PI3 kinase blockade by Ad-PTEN inhibits invasion and induces apoptosis in RGP and metastatic melanoma cells

BACKGROUND: Melanoma is an aggressive tumor with a propensity to rapidly metastasize. The PTEN gene encodes a phosphatase with an unusual dual specificity for proteins and lipids. Mutations of PTEN have been found in various human cancers, including glioblastoma, prostate, breast, lung, and melanoma. Here we investigate in vitro the effects of blocking PI3K signaling using adenoviral-delivered PTEN (Ad-PTEN) in cell lines derived from both early- and late-stage melanoma. MATERIALS AND METHODS: Ad-PTEN transduced melanoma cell lines or normal cells were assayed for cell death, apoptosis, gene expression, invasion and migration, and regulation of angiogenesis. RESULTS: The PTEN locus from RGP and metastatic melanoma cell lines was sequenced; no coding region mutations were found. Adenoviral transfer of PTEN into melanoma cells containing wild-type PTEN alleles led to tumor-specific apoptosis and growth inhibition, with coordinate inhibition of AKT phosphorylation. Ad-PTEN suppressed cell migration by metastatic melanoma cells with concomitant increase in the level of cell surface E-cadherin. Immunohistochemical and confocal analyses localized PTEN to the cytoplasm and demonstrated enrichment at the cell membrane. Ad-PTEN inhibited angiogenesis as demonstrated by the tube formation assay using human vascular endothelial cells. CONCLUSIONS: These studies indicate that Ad-PTEN can inhibit tumor cells via multiple mechanisms and has pro-apoptotic, anti-metastatic, and anti-angiogenic properties. Thus, PI3K blockade via Ad-PTEN may be a promising approach for the treatment of early- and late-stage melanoma, even in tumors that do not harbor PTEN mutations.     

Why is melanoma so metastatic?

Malignant melanoma is one of the most aggressive cancers and can disseminate from a relatively small primary tumor and metastasize to multiple sites, including the lung, liver, brain, bone, and lymph nodes. Elucidating the molecular and genetic changes that take place during the metastatic process has led to a better understanding of why melanoma is so metastatic. Herein, we describe the unique features that distinguish melanoma from other solid tumors and contribute to the malignant phenotype of melanoma cells. For example, although melanoma cells are highly antigenic, they are extremely efficient at evading host immune response. Melanoma cells share numerous cell surface molecules with vascular cells, are highly angiogenic, are mesenchymal in nature, and possess a higher degree of ‘stemness’ than do other solid tumors. Finally, analysis of melanoma mutations has revealed that the gene expression profile of malignant melanoma is different from that of other cancers. Elucidating these molecular and genetic processes in highly metastatic melanoma can lead to the development of improved treatment and individualized therapy options.     

iTRAQ Quantitative Proteomic Comparison of Metastatic and Non-Metastatic Uveal Melanoma Tumors

Background

Uveal melanoma is the most common malignancy of the adult eye. The overall mortality rate is high because this aggressive cancer often metastasizes before ophthalmic diagnosis. Quantitative proteomic analysis of primary metastasizing and non-metastasizing tumors was pursued for insights into mechanisms and biomarkers of uveal melanoma metastasis.

Methods

Eight metastatic and 7 non-metastatic human primary uveal melanoma tumors were analyzed by LC MS/MS iTRAQ technology with Bruch’s membrane/choroid complex from normal postmortem eyes as control tissue. Tryptic peptides from tumor and control proteins were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Protein identification utilized the Mascot search engine and the human Uni-Prot/Swiss-Protein database with false discovery ≤ 1%; protein quantitation utilized the Mascot weighted average method. Proteins designated differentially expressed exhibited quantitative differences (p ≤ 0.05, t-test) in a training set of five metastatic and five non-metastatic tumors. Logistic regression models developed from the training set were used to classify the metastatic status of five independent tumors.

Results

Of 1644 proteins identified and quantified in 5 metastatic and 5 non-metastatic tumors, 12 proteins were found uniquely in ≥ 3 metastatic tumors, 28 were found significantly elevated and 30 significantly decreased only in metastatic tumors, and 31 were designated differentially expressed between metastatic and non-metastatic tumors. Logistic regression modeling of differentially expressed collagen alpha-3(VI) and heat shock protein beta-1 allowed correct prediction of metastasis status for each of five independent tumor specimens.

