Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Apaf-1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf-1, an important mediator of mitochondrial-dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf-1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf-1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf-1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf-1 mRNA by fluorescence in situ hybridization and of Apaf-1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf-1. A significant decrease in Apaf-1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf-1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf-1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf-1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf-1 may be an important contributory factor in the development of the disease.     

Apaf-1XL is an inactive isoform compared with Apaf-1L

Apaf-1 plays a crucial role in the cytochrome c/dATP-dependent activation of caspase-9 and -3. We found that the human myeloid leukemic K562 cells were more resistant to cytochrome c-induced activation of caspase-9 and -3 in a cell-free system compared with the human T-lymphoblastic subclone CEM/VLB(100) cells. Apaf-1 cDNA sequencing revealed an additional insert of 11 aa between the CARD and CED-4 (ATPase) domains in K562 cells, which was identical to the sequence of Apaf-1XL. Immunoprecipitation of Apaf-1 with caspase-9 after a cell-free reaction demonstrated that Apaf-1XL in the K562 cell line showed a lower binding ability to caspase-9 compared with Apaf-1L protein. The resistance of K562 cells to cytochrome c-dependent apoptosis may be partly due to this Apaf-1XL form. These results suggest that the additional insert between CARD and CED-4 domains might affect Apaf-1 recruitment of caspase-9 during apoptosis.     

Apaf‐1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf‐1, an important mediator of mitochondrial‐dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf‐1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf‐1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf‐1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf‐1 mRNA by fluorescence in situ hybridization and of Apaf‐1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf‐1. A significant decrease in Apaf‐1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf‐1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf‐1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf‐1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf‐1 may be an important contributory factor in the development of the disease.     

HDAC6 interacts with PTPN1 to enhance melanoma cells progression

Histone deacetylase 6 (HDAC6) plays an important role in oncogenic transformation and cancer metastasis. Our previous study has demonstrated that HDAC6 was highly expressed in melanoma cells, and contributed to the proliferation and metastasis of melanoma cells. However, the underlying mechanism of HDAC6 in melanoma metastasis and progression remains largely unclear. In this study, we reported that HDAC6 directly interacted with Tyrosine-protein phosphatase non-receptor type 1 (PTPN1) by performing co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). HDAC6 increased the protein level of PTPN1 independent of histone modifying activity. In addition, PTPN1 promoted proliferation, colony formation and migration while decreased apoptosis of melanoma cells through activating extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, we found that matrix metallopeptidase 9 (MMP9) was increased by HDAC6/PTPN1/ERK1/2 axis, which might serve as a mechanism for melanoma invasion and metastasis. In conclusion, HDAC6 might enhance aggressive melanoma cells progression via interacting with PTPN1, which was independent of its histone modifying activity.     

Glycosylation profile of integrin alpha 3 beta 1 changes with melanoma progression

Glycosylation of integrins has been implicated in the modulation of their function. Characterisation of carbohydrate moieties of alpha(3) and beta(1) subunits from non-metastatic (WM35) and metastatic (A375) human melanoma cell lines was carried out on peptide-N-glycosidase F-released glycans using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). beta(1) integrin subunit from both cell lines displayed tri- and tetraantennary oligosaccharides complex type glycans, but only in A375 cell line was the sialylated tetraantennary complex type glycan (Hex(7)HexNAc(6)FucSia(4)) present. In contrast, only alpha(3) subunit from metastatic cells possessed beta1-6 branched structures. Our data indicate that the beta(1) and alpha(3) subunits expressed by the metastatic A375 cell line carry beta1-6 branched structures, suggesting that these cancer-associated glycan chains may modulate tumor cell adhesion by affecting the ligand binding properties of alpha(3)beta(1) integrin. In direct ligand binding assays, alpha(3)beta(1) integrin from both cell lines binds strongly to fibronectin and to much lesser degree to placental laminin. No binding to collagen IV was observed. Enzymatic removal of sialic acid residues from purified alpha(3)beta(1) integrin stimulates its adhesion to all examined ECM proteins. Our data suggest that the glycosylation profile of alpha(3)beta(1) integrin in human melanoma cells correlates with the acquisition of invasive capacity during melanoma progression.     

