A heterodimer of α and ß hemoglobin chains functions as an innate anticancer agent.

Metastatic (as well as tumor) microenvironments contain both cancer-promoting and cancer-restraining factors. The balance between these opposing forces determines the fate of cancer cells that disseminate to secondary organ sites. In search for microenvironmental drivers or inhibitors of metastasis, we identified, in a previous study, the beta subunit of hemoglobin (HBB) as a lung-derived antimetastatic factor. In the present study, exploring mechanisms regulating melanoma brain metastasis, we discovered that brain-derived factors restrain proliferation and induce apoptosis and necrosis of brain-metastasizing melanoma cells. Employing various purification procedures, we identified a heterodimer composed of hemoglobin alpha and beta chains that perform these antimetastatic functions. Neither the alpha nor the beta subunit alone was inhibitory. An alpha/beta chain dimer chemically purified from human hemoglobin inhibited the cell viability of primary melanomas, melanoma brain metastasis (MBM), and breast cancer cell lines. The dimer-induced DNA damage, cell cycle arrest at the SubG1 phase, apoptosis, and significant necrosis in four MBM cell lines. Proteomic analysis of dimer-treated MBM cells revealed that the dimer downregulates the expression of BRD4, GAB2, and IRS2 proteins, playing crucial roles in cancer cell sustainability and progression. Thus, we hypothesize that the hemoglobin dimer functions as a resistance factor against brain-metastasizing cancer cells.

Heterogeneity in the Metastatic Microenvironment: JunB-Expressing Microglia Cells as Potential Drivers of Melanoma Brain Metastasis Progression.

Reciprocal signaling between melanoma brain metastatic (MBM) cells and microglia reprograms the phenotype of both interaction partners, including upregulation of the transcription factor JunB in microglia. Here, we aimed to elucidate the impact of microglial JunB upregulation on MBM progression. For molecular profiling, we employed RNA-seq and reverse-phase protein array (RPPA). To test microglial JunB functions, we generated microglia variants stably overexpressing JunB (JunBhi) or with downregulated levels of JunB (JunBlo). Melanoma-derived factors, namely leukemia inhibitory factor (LIF), controlled JunB upregulation through Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling. The expression levels of JunB in melanoma-associated microglia were heterogeneous. Flow cytometry analysis revealed the existence of basal-level JunB-expressing microglia alongside microglia highly expressing JunB. Proteomic profiling revealed a differential protein expression in JunBhi and JunBlo cells, namely the expression of microglia activation markers Iba-1 and CD150, and the immunosuppressive molecules SOCS3 and PD-L1. Functionally, JunBhi microglia displayed decreased migratory capacity and phagocytic activity. JunBlo microglia reduced melanoma proliferation and migration, while JunBhi microglia preserved the ability of melanoma cells to proliferate in three-dimensional co-cultures, that was abrogated by targeting leukemia inhibitory factor receptor (LIFR) in control microglia-melanoma spheroids. Altogether, these data highlight a melanoma-mediated heterogenous effect on microglial JunB expression, dictating the nature of their functional involvement in MBM progression. Targeting microglia highly expressing JunB may potentially be utilized for MBM theranostics.

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

LY6S, a New IFN-Inducible Human Member of the Ly6a Subfamily Expressed by Spleen Cells and Associated with Inflammation and Viral Resistance.

Syntenic genomic loci on human chromosome 8 and mouse chromosome 15 (mChr15) code for LY6/Ly6 (lymphocyte Ag 6) family proteins. The 23 murine Ly6 family genes include eight genes that are flanked by the murine Ly6e and Ly6l genes and form an Ly6 subgroup referred to in this article as the Ly6a subfamily gene cluster. Ly6a, also known as Stem Cell Ag-1 and T cell-activating protein, is a member of the Ly6a subfamily gene cluster. No LY6 genes have been annotated within the syntenic LY6E to LY6L human locus. We report in this article on LY6S, a solitary human LY6 gene that is syntenic with the murine Ly6a subfamily gene cluster, and with which it shares a common ancestry. LY6S codes for the IFN-inducible GPI-linked LY6S-iso1 protein that contains only 9 of the 10 consensus LY6 cysteine residues and is most highly expressed in a nonclassical spleen cell population. Its expression leads to distinct shifts in patterns of gene expression, particularly of genes coding for inflammatory and immune response proteins, and LY6S-iso1-expressing cells show increased resistance to viral infection. Our findings reveal the presence of a previously unannotated human IFN-stimulated gene, LY6S, which has a 1:8 ortholog relationship with the genes of the Ly6a subfamily gene cluster, is most highly expressed in spleen cells of a nonclassical cell lineage, and whose expression induces viral resistance and is associated with an inflammatory phenotype and with the activation of genes that regulate immune responses.