Conclusions

The present data provide new clues to molecular differences in metastatic and non-metastatic uveal melanoma tumors. While sample size is limited and validation required, the results support collagen alpha-3(VI) and heat shock protein beta-1 as candidate biomarkers of uveal melanoma metastasis and establish a quantitative proteomic database for uveal melanoma primary tumors.     

Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer

Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.     

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10⁶ cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.     

Constitutively Active Inflammasome in Human Melanoma Cells Mediating Autoinflammation via Caspase-1 Processing and Secretion of Interleukin-1��

Interleukin-1β (IL-1β) is a pleiotropic cytokine promoting inflammation, angiogenesis, and tissue remodeling as well as regulation of immune responses. Although IL-1β contributes to growth and metastatic spread in experimental and human cancers, the molecular mechanisms regulating the conversion of the inactive IL-1β precursor to a secreted and active cytokine remains unclear. Here we demonstrate that NALP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 in human melanoma cells. Late stage human melanoma cells spontaneously secrete active IL-1β via constitutive activation of the NALP3 inflammasome and IL-1 receptor signaling, exhibiting a feature of autoinflammatory diseases. Unlike human blood monocytes, these melanoma cells require no exogenous stimulation. In contrast, NALP3 functionality in intermediate stage melanoma cells requires activation of the IL-1 receptor to secrete active IL-1β; cells from an early stage of melanoma require stimulation of the IL-1 receptor plus the co-stimulant muramyl dipeptide. The spontaneous secretion of IL-1β from melanoma cells was reduced by inhibition of caspase-1 or the use of small interfering RNA directed against ASC. Supernatants from melanoma cell cultures enhanced macrophage chemotaxis and promoted in vitro angiogenesis, both prevented by pretreating melanoma cells with inhibitors of caspases-1 and -5 or IL-1 receptor blockade. These findings implicate IL-1-mediated autoinflammation as contributing to the development and progression of human melanoma and suggest that inhibiting the inflammasome pathway or reducing IL-1 activity can be a therapeutic option for melanoma patients.     

Biochemical analysis of metastasis-related Ax actin in B16 mouse melanoma cells after chemical reversional modulation and of tumor progression-related A' actin in the ontogeny of human malignant melanoma

To examine the correlation between tumor metastasis and Ax actin in mouse melanoma and between tumor progression and A'.actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of Ax actin. Will an induced decrease in metastasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of Ax actin? We also examined the appearance of A' actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented Ax actin in vitro. These results suggest that the appearance of Ax actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, Ax actin appearance remained unchanged. Appearance of A' actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A' actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.     

CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells

Background

A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+.

Methods/Findings

We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone.

Conclusions/Significance

The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.     

Biochemical Analysis of Metastasis-Related Ax Actin in B16 Mouse Melanoma Cells After Chemical Reversional Modulation and of Tumor Progression-Related A′ Actin in the Ontogeny of Human Malignant Melanoma

To examine the correlation between tumor metastasis and A^x actin in mouse melanoma and between tumor progression and A′, actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of A^x actin. Will an induced decrease in metasasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of A^x actin? We also examined the appearance of A′ actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented A^x actin in vitro. These results suggest that the appearance of A^x actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, A^x actin appearance remained unchanged. Appearance of A′ actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A′ actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.     

Expression of Zonulin,c-kit,and Glial Fibrillary Acidic Protein in Human Gliomas

The hallmarks of human malignant gliomas are their marked invasiveness and vascularity. Because angiogenesis and tumor invasion have been associated with extracellular matrix degradation and intercellular tight junctions, the involvement of zonulin in glioma biology is in the focus. We selected for histological examination five cases of glioblastoma WHO IV (nomenclature of the World Health Organization) and one case each from astrocytoma WHO III, meningioma WHO III, and meningioma WHO I as control samples. The meningioma WHO I is regarded as benign, whereas the meningioma WHO III is recognized as the transition form of malignant tumors in humans. The visualization of a newly designed antibody against human zonulin was studied in triple-labeling studies using fluorescence immunocytochemistry and compared with the expression of c-kit and glial fibrillary acidic protein in differently developed human gliomas. We found that increasing the expression of c-kit is accompanied by an increase of zonulin expression. Both are correlated to the degree of malignancy of human brain tumors. The expression of zonulin is correlated to the degradation of the blood-brain barrier as revealed by Griffonia simplicifolia lectin. In differently graded tumors, we found differently graded involvement of blood vessels in the tumor development, explaining patients'' survival.     