Apaf-1 localization is modulated indirectly by Bcl-2 expression.

Apoptotic protease activating factor-1 (Apaf-1) is an adaptor molecule essential for caspase-9 activation. Subcellular analysis of Apaf-1 in NIH-3T3 fibroblasts and the immature murine B cell lymphoma WEHI-231 indicates that Apaf-1 is localized in the Golgi apparatus and cytoplasm. Bcl-2 overexpression in WEHI-231 cells disrupts Apaf-1 localization in Golgi, causing a perinuclear Apaf-1 redistribution. Bcl-2 overexpression in NIH-3T3 fibroblasts however does not cause similar Apaf-1 redistribution, suggesting that cell type factors are involved in the redistribution process. The ability of Bcl-2 to modify Apaf-1 subcellular localization is not explained by direct interaction between Apaf-1 and Bcl-2. These data may help to clarify the anti-apoptotic Bcl-2 function.     

Decreased P27 protein expression is correlated with the progression and poor prognosis of nasopharyngeal carcinoma

Background

To determine the correlation of cyclin-dependent kinase inhibitor 1B (p27) expression with clinicopathologic features in nasopharyngeal carcinoma (NPC), including patient prognosis.

Methods

Real-time PCR and immunohistochemistry were used to examine the mRNA and protein expressions of p27 in NPC and nasopharyngeal tissues. The relationship of p27 expression levels with clinical features and prognosis of NPC patients was analyzed.

Results

The expression level of p27 mRNA was markedly lower in NPC tissues than that in the nasopharyngeal tissues (P?=?0.0006). Specific p27 protein staining by immunohistochemistry was found in the nuclei and cytoplasm of nasopharyngeal and malignant epithelial cells but decreased expression was observed in NPC samples compared to normal epithelium samples (P?=?0.002). In addition, low levels of p27 protein were inversely correlated with the status of T classification (p?=?0.002) and clinical stage (p?=?0.019) of NPC patients. Patients with lower p27 expression had a significantly shorter overall survival time than did patients with high p27 expression. Multivariate analysis suggested that the level of p27 expression was not an independent prognostic indicator (p?=?0.682) for NPC survival.

Conclusion

Low level of p27 expression is a potential unfavorable prognostic factor for patients with NPC.

Virtual slides

The virtual slide (s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1915282782109343.

     

Toward predicting metastatic progression of melanoma based on gene expression data

Primary and metastatic melanoma tumors share the same cell origin, making it challenging to identify genomic biomarkers that can differentiate them. Primary tumors themselves can be heterogeneous, reflecting ongoing genomic changes as they progress toward metastasizing. We developed a computational method to explore this heterogeneity and to predict metastatic progression of the primary tumors. We applied our method separately to gene expression and to microRNA (miRNA) expression data from ~450 primary and metastatic skin cutaneous melanoma (SKCM) samples from the Cancer Genome Atlas (TCGA). Metastatic progression scores from RNA‐seq data were significantly associated with clinical staging of patients’ lymph nodes, whereas scores from miRNA‐seq data were significantly associated with Clark's level. The loss of expression of many characteristic epithelial lineage genes in primary SKCM tumor samples was highly correlated with predicted progression scores. We suggest that those genes/miRNAs might serve as putative biomarkers for SKCM metastatic progression.     

Microsomal PGE2 synthase‐1 regulates melanoma cell survival and associates with melanoma disease progression

COX‐2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase‐1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels, and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS‐mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1‐specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy.     

Apaf-1 Deficiency Causes Chromosomal Instability

Apaf-1 is an essential component of the apoptosome, the molecular complex assembled in response to mitochondrial cytochrome c release that promotes caspase activation. Apaf-1 expression is suppressed in some malignant tumors, in particular melanoma as well as cervical and colorectal carcinoma, in which the loss of Apaf-1 expression marks tumor progression and poor prognosis. Recent results from our laboratory demonstrate that Apaf-1 has an apoptosis-unrelated function that may well account for its role as a tumor suppressor. The knockout of apaf-1 (in mice), the knockdown of Apaf-1 (in human cells) and loss of function mutations of ced-4 (the Caenorhabditis elegans ortholog of Apaf-1) compromise the arrest of DNA synthesis in response to DNA damage, in a context in which apoptosis does not occur. Here, we show that the depletion of Apaf-1 also sensitizes cells to chromosomalinstability induced by different types of DNA damage such as cisplatin, UVC light and &gamma;-irradiation. These results unravel a hitherto unsuspected role for Apaf-1 in the maintenance of genomic stability, independently from its function in the cell death machinery.     

