta cells. We hypothesised that loss of PC2 would promote beta cell death and dysfunction in a rodent model of human beta cell proIAPP overexpression. METHODS: We generated an islet transplant model wherein immune-deficient mouse models of diabetes received islets expressing amyloidogenic human proIAPP and lacking PC2, leading to restoration of normoglycaemia accompanied by increased secretion of human proIAPP. Blood glucose levels were analysed for up to 16 weeks in transplant recipients and grafts were assessed for islet amyloid and beta cell number and death. RESULTS: Hyperglycaemia (blood glucose >16.9 mmol/l) returned in 94% of recipients of islets expressing human proIAPP and lacking PC2, whereas recipients of islets that express human proIAPP and normal PC2 levels remained normoglycaemic for at least 16 weeks. Islet graft failure was accompanied by a ∼20% reduction in insulin-positive cells, yet the degree of amyloid deposition and beta cell apoptosis was similar to those of controls expressing human proIAPP with functional PC2 levels. CONCLUSIONS/INTERPRETATION: PC2 deficiency in transplanted mouse islets expressing human proIAPP promotes beta cell loss and graft failure. Our data suggest that impaired NH2-terminal processing and increased secretion of human proIAPP promote beta cell failure.

CCL22 Prevents Rejection of Mouse Islet Allografts and Induces Donor-Specific Tolerance.

Manipulation of regulatory T cell (Treg) migration by islet expression of the chemokine CCL22 prevents diabetes in NOD mice and delays recurrent autoimmunity in syngeneic islet transplants. We sought to determine whether attracting Tregs with CCL22 also prevents islet allograft rejection. Isolated Bl/6 mouse islets were transduced overnight with adenovirus expressing CCL22 (Ad-CCL22) downstream of the CMV promoter. Islets were transplanted under the renal capsule of Balb/c recipients made diabetic by streptozotocin. To assess immunologic tolerance, graft-bearing kidneys from recipients of CCL22-expressing islet grafts were removed, and mice received a second transplant of naive islets from the same donor strain or third-party islets into the contralateral kidney. Adenoviral expression of CCL22 conferred prolonged protection of islet allografts in MHC-mismatched, diabetic recipients, maintaining normoglycemia in 75% of recipients for at least 80 days. Increased frequency of Treg cells was observed in islet grafts transduced with Ad-CCL22 compared with untreated grafts. Normoglycemic recipients of CCL22-expressing islet grafts showed complete absence of antidonor antibodies and no lymphocyte proliferation after exposure to donor splenocytes. After removal of the primary graft at day 80, mice that received a second transplant with untreated islets from the same donor strain did not reject the grafts, suggesting the development of tolerance. Expression of CCL22 recruits Treg cells to transplanted islets, prevents activation of alloreactive T-cells and islet allograft failure and induces alloantigen-specific tolerance. Manipulation of Treg cells by CCL22 in transplanted islets may be a novel therapeutic strategy for diabetes.

Cellular mechanisms of CCL22-mediated attenuation of autoimmune diabetes.

Autoimmune destruction of insulin-producing ß cells in type 1 diabetes and islet transplantation involves a variety of immune pathways but is primarily mediated by self-reactive T cells. Chemokines can modulate local immune responses in inflammation and tumors by recruiting immune cells. We have reported that expression of the chemokine CCL22 in pancreatic ß cells in the NOD mouse prevents autoimmune attack by recruiting T regulatory cells (Tregs), protecting mice from diabetes. In this study we show that invariant NKT cells are also recruited to CCL22-expressing islet transplants and are required for CCL22-mediated protection from autoimmunity. Moreover, CCL22 induces an influx of plasmacytoid dendritic cells, which correlates with higher levels of IDO in CCL22-expressing islet grafts. In addition to its chemotactic properties, we found that CCL22 activates Tregs and promotes their ability to induce expression of IDO by dendritic cells. Islet CCL22 expression thus produces a tolerogenic milieu through the interplay of Tregs, invariant NKT cells, and plasmacytoid dendritic cells, which results in suppression of effector T cell responses and protection of ß cells. The immunomodulatory properties of CCL22 could be harnessed for prevention of graft rejection and type 1 diabetes as well as other autoimmune disorders.