Cancer microenvironment and genomics: evolution in process.

Cancer heterogeneity is a result of genetic mutations within the cancer cells. Their proliferation is not only driven by autocrine functions but also under the influence of cancer microenvironment, which consists of normal stromal cells such as infiltrating immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, vascular and lymphatic channels. The relationship between cancer cells and cancer microenvironment is a critical one and we are just on the verge to understand it on a molecular level. Cancer microenvironment may serve as a selective force to modulate cancer cells to allow them to evolve into more aggressive clones with ability to invade the lymphatic or vascular channels to spread to regional lymph nodes and distant sites. It is important to understand these steps of cancer evolution within the cancer microenvironment towards invasion so that therapeutic strategies can be developed to control or stop these processes.

Cancer drug resistance induced by EMT: novel therapeutic strategies.

Over the last decade, important clinical benefits have been achieved in cancer patients by using drug-targeting strategies. Nevertheless, drug resistance is still a major problem in most cancer therapies. Epithelial-mesenchymal plasticity (EMP) and tumour microenvironment have been described as limiting factors for effective treatment in many cancer types. Moreover, epithelial-to-mesenchymal transition (EMT) has also been associated with therapy resistance in many different preclinical models, although limited evidence has been obtained from clinical studies and clinical samples. In this review, we particularly deepen into the mechanisms of which intermediate epithelial/mesenchymal (E/M) states and its interconnection to microenvironment influence therapy resistance. We also describe how the use of bioinformatics and pharmacogenomics will help to figure out the biological impact of the EMT on drug resistance and to develop novel pharmacological approaches in the future.

The melanoma brain metastatic microenvironment: aldolase C partakes in shaping the malignant phenotype of melanoma cells - a case of inter-tumor heterogeneity.

Previous studies indicated that microglia cells upregulate the expression of aldolase C (ALDOC) in melanoma cells. The present study using brain-metastasizing variants from three human melanomas explores the functional role of ALDOC in the formation and maintenance of melanoma brain metastasis (MBM). ALDOC overexpression impacted differentially the malignant phenotype of these three variants. In the first variant, ALDOC overexpression promoted cell viability, adhesion to and transmigration through a layer of brain endothelial cells, and amplified brain micrometastasis formation. The cross-talk between this MBM variant and microglia cells promoted the proliferation and migration of the latter cells. In sharp contrast, ALDOC overexpression in the second brain-metastasizing melanoma variant reduced or did not affect the same malignancy features. In the third melanoma variant, ALDOC overexpression augmented certain characteristics of malignancy and reduced others. The analysis of biological functions and disease pathways in the ALDOC overexpressing variants clearly indicated that ALDOC induced the expression of tumor progression promoting genes in the first variant and antitumor progression properties in the second variant. Overall, these results accentuate the complex microenvironment interactions between microglia cells and MBM, and the functional impact of intertumor heterogeneity. Since intertumor heterogeneity imposes a challenge in the planning of cancer treatment, we propose to employ the functional response of tumors with an identical histology, to a particular drug or the molecular signature of this response, as a predictive indicator of response/nonresponse to this drug.

Constitutive low expression of antiviral effectors sensitizes melanoma cells to a novel oncolytic virus.

STAT1 is a critical effector and a target gene of interferon (IFN) signaling, and thus a central mediator of antiviral responses. As both a mediator and a target of IFN signals, STAT1 expression reports on, and determines IFN activity. Gene expression analyses of melanoma patient samples revealed varied levels of STAT1 expression, which highly correlated with expression of >700 genes. The ability of oncolytic viruses to exploit tumor-induced defects to antiviral responses suggests that oncolytic viruses may efficiently target a subset of melanomas, yet these should be defined. We modeled this scenario with murine B16F10 melanomas, immortalized skin fibroblasts as controls and a novel oncolytic virus, EHDV-TAU. In B16F10 cells, constitutive low expression of STAT1 and its target genes, which included intracellular pattern recognition receptors (PRRs), correlated with their inability to mount IFN-based antiviral responses upon EHDV-TAU challenge, and with potency of EHDV-TAU-induced oncolysis. This underexpression of interferon stimulated genes (ISGs) and PRRs, and the inability of EHDV-TAU to induce their expression, were reversed by epigenetic modifiers, suggesting epigenetic silencing as a basis for their underexpression. Despite their inability to mount IFN/STAT-based responses upon viral infection, EHDV-TAU infected B16F10 cells secreted immune-stimulatory chemokines. Accordingly, in vivo, EHDV-TAU enhanced intratumoral infiltration of cytotoxic T-cells and reduced growth of local and distant tumors. We propose that "STAT1 signatures" should guide melanoma virotherapy treatments, and that oncolytic viruses such as EHDV-TAU have the potential to exploit the cellular context of low-STAT1 tumors.

