Megakaryocytes Mediate Hyperglycemia-Induced Tumor Metastasis.

High blood glucose has long been established as a risk factor for tumor metastasis, yet the molecular mechanisms underlying this association have not been elucidated. Here we describe that hyperglycemia promotes tumor metastasis via increased platelet activity. Administration of glucose, but not fructose, reprogrammed the metabolism of megakaryocytes to indirectly prime platelets into a prometastatic phenotype with increased adherence to tumor cells. In megakaryocytes, a glucose metabolism-related gene array identified the mitochondrial molecular chaperone glucose-regulated protein 75 (GRP75) as a trigger for platelet activation and aggregation by stimulating the Ca2+-PKCα pathway. Genetic depletion of Glut1 in megakaryocytes blocked MYC-induced GRP75 expression. Pharmacologic blockade of platelet GRP75 compromised tumor-induced platelet activation and reduced metastasis. Moreover, in a pilot clinical study, drinking a 5% glucose solution elevated platelet GRP75 expression and activated platelets in healthy volunteers. Platelets from these volunteers promoted tumor metastasis in a platelet-adoptive transfer mouse model. Together, under hyperglycemic conditions, MYC-induced upregulation of GRP75 in megakaryocytes increases platelet activation via the Ca2+-PKCα pathway to promote cancer metastasis, providing a potential new therapeutic target for preventing metastasis. SIGNIFICANCE: This study provides mechanistic insights into a glucose-megakaryocyte-platelet axis that promotes metastasis and proposes an antimetastatic therapeutic approach by targeting the mitochondrial protein GRP75.

Psychologic Stress Drives Progression of Malignant Tumors via DRD2/HIF1α Signaling.

Although it is established that the sustained psychologic stress conditions under which patients with tumors often reside accelerates malignant progression of tumors, the molecular mechanism behind this association is unclear. In this work, the effect of psychologic stress on tumor progression was verified using a stress-stimulated tumor-bearing mouse model (Str-tumor). Both D2 dopamine receptor (DRD2) and hypoxia-inducible factor-1α (HIF1α) were highly expressed in the nucleus of Str-tumors. Treatment with trifluoperazine (TFP), a DRD2 inhibitor, elicited better antitumor effects in Str-tumors than the control group. These results indicate that DRD2 may mediate stress-induced malignant tumor progression. DRD2 interacted with von Hippel-Lindau (VHL) in the nucleus, and competitive binding of DRD2 and HIF1α to VHL resulted in reduced ubiquitination-mediated degradation of HIF1α, enhancing the epithelial-mesenchymal transition of tumor cells. TFP acted as an interface inhibitor between DRD2 and VHL to promote the degradation of HIF1α. In conclusion, DRD2 may promote the progression of malignant tumors induced by psychologic stress via activation of the oxygen-independent HIF1α pathway, and TFP may serve as a therapeutic strategy for stress management in patients with cancer. SIGNIFICANCE: This work identifies DRD2 regulation of HIF1α as a mechanism underlying the progression of malignant tumors stimulated by psychologic stress and suggests that DRD2 inhibition can mitigate these stress conditions in patients.See related commentary by Bernabé, p. 5144.

Monoclonal Antibodies to PRAME Protein Slow the Development of PRAME-Expressing Tumor.

Two monoclonal antibodies recognizing non-overlapping epitopes of the PRAME protein were injected into immunocompetent mice to study their influence on the growth of subcutaneous tumor nodes. The B16F10 murine melanoma line, either expressing human PRAME protein or bearing only a vector without PRAME gene, were used as transplants. Each of the antibodies showed the ability to suppress tumor growth of a PRAME-expressing tumour, but not a tumor without PRAME.

Dysfunction of CD8 + PD-1 + T cells in type 2 diabetes caused by the impairment of metabolism-immune axis.

The metabolic changes and dysfunction in CD8 + T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8 + splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8 + splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8 + PD-1 + T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8 + PD-1 + T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity.

Protective role of histone deacetylase 4 from ultraviolet radiation-induced DNA lesions.

Ultraviolet B (UVB) exposure is a core factor that leads to skin disease or carcinogenesis through the insufficient repair of DNA lesions. UVB-induced DNA lesions are mainly removed by the nucleotide excision repair (NER) mechanism. The expression of histone deacetylase 4 (HDAC4) is altered in the skin upon UVB exposure, indicating its possible implication in UVB-induced DNA lesions repair. Here, we investigated the role of HDAC4 in the NER removal of the main classes of UVB-induced DNA lesions consisting of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). We found that UVB irradiation increased HDAC4 expression at both the mRNA and protein levels. HDAC4 interacted with NER factor XPC, which played an important role in effectively removing the UVB-induced DNA lesions. This study provides an understanding of the HDAC4 function in DNA repair, which will allow the development of efficient strategies to protect the skin from UVR-induced diseases.