IL-1 blockade attenuates islet amyloid polypeptide-induced proinflammatory cytokine release and pancreatic islet graft dysfunction.

Islets from patients with type 2 diabetes exhibit ß 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-1ß, 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 ß cell dysfunction in type 2 diabetes and islet transplantation.

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

Type 1 diabetes is characterized by destruction of insulin-producing ß 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 ß cell destruction by autoreactive T cells and thereby delayed recurrence of diabetes. CCL22 expression increased the frequency of Tregs, produced higher levels of TGF-ß in the CD4+ T cell population near islets, and decreased the frequency of circulating autoreactive CD8+ T cells and CD8+ IFN-γ­producing 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 ß cells. CCL22-mediated recruitment of Tregs to islets may be a novel therapeutic strategy for type 1 diabetes.

Bim expression is reduced in human cutaneous melanomas.

Bim is a BH3-only protein belonging to the Bcl-2 family of apoptotic regulators. Upon activation, Bim can antagonize all the prosurvival Bcl-2 proteins, leading to apoptosis. To investigate whether Bim plays a role in melanoma progression, we used tissue microarray and immunohistochemistry to measure Bim expression in 52 cases of dysplastic nevi, 159 cases of primary melanomas and 52 cases of melanoma metastases, and evaluated the prognostic value of Bim expression. Our results showed that Bim expression is reduced as melanoma progresses. Significant differences for Bim staining pattern were observed between dysplastic nevi and metastatic melanomas (P<0.001, chi2 test), and between primary melanomas and metastatic melanomas (P<0.001, chi2 test). Moreover, reduced Bim expression is significantly correlated with poor 5-year survival of melanoma patients but failed to be an independent prognostic factor by Cox regression analysis. Our data suggest that Bim loss may play an important role in melanoma progression.

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 >or=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.

Prognostic significance of nuclear factor-kappaB p105/p50 in human melanoma and its role in cell migration.

Transcriptional factor nuclear factor-kappaB (NF-kappaB) family has been shown to play an important role in tumor pathogenesis and serve as a potential target in cancer therapy. However, it is necessary to clarify the specific functions of NF-kappaB members, which would provide the basis for the selective blockade and reduction of therapeutic side effects resulting from unspecific inhibition of NF-kappaB members. In this study, we explored the role of NF-kappaB p105/p50 in melanoma pathogenesis in vitro and in vivo. We found that the expression of NF-kappaB p105/p50 significantly increased in dysplastic nevi, primary melanoma, and metastatic melanoma compared with normal nevi (P = 0.0004, chi(2) test). Furthermore, NF-kappaB p105/p50 nuclear staining increased with melanoma progression and strong NF-kappaB p105/p50 nuclear staining was inversely correlated with disease-specific 5-year survival of patients with tumor thickness >2.0 mm (P = 0.014, log-rank test). Multivariate Cox regression analysis revealed that nuclear expression of NF-kappaB p105/p50 is an independent prognostic factor in this subgroup. Moreover, we found that up-regulation of NF-kappaB p50 enhanced melanoma cell migration, whereas small interfering RNA knockdown inhibited cell migration. In addition, overexpression of NF-kappaB p50 induced RhoA activity and Rock-mediated formation of stress fiber in melanoma cells. Taken together, our data indicate that NF-kappaB p105/p50 may be an important marker for human melanoma progression and prognosis as well as a potentially selective therapeutic target.

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.

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 CTHRC(1) 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, chi(2) test). There was a further increase of CTHRC1 protein in metastatic melanoma specimens compared with nonmetastatic lesions (P < 0.01, chi(2) 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.