Site-specific metastasis: A cooperation between cancer cells and the metastatic microenvironment.

The conclusion derived from the information provided in this review is that disseminating tumor cells (DTC) collaborate with the microenvironment of a future metastatic organ site in the establishment of organ-specific metastasis. We review the basic principles of site-specific metastasis and the contribution of the cross talk between DTC and the microenvironment of metastatic sites (metastatic microenvironment [MME]) to the establishment of the organ-specific premetastatic niche; the targeted migration of DTC to the endothelium of the future organ-specific metastasis; the transmigration of DTC to this site and the seeding and colonization of DTC in their future MME. We also discuss the role played by DTC-MME interactions on tumor dormancy and on the differential response of tumor cells residing in different MMEs to antitumor therapy. Finally, we summarize some studies dealing with the effects of the MME on a unique site-specific metastasis-brain metastasis.

Inter-Tumor Heterogeneity-Melanomas Respond Differently to GM-CSF-Mediated Activation.

Granulocyte-monocyte colony stimulating factor (GM-CSF) is used as an adjuvant in various clinical and preclinical studies with contradictory results. These were attributed to opposing effects of GM-CSF on the immune or myeloid systems of the treated patients or to lack of optimal dosing regimens. The results of the present study point to inter-tumor heterogeneity as a possible mechanism accounting for the contrasting responses to GM-CSF incorporating therapies. Employing xenograft models of human melanomas in nude mice developed in our lab, we detected differential functional responses of melanomas from different patients to GM-CSF both in vitro as well as in vivo. Whereas cells of one melanoma acquired pro metastatic features following exposure to GM-CSF, cells from another melanoma either did not respond or became less malignant. We propose that inter-melanoma heterogeneity as manifested by differential responses of melanoma cells (and perhaps also of other tumor) to GM-CSF may be developed into a predictive marker providing a tool to segregate melanoma patients who will benefit from GM-CSF therapy from those who will not.

Upregulation of cell surface GD3 ganglioside phenotype is associated with human melanoma brain metastasis.

Melanoma metastasis to the brain is one of the most frequent extracranial brain tumors. Cell surface gangliosides are elevated in melanoma metastasis; however, the metabolic regulatory mechanisms that govern these specific changes are poorly understood in melanoma particularly brain metastases (MBM) development. We found ganglioside GD3 levels significantly upregulated in MBM compared to lymph node metastasis (LNM) but not for other melanoma gangliosides. Moreover, we demonstrated an upregulation of ST8SIA1 (GD3 synthase) as melanoma progresses from melanocytes to MBM cells. Using RNA-ISH on FFPE specimens, we evaluated ST8SIA1 expression in primary melanomas (PRM) (n = 23), LNM and visceral metastasis (n = 45), and MBM (n = 39). ST8SIA1 was significantly enhanced in MBM compared to all other specimens. ST8SIA1 expression was assessed in clinically well-annotated melanoma patients from multicenters with AJCC stage III B-D LNM (n = 58) with 14-year follow-up. High ST8SIA1 expression was significantly associated with poor overall survival (HR = 3.24; 95% CI, 1.19-8.86, P = 0.02). In a nude mouse human xenograft melanoma brain metastasis model, MBM variants had higher ST8SIA1 expression than their respective cutaneous melanoma variants. Elevated ST8SIA1 expression enhances levels of cell surface GD3, a phenotype that favors MBM development, hence associated with very poor prognosis. Functional assays demonstrated that ST8SIA1 overexpression enhanced cell proliferation and colony formation, whereby ST8SIA1 knockdown had opposite effects. Icaritin a plant-derived phytoestrogen treatment significantly inhibited cell growth in high GD3-positive MBM cells through targeting the canonical NFκB pathway. The study demonstrates GD3 phenotype associates with melanoma progression and poor outcome.

The Challenge of Classifying Metastatic Cell Properties by Molecular Profiling Exemplified with Cutaneous Melanoma Cells and Their Cerebral Metastasis from Patient Derived Mouse Xenografts.