Lack of Apaf-1 expression confers resistance to cytochrome c-driven apoptosis in cardiomyocytes

Apoptosis plays a role in cardiomyocyte death in several cardiovascular disorders. Here, we show that primary postnatal cardiomyocytes did not die upon activation of the intrinsic (cytochrome c-dependent) apoptotic pathway. Release of cytochrome c from mitochondria to the cytosol occurred, but did not activate the effector phase of apoptosis. Myocardial cells did not express apoptotic protease-activating factor-1 (Apaf-1), the allosteric activator of caspase-9 acting downstream of cytochrome c release. Forced expression of Apaf-1 restored the competence to complete the cytochrome c-induced apoptotic program and this effect was prevented by overexpression of Bcl-X(L). However, cardiomyocytes were able to enter the apoptotic program when it was initiated by activation of death receptors, as observed during serum deprivation and metabolic inhibition. Our results indicate that regulation of Apaf-1 expression may be a new regulatory mechanism developed in postmitotic cells in order to prevent irreversible commitment to die after release of cytochrome c.     

Fli-1 expression in malignant melanoma

Friend leukemia integration site 1 (Fli-1) has been reported as the first nuclear marker of endothelial differentiation; it is expressed in leukocytes and recently demonstrated in melanomas. Formalin-fixed, paraffin-embedded tissue sections from 97 melanomas including 69 cases of primary and 28 metastatic melanomas were evaluated by immunohistochemistry. Five melanoma cell lines were evaluated by Western blot and immunocytochemistry. Fli-1 expression was observed in all cell lines. Fli-1 expression was higher in metastatic than in primary tumors (r=0.208, p=0.041, Spearman correlation), it positively correlated with Ki-67 expression (r=0.233, p=0.022, Spearman correlation), and the presence of an ulcer in the primary tumor (r=0.267, p=0.030, Spearman correlation). Therefore, the expression of Fli-1 in malignant melanoma appears to be associated with biologically more aggressive tumors.     

Laboratory markers of melanoma progression

Extracellular tumour markers may have potential role in the follow-up of patients with malignant melanoma, in therapy monitoring and in prediction of prognosis. In our article circulating tumour markers in melanoma (melanoma inhibitory activity, lipid bound sialic acid, neuron specific enolase, TA90 immune complex, S-100B protein, 5-S-cysteinyldopa, tyrosinase, cytokines, metalloproteinases, LDH) were reviewed. Among laboratory melanoma markers the S-100B protein is the most investigated. S-100B protein has high specificity, appropriate sensitivity and proved to be significant prognostic factor independent from stages. High serum values are associated with shorter survival. However, before S-100B monitoring immunohistochemistry for the detection of S-100B is required. In the case of malignant melanomas with low expression serum S-100B monitoring may not be sensitive enough to follow disease progression. Although the serum concentration of 5-S-cysteinyldopa did not prove to be independent prognostic factor in our previous studies comprising the highest patient number in the literature, the marker was suggested for therapy monitoring. The survival analysis indicated that the elevated 5-S-cysteinyldopa level predicts shorter survival. In spite of the calculated low correlation between the two markers, parallel elevation of S-100B protein and 5-S-cysteinyldopa indicated shorter survival. On the basis of the literature LDH is the most appropriate tumour marker in stage IV to predict prognosis, but its sensitivity and specificity could not achieve that of S-100B protein. S-100B and LDH proved to be similarly reliable in respect to the clinical outcome. Determination of serum concentration of MIA and tyrosinase are also reliable markers in malignant melanoma. The other investigated markers are not well known yet or do not provide useful information to the clinicians.     

Tissue factor expression and serum level in patients with melanoma does not correlate with disease progression.

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.