Intravital Imaging of Adoptive T-Cell Morphology, Mobility and Trafficking Following Immune Checkpoint Inhibition in a Mouse Melanoma Model.

Efficient T-cell targeting, infiltration and activation within tumors is crucial for successful adoptive T-cell therapy. Intravital microscopy is a powerful tool for the visualization of T-cell behavior within tumors, as well as spatial and temporal heterogeneity in response to immunotherapy. Here we describe an experimental approach for intravital imaging of adoptive T-cell morphology, mobility and trafficking in a skin-flap tumor model, following immune modulation with immune checkpoint inhibitors (ICIs) targeting PD-L1 and CTLA-4. A syngeneic model of ovalbumin and mCherry-expressing amelanotic mouse melanoma was used in conjunction with adoptively transferred OT-1+ cytotoxic T-cells expressing GFP to image antigen-specific live T-cell behavior within the tumor microenvironment. Dynamic image analysis of T-cell motility showed distinct CD8+ T-cell migration patterns and morpho-dynamics within different tumor compartments in response to ICIs: this approach was used to cluster T-cell behavior into four groups based on velocity and meandering index. The results showed that most T-cells within the tumor periphery demonstrated Lévy-like trajectories, consistent with tumor cell searching strategies. T-cells adjacent to tumor cells had reduced velocity and appeared to probe the local environment, consistent with cell-cell interactions. An increased number of T-cells were detected following treatment, traveling at lower mean velocities than controls, and demonstrating reduced displacement consistent with target engagement. Histogram-based analysis of immunofluorescent images from harvested tumors showed that in the ICI-treated mice there was a higher density of CD31+ vessels compared to untreated controls and a greater infiltration of T-cells towards the tumor core, consistent with increased cellular trafficking post-treatment.

La inflamación inducida por formalina favorece el desarrollo del melanoma B16 en ratones Balb/C / inflamation induced by formaline promotes the development of melanoma B16 in Balb/C mice

El cáncer es la segunda causa de muerte en el mundo representando el melanoma 1% de todos los tipos de cáncer. Se ha planteado que el cáncer es una enfermedad inflamatoria sistémica que genera radicales libres causando mutaciones y liberan factores tróficos que favorecen la iniciación tumoral y la proliferación celular, respectivamente. Con el objetivo de estudiar el efecto de la inflamación inducida por formalina sobre el desarrollo del melanoma B16, 22 ratones Balb/C fueron distribuidos en tres grupos: Control Melanoma, Melanoma-Formalina y Control Formalina. A los grupos CF y MF se les aplicó 20 µl de Formalina al 2% en el dorso de la pata trasera derecha a nivel subcutáneo; a los grupos CM y MF se le trasplantaron 100.000 células melanocíticas vía subcutánea en la superficie plantar de la pata derecha, 24 horas posteriores a la formalina. Los ratones del grupo CF desarrollaron una inflamación que fue máxima entre la primera y segunda semana y luego cedió progresivamente hasta desaparecer a la sexta semana. Los ratones del grupo CM desarrollaron máculas tumorales hasta 30 mm² que involucionaron espontáneamente. Los ratones del grupo MF desarrollaron masas tumorales que alcanzaron hasta 300 mm³ entre la 3-4 semanas post-trasplante y luego disminuyeron progresivamente de volumen. Los ratones de los grupos CF y MF disminuyeron significativamente de peso respecto al grupo CM. En conclusión, la inflamación inducida por formalina favorece el desarrollo tumoral en un modelo alogénico de melanoma maligno.