The prediction of metastatic properties from molecular analyses still poses a major challenge. Here we aimed at the classification of metastasis-related cell properties by proteome profiling making use of cutaneous and brain-metastasizing variants from single melanomas sharing the same genetic ancestry. Previous experiments demonstrated that cultured cells derived from these xenografted variants maintain a stable phenotype associated with a differential metastatic behavior: The brain metastasizing variants produce more spontaneous micro-metastases than the corresponding cutaneous variants. Four corresponding pairs of cutaneous and metastatic cells were obtained from four individual patients, resulting in eight cell-lines presently investigated. Label free proteome profiling revealed significant differences between corresponding pairs of cutaneous and cerebellar metastases from the same patient. Indeed, each brain metastasizing variant expressed several apparently metastasis-associated proteomic alterations as compared with the corresponding cutaneous variant. Among the differentially expressed proteins we identified cell adhesion molecules, immune regulators, epithelial to mesenchymal transition markers, stem cell markers, redox regulators and cytokines. Similar results were observed regarding eicosanoids, considered relevant for metastasis, such as PGE2 and 12-HETE. Multiparametric morphological analysis of cells also revealed no characteristic alterations associated with the cutaneous and brain metastasis variants. However, no correct classification regarding metastatic potential was yet possible with the present data. We thus concluded that molecular profiling is able to classify cells according to known functional categories but is not yet able to predict relevant cell properties emerging from networks consisting of many interconnected molecules. The presently observed broad diversity of molecular patterns, irrespective of restricting to one tumor type and two main classes of metastasis, highlights the important need to develop meta-analysis strategies to predict cell properties from molecular profiling data. Such base knowledge will greatly support future individualized precision medicine approaches.

Regeneration Enhances Metastasis: A Novel Role for Neurovascular Signaling in Promoting Melanoma Brain Metastasis.

Neural repair after stroke involves initiation of a cellular proliferative program in the form of angiogenesis, neurogenesis, and molecular growth signals in the surrounding tissue elements. This cellular environment constitutes a niche in which regeneration of new blood vessels and new neurons leads to partial tissue repair after stroke. Cancer metastasis has similar proliferative cellular events in the brain and other organs. Do cancer and CNS tissue repair share similar cellular processes? In this study, we identify a novel role of the regenerative neurovascular niche induced by stroke in promoting brain melanoma metastasis through enhancing cellular interactions with surrounding niche components. Repair-mediated neurovascular signaling induces metastatic cells to express genes crucial to metastasis. Mimicking stroke-like conditions in vitro displays an enhancement of metastatic migration potential and allows for the determination of cell-specific signals produced by the regenerative neurovascular niche. Comparative analysis of both in vitro and in vivo expression profiles reveals a major contribution of endothelial cells in mediating melanoma metastasis. These results point to a previously undiscovered role of the regenerative neurovascular niche in shaping the tumor microenvironment and brain metastatic landscape.

The metastatic microenvironment: Melanoma-microglia cross-talk promotes the malignant phenotype of melanoma cells.

Melanoma has the highest propensity to metastasize to the brain compared to other cancers, as brain metastases are found frequently high in patients who have prolonged survival with visceral metastasis. Once disseminated in the brain, melanoma cells communicate with brain resident cells that include astrocytes and microglia. Microglia cells are the resident macrophages of the brain and are the main immunological cells in the CNS involved in neuroinflammation. Data on the interactions between brain metastatic melanoma cells and microglia and on the role of microglia-mediated neuroinflammation in facilitating melanoma brain metastasis are lacking. To elucidate the role of microglia in melanoma brain metastasis progression, we examined the bidirectional interactions between microglia and melanoma cells in the tumor microenvironment. We identified the molecular and functional modifications occurring in brain-metastasizing melanoma cells and microglia cells after the treatment of each cell type with supernatants of the counter cell type. Both cells induced alteration in gene expression programs, cell signaling, and cytokine secretion in the counter cell type. Moreover, melanoma cells exerted significant morphological changes on microglia cells, enhanced proliferation, induced matrix metalloproteinase-2 (MMP-2) activation, and cell migration. Microglia cells induced phenotypic changes in melanoma cells increasing their malignant phenotype: increased melanoma proliferation, MMP-2 activity, cell migration, brain endothelial penetration, and tumor cells ability to grow as spheroids in 3D cultures. Our work provides a novel insight into the bidirectional interactions between melanoma and micoglia cells, suggesting the contribution of microglia to melanoma brain metastasis formation.

Cystatin C takes part in melanoma-microglia cross-talk: possible implications for brain metastasis.