Osteopontin expression correlates with melanoma invasion.

Melanoma is one of the most aggressive cancers affecting humans. Although early melanomas are curable with surgical excision, metastatic melanomas are associated with high mortality. The mechanism of melanoma development, progression, and metastasis is largely unknown. In order to uncover genes unique to melanoma cells, we used high-density DNA microarrays to examine the gene expression profiles of metastatic melanoma nodules using benign nevi as controls. Over 190 genes were significantly overexpressed in metastatic melanomas compared with normal nevi by at least 2-fold. One of the most abundantly expressed genes in metastatic melanoma nodules is osteopontin (OPN). Immunohistochemistry staining on tissue microarrays and individual skin biopsies representing different stages of melanoma progression revealed that OPN expression is first acquired at the step of melanoma tissue invasion. In addition, blocking of OPN expression by RNA interference reduced melanoma cell numbers in vitro. Our observations suggest that OPN may be acquired early in melanoma development and progression, and may enhance tumor cell growth in invasive melanoma.

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.

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.

Mutations of the ING1 tumor suppressor gene detected in human melanoma abrogate nucleotide excision repair.

Epidemiological evidence indicates that ultraviolet radiation (UVR) is the primary environmental cause of the rapid increase in the incidence of human cutaneous melanoma observed in the past decades. However, the genetic changes caused by UVR that lead to melanoma formation remain unclear. The ING1 (inhibitor of growth 1) tumor suppressor plays an important role in cellular stress response to UVR. To further investigate whether ING1 is involved in melanoma development, we examined the mutational status of the ING1 gene in 46 human cutaneous melanoma biopsies and characterized the biological importance of ING1 mutations in nucleotide excision repair. Single-strand conformation polymorphism and DNA sequencing were used to detect the mutational status of the ING1 gene. The host-cell-reactivation assay and radioimmunoassay were used to determine the role of ING1 mutations in nucleotide excision repair. We show that 20% of the melanoma primaries contained missense mutations in the SAP30-interacting domain and PHD finger motif of the ING1 gene with the R102L and N260S alterations observed more than once. Furthermore, our data indicate that patients that harbor ING1 mutations in the tumors have a higher risk to die from the disease within 5 years (50%) compared to patients with no ING1 mutation (18%). Moreover, we demonstrated that mutations at codon 102 or 260 as well as deletion of the PHD finger motif are detrimental to p33ING1-mediated enhancement of DNA repair. Taken together, our data indicate that ING1 mutations abrogate its enhancement in nucleotide excision repair.

Increased expression of integrin-linked kinase is correlated with melanoma progression and poor patient survival.

PURPOSE: Integrin-linked kinase (ILK), a key component of the extracellular matrix adhesion, has been studied extensively in recent years. Overexpression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression. Furthermore, increased ILK expression is correlated with progression of several human tumor types, including breast, prostate, and colon carcinomas. However, the role of ILK overexpression in human melanoma pathogenesis is not known. To investigate whether ILK plays a role in melanoma progression, we measured ILK expression in primary melanoma biopsies at various stages of invasion and evaluated the prognostic value of ILK expression in human melanoma. EXPERIMENTAL DESIGN: We used tissue microarray and immunohistochemistry to determine ILK expression in 67 primary melanomas and analyzed the correlation between ILK expression and melanoma progression and 5-year patient survival. RESULTS: We show that strong ILK expression is significantly associated with melanoma thickness. Strong ILK expression was observed in 0, 22, 33, and 63% in melanoma biopsies 3.0 mm in thickness, respectively. Furthermore, strong ILK expression was detected in 83% of the tumors with lymph node invasion compared with only 18% for tumors without lymph node invasion (P < 0.01). Strikingly, our data revealed that strong ILK expression is inversely correlated with 5-year patient survival (P < 0.05). CONCLUSION: ILK expression increases dramatically with melanoma invasion and progression and is inversely correlated with patient survival.