Cancer is the second cause of death around the world, representing melanoma 1% of all cancer. It has been suggested that cancer is a systemic inflammatory disease that generates free radicals causing mutations and releasing trophic factors that favors tumor initiation and cell proliferation. In order to study the effect of formalin-induced inflammation on the development of B16 melanoma, 22 Balb/C mice were divided into three groups: Control Melanoma (CM), Melanoma-Formalin (MF) and Control Formalin (CF). CF and MF groups were injected with 20 µl of 2% formalin on the back of the right paw at the subcutaneous level; CM and MF groups were transplanted with 100,000 melanocytic cells subcutaneously in the plantar surface of the right paw, 24 hours after formalin. CF group mice developed an inflammation that was maximal between the first and second week, then progressively diminished until disappearance by the sixth week. CM group mice developed tumoral macules up to 30 mm², which involute spontaneously. MF group mice developed tumor masses that reached up to 300 mm³ between 3-4 weeks post-transplant and then progressively decreased in volume. CF and MF mice significantly decreased in weight with respect to CM group. In conclusion, inflammation induced by formalin favors tumor development in an allogenic model of malignant melanoma, indicating that anti-inflammatory treatments may be useful in the management of melanoma.

Melanoma asociado a nevo melanocítico / Melanoma Arising in a Melanocytic Nevus

La asociación clínica o histológica de un melanoma con un nevo melanocítico previo varía entre las series previamente publicadas de forma prominente. Esta variación se produce tanto en función de si se tienen en cuenta los restos histológicos (4-72%) como en función de la presencia de una lesión clínicamente evidente (42-85%). La asociación histológica con un nevo se ha correlacionado con factores pronósticos favorables, mientras que la asociación clínica por el contrario lo hace con factores desfavorables. Esta revisión pretende abordar las características vinculadas con el melanoma asociado a nevo, en relación con: la teoría de las vías divergentes para el desarrollo de un melanoma cutáneo de Whiteman, los factores vinculados a nevogenicidad y la genética y biología molecular del melanoma y sus lesiones precursoras. Adicionalmente, basado en el análisis agregado de un total de 16.162 pacientes publicados en la literatura hasta la fecha, se ha calculado la proporción total de melanomas histológicamente asociados a nevo melanocítico, cifrándose en el 29,8%

The association of melanoma with a preexisting melanocytic nevus varies considerably between series, depending on whether the association is based on histological signs (4%-72%) or a clinically evident lesion (42%-85%). Histological association with a nevus correlates with favorable prognostic factors, whereas a clinical association correlates with unfavorable factors. In this review, we discuss the characteristics of nevus-associated melanoma from different perspectives: Whiteman's divergent pathway hypothesis for the development of cutaneous melanoma; and the factors involved in nevogenicity, including both the genetic and molecular factors involved in the development of the melanoma and its precursor lesions. Finally, a cumulative analysis of the 16 162 cases reported in the literature revealed that 29.8% of melanomas are histologically associated with a melanocytic nevus

Tumor suppressor ARF regulates tissue microenvironment and tumor growth through modulation of macrophage polarization.

Tumor microenvironment has been described to play a key role in tumor growth, progression, and metastasis. Macrophages are a major cellular constituent of the tumor stroma, and particularly tumor associated macrophages (TAMs or M2-like macrophages) exert important immunosuppressive activity and a pro-tumoral role within the tumor microenvironment. Alternative-reading frame (ARF) gene is widely inactivated in human cancer. We have previously demonstrated that ARF deficiency severely impairs inflammatory response establishing a new role for ARF in the regulation of innate immunity. On the basis of these observations, we hypothesized that ARF may also regulates tumor growth through recruitment and modulation of the macrophage phenotype in the tumor microenvironment. Xenograft assays of B16F10 melanoma cells into ARF-deficient mice resulted in increased tumor growth compared to those implanted in WT control mice. Tumors from ARF-deficient mice exhibited significantly increased number of TAMs as well as microvascular density. Transwell assays showed crosstalk between tumor cells and macrophages. On the one hand, ARF-deficient macrophages modulate migratory ability of the tumor cells. And on the other, tumor cells promote the skewing of ARF-/- macrophages toward a M2-type polarization. In conclusion, these results demonstrate that ARF deficiency facilitates the infiltration of macrophages into the tumor mass and favors their polarization towards a M2 phenotype, thus promoting tumor angiogenesis and tumor growth. This work provides novel information about the critical role of ARF in the modulation of tumor microenvironment.

Acute exposure to ultraviolet-B radiation modulates sex steroid hormones and receptor expression in the skin and may contribute to the sex bias of melanoma in a fish model.

Using the Xiphophorus fish melanoma model, we show a strong male bias for sunlight-induced malignant melanoma, consistent with that seen in the human population. To examine underlying factors, we exposed adult X. couchianus fish to a single, sublethal dose of UVB and measured circulating sex steroid hormones and expression of associated hormone receptor genes over a 24-h period. We found that a single exposure had profound effects on circulating levels of steroid hormones with significant decreases for all free sex steroids at 6 and 24 h and increases in conjugated 2-estradiol and 11-ketotestosterone at 6 and 24 h, respectively. Whereas ARα expression increased in male and female skin, neither ARß nor either of the ERs showed significant responses to UVB in either sex. The rapid response of male androgens and their receptors in the skin after UVB irradiation implicates hormones in the male bias of skin cancer and suggests that the photoendocrine response immediately after UV exposure may be relevant to melanomagenesis.

Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma.

Malignant melanoma is frequently driven by mutational activation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) accompanied by silencing of the phosphatase and tensin homology (PTEN) tumor suppressor. Despite the implied importance of PI3K signaling in PTENNull melanomas, mutational activation of the gene encoding the catalytic subunit of PI3Kα (PIK3CA), is rarely detected. Since PTEN has both PI3-lipid phosphatase-dependent and -independent tumor suppressor activities, we investigated the contribution of PI3K signaling to BRAFV600E-induced melanomagenesis using mouse models, cultured melanoma cells, and PI3K pathway-targeted inhibitors. These experiments revealed that mutationally activated PIK3CAH1047R cooperates with BRAFV600E for melanomagenesis in mice. Moreover, pharmacological inhibition of PI3Ks prevented growth of BRAFV600E/PTENNull melanomas in vivo and in tissue culture. Combined inhibition of BRAFV600E and PI3K had more potent effects on the regression of established BRAFV600E/PTENNull melanomas and cultured melanoma cells than individual blockade of either pathway. Surprisingly, growth of BRAFV600E/PIK3CAH1047R melanomas was dependent on the protein kinase AKT; however, AKT inhibition had no effect on growth of BRAFV600E/PTENNull melanomas. These data indicate that PTEN silencing contributes a PI3K-dependent, but AKT-independent, function in melanomagenesis. Our findings enhance our knowledge of how BRAFV600E and PI3K signaling cooperate in melanomagenesis and provide preclinical validation for combined pathway-targeted inhibition of PI3K and BRAFV600E in the therapeutic management of BRAFV600E/PTENNull melanomas.

Hmgb1-IL-23-IL-17-IL-6-Stat3 axis promotes tumor growth in murine models of melanoma.

In order to understand how tumor cells can escape immune surveillance mechanisms and thus develop antitumor therapies, it is critically important to investigate the mechanisms by which the immune system interacts with the tumor microenvironment. In our current study, IL-17 deficiency results in reduced melanoma tumor size, diminished numbers of proliferating cells and blood vessels, and decreased percentage of CD11b(+)Gr-1(+) MDSCs in tumor tissues. IL-17 promotes IL-6 induction and Stat3 activation. Treatment of Stat3 inhibitor WP1066 in B16-F10 tumor cells inoculated wild-type mice inhibits tumor growth. Additional administration of recombinant IL-6 into B16-F10 tumor-bearing IL-17(-/-) mice results in markedly increased tumor size and p-Stat3 expression, whereas additional recombinant IL-17 administration into B16-F10 tumor-bearing wild-type mice treated with anti-IL-6 mAb does not significantly alter the tumor growth and p-Stat3 expression. In our further study, blockade of Hmgb1-RAGE pathway inhibits melanoma tumor growth and reduces production of IL-23 and IL-17. All these data suggest that Hmgb1-IL-23-IL-17-IL-6-Stat3 axis plays a pivotal role in tumor development in murine models of melanoma, and blocking any portion of this axis will attenuate melanoma tumor growth.

MDM4 is a key therapeutic target in cutaneous melanoma.

The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma-a highly chemotherapy-resistant disease-TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (∼65%) of stage I-IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras. MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy.

Recent advances in sunlight-induced carcinogenesis using the Xiphophorus melanoma model.

Unlike breast and prostate cancers, the nature and sequence of critical genetic and epigenetic events involved in the initiation and progression of melanoma are not well understood. A contributing factor to this dilemma, especially given our current understanding of the importance of UV light in melanoma etiology, is the lack of quality UV-inducible melanoma animal models. In this study we elaborate on the capability of UV light to induce cutaneous malignant melanomas (CMM) in Xiphophorus fishes, which were previously found to develop melanomas after acute neonatal UVB irradiation. In two separate tumorigenesis experiments, we exposed adult Xiphophorus hybrids to either acute UVB irradiations (5 consecutive daily treatments) or chronic solar irradiations (continuous UVA/UVB treatment for 9 months). Acute adult UVB irradiation resulted in the significant induction of melanomas, and moreover, this induction rate is equivalent to that of animals exposed to acute neonatal UVB irradiation. This study represents the first evidence that acute adult UVB irradiation, in the absence of any early life exposures, induces CMM. Similar to the findings conducted on other divergent melanoma models, including HGF/SF transgenic mice and Monodelphis domestica, prolonged chronic solar UV was not a factor in melanomagenesis.

Metastasis suppressor NM23-H1 promotes repair of UV-induced DNA damage and suppresses UV-induced melanomagenesis.

Reduced expression of the metastasis suppressor NM23-H1 is associated with aggressive forms of multiple cancers. Here, we establish that NM23-H1 (termed H1 isoform in human, M1 in mouse) and two of its attendant enzymatic activities, the 3'-5' exonuclease and nucleoside diphosphate kinase, are novel participants in the cellular response to UV radiation (UVR)-induced DNA damage. NM23-H1 deficiency compromised the kinetics of repair for total DNA polymerase-blocking lesions and nucleotide excision repair of (6-4) photoproducts in vitro. Kinase activity of NM23-H1 was critical for rapid repair of both polychromatic UVB/UVA-induced (290-400 nm) and UVC-induced (254 nm) DNA damage, whereas its 3'-5' exonuclease activity was dominant in the suppression of UVR-induced mutagenesis. Consistent with its role in DNA repair, NM23-H1 rapidly translocated to sites of UVR-induced (6-4) photoproduct DNA damage in the nucleus. In addition, transgenic mice hemizygous-null for nm23-m1 and nm23-m2 exhibited UVR-induced melanoma and follicular infundibular cyst formation, and tumor-associated melanocytes displayed invasion into adjacent dermis, consistent with loss of invasion-suppressing activity of NM23 in vivo. Taken together, our data show a critical role for NM23 isoforms in limiting mutagenesis and suppressing UVR-induced melanomagenesis.

Ob/ob serum promotes a mesenchymal cell phenotype in B16BL6 melanoma cells.

In 2009, malignant melanoma was responsible for approximately 9,000 deaths in the US. These deaths are often associated with aggressive metastasis to secondary sites such as the lungs. Epidemiological and animal studies suggest that obesity is a risk factor for melanoma. Others have shown that B16BL6 melanoma cells metastasize more aggressively in obese ob/ob than in lean mice. However, the mechanism by which obesity promotes B16BL6 melanoma metastasis in ob/ob mice has not been identified. In the present study, we used serum obtained from control and ob/ob leptin-deficient obese mice to determine if obese serum increases the aggressive phenotype of melanoma cells. Results showed that ob/ob serum has higher levels of resistin, insulin, tPAI1, IL-6, TNF-α, and MCP-1 compared to control serum. We showed that ob/ob serum increases the invasive ability of B16BL6 melanomas. To further determine the mechanism by which ob/ob serum increases the invasive ability of melanomas, we determined the effect of ob/ob and control serum on genes associated with the epithelial-to-mesenchymal transition (EMT). Cancer cells with a mesenchymal phenotype have a higher metastatic ability. Snai1 and Twist are genes that are strongly associated with EMT and metastasis of melanomas. Our results showed that ob/ob serum increases the expression of Snai1 and Twist. Moreover, ob/ob serum increased matrix metalloproteast 9 (MMP9) activity and decreased the expression of E-cadherin and the metastasis suppressor gene Kiss1. In summary, results suggest that obesity may increase the metastatic ability of melanoma by promoting a mesenchymal cell phenotype.

The roles of P- and E-selectins and P-selectin glycoprotein ligand-1 in primary and metastatic mouse melanomas.

BACKGROUND: Malignant melanoma is often accompanied by a host response of inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. OBJECTIVE: To evaluate the role of adhesion molecules, including P-selectin glycoprotein ligand-1 (PSGL-1), P-selectin, and E-selectin. METHODS: Subcutaneous primary growth and metastasis to the lung of B16 melanoma cells were examined in mice lacking PSGL-1, P-selectin, or E-selectin. RESULTS: Primary subcutaneous growth of B16 melanoma was augmented by loss of PSGL-1, P-selectin, or E-selectin, while pulmonary metastasis was reduced by the loss of E-selectin. The enhancement of subcutaneous tumor growth was associated with a reduced accumulation of natural killer cells, CD4(+) T cells and CD8(+) T cells, while the attenuation of pulmonary metastasis was related to the numbers of CD8(+) T cells. The expressions of transforming growth factor (TGF)-ß and interleukin (