The development of melanoma brain metastasis is largely dependent on mutual interactions between the melanoma cells and cells in the brain microenvironment. Here, we report that the extracellular cysteine protease inhibitor cystatin C (CysC) is involved in these interactions. Microglia-derived factors upregulated CysC secretion by melanoma. Similarly, melanoma-derived factors upregulated CysC secretion by microglia. Whereas CysC enhanced melanoma cell migration through a layer of brain endothelial cells, it inhibited the migration of microglia cells toward melanoma cells. CysC was also found to promote the formation of melanoma three-dimensional structures in matrigel. IHC analysis revealed increased expression levels of CysC in the brain of immune-deficient mice bearing xenografted human melanoma brain metastasis compared to the brain of control mice. Based on these in vitro and in vivo experiments we hypothesize that CysC promotes melanoma brain metastasis. Increased expression levels of CysC were detected in the regenerating brain of mice after stroke. Post-stroke brain with melanoma brain metastasis showed an even stronger expression of CysC. The in vitro induction of stroke-like conditions in brain microenvironmental cells increased the levels of CysC in the secretome of microglia cells, but not in the secretome of brain endothelial cells. The similarities between melanoma brain metastasis and stroke with respect to CysC expression by and secretion from microglia cells suggest that CysC may be involved in shared pathways between brain metastasis and post-stroke regeneration. This manifests the tendency of tumor cells to highjack physiological molecular pathways in their progression.

A history of exploring cancer in context.

The concept that progression of cancer is regulated by interactions of cancer cells with their microenvironment was postulated by Stephen Paget over a century ago. Contemporary tumour microenvironment (TME) research focuses on the identification of tumour-interacting microenvironmental constituents, such as resident or infiltrating non-tumour cells, soluble factors and extracellular matrix components, and the large variety of mechanisms by which these constituents regulate and shape the malignant phenotype of tumour cells. In this Timeline article, we review the developmental phases of the TME paradigm since its initial description. While illuminating controversies, we discuss the importance of interactions between various microenvironmental components and tumour cells and provide an overview and assessment of therapeutic opportunities and modalities by which the TME can be targeted.

ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis.

In an ongoing effort to identify molecular determinants regulating melanoma brain metastasis, we previously identified Angiopoietin-like 4 (ANGPTL4) as a component of the molecular signature of such metastases. The aim of this study was to determine the functional significance of ANGPTL4 in the shaping of melanoma malignancy phenotype, especially in the establishment of brain metastasis. We confirmed that ANGPTL4 expression is significantly higher in cells metastasizing to the brain than in cells from the cutaneous (local) tumor from the same melanoma in a nude mouse xenograft model, and also in paired clinical specimens of melanoma metastases than in primary melanomas from the same patients. In vitro experiments indicated that brain-derived soluble factors and transforming growth factor ß1 (TGFß1) up-regulated ANGPTL4 expression by melanoma cells. Forced over-expression of ANGPTL4 in cutaneous melanoma cells promoted their ability to adhere and transmigrate brain endothelial cells. Over-expressing ANGPTL4 in cells derived from brain metastases resulted in the opposite effects. In vivo data indicated that forced overexpression of ANGPTL4 promoted the tumorigenicity of cutaneous melanoma cells but did not increase their ability to form brain metastasis. This finding can be explained by inhibitory activities of brain-derived soluble factors. Taken together these findings indicate that ANGPTL4 promotes the malignancy phenotype of primary melanomas of risk to metastasize to the brain.

P-REX1 amplification promotes progression of cutaneous melanoma via the PAK1/P38/MMP-2 pathway.

P-REX1 (PIP3-dependent Rac exchange factor-1) is a guanine nucleotide exchange factor that activates Rac by catalyzing exchange of GDP for GTP bound to Rac. Aberrant up-regulation of P-REX1 expression has a role in metastasis however, copy number (CN) and function of P-REX1 in cutaneous melanoma are unclear. To explore the role of P-REX1 in melanoma, SNP 6.0 and Exon 1.0 ST microarrays were assessed. There was a higher CN (2.82-fold change) of P-REX1 in melanoma cells than in melanocytes, and P-REX1 expression was significantly correlated with P-REX1 CN. When P-REX1 was knocked down in cells by P-REX1 shRNA, proliferation, colony formation, 3D matrigel growth, and migration/invasiveness were inhibited. Loss of P-REX1 inhibited cell proliferation by inhibiting cyclin D1, blocking cell cycle, and increased cell apoptosis by reducing expression of the protein survivin. Knockdown of P-REX1 expression inhibited cell migration/invasiveness by disrupting P-REX1/RAC1/PAK1/p38/MMP-2 pathway. Assessment of patient tumors and disease outcome demonstrated lower distant metastasis-free survival among AJCC stage I/II/III patients with high P-REX1 expression compared to patients with low P-REX1 expression. These results suggest P-REX1 plays an important role in tumor progression and a potential theranostic target.