Selective Expression of PNA-Binding Glycoconjugates by Invasive Human Melanomas: A New Marker of Metastatic Potential

Alterations of cell-surface glycoconjugates have been associated with invasiveness and metastatic capacity in a number of experimental and human tumors (bladder and colon cancer). We have recently shown that human melanoma cells from variants selected for high metastatic potential in an animal model bind the lectin peanut agglutinin (PNA), and that human melanoma cell populations enriched for PNA binding cells generate a higher frequency of metastases when xenografted into immune suppressed neonatal rats. We have therefore sought cells binding PNA in biopsied human melanocytic tumors and compared frequencies of PNA binding by cells from benign nevi, early and late primary melanomas, and metastatic melanomas. Sections of conventionally processed tissues were deparaffinised and exposed to biotinylated PNA; PNA fixation was revealed by the avidine/peroxidase/AEC technique. In 51 specimens tested, PNA appears to react electively with invasive tumors, since only one of the 7 early primary melanomas (Clark III) reacted while 13/23 late primary melanomas (Clark III-V), and 4/21 melanoma metastases were reactive. In addition, only 1/17 benign nevi bound PNA. In primary tumors, the reactive cells were exclusively invasive tumors cells in the dermis. PNA reactive material was observed in the cytoplasm and plasma membrane of reactive cells. Hence, alterations in composition and cellular localisation of glycoconjugates detectable by lectin histochemistry in melanoma cells may be markers of metastatic potential that may be applicable on an individual patient basis.     

Up-Regulation of Hepatoma-Derived Growth Factor Facilities Tumor Progression in Malignant Melanoma

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16–F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.     

Cancer cells copy migratory behavior and exchange signaling networks via extracellular vesicles

Recent data showed that cancer cells from different tumor subtypes with distinct metastatic potential influence each other's metastatic behavior by exchanging biomolecules through extracellular vesicles (EVs). However, it is debated how small amounts of cargo can mediate this effect, especially in tumors where all cells are from one subtype, and only subtle molecular differences drive metastatic heterogeneity. To study this, we have characterized the content of EVs shed in vivo by two clones of melanoma (B16) tumors with distinct metastatic potential. Using the Cre‐LoxP system and intravital microscopy, we show that cells from these distinct clones phenocopy their migratory behavior through EV exchange. By tandem mass spectrometry and RNA sequencing, we show that EVs shed by these clones into the tumor microenvironment contain thousands of different proteins and RNAs, and many of these biomolecules are from interconnected signaling networks involved in cellular processes such as migration. Thus, EVs contain numerous proteins and RNAs and act on recipient cells by invoking a multi‐faceted biological response including cell migration.     

Biological processes, properties and molecular wiring diagrams of candidate low-penetrance breast cancer susceptibility genes

Background

The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.

Methods

We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.

Results

We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.

Conclusion

The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.     

Targeting of HLA-DR molecules transduces agonistic functional signals in cutaneous melanoma

The role of human leukocyte antigens (HLA) class II molecules in transducing intracellular signals in immune cells is well established. Solid tumors of different histotype can also express HLA class II antigens; however, their intracellular signaling ability is essentially unknown. Due to the frequent expression of HLA class II molecules in primary and metastatic lesions, cutaneous melanoma was utilized to investigate whether the engagement of HLA-DR molecules transduces functional intracellular signal(s). Triggering of HLA-DR molecules by the anti-HLA-DR monoclonal antibody (mAb) L243 induced a significant (P < 0.05) and dose-dependent growth-inhibition of metastatic melanoma cells Mel 120, as well as their homotypic aggregation. Furthermore, an increase in tyrosine phosphorylation of multiple cellular proteins with a molecular weight ranging from 66 to 130 kD, including p125 focal adhesion kinase, was observed. Lastly, the engagement of HLA-DR molecules by mAb L243 inhibited activator protein-1-DNA binding. Thus, HLA-DR molecules expressed on melanoma cells can transduce functional intracellular signals. This finding is consistent with evidences obtained in hematological malignancies, and suggests the potential usefulness of HLA-DR molecules to set-up new approaches of targeted therapy in metastatic melanoma.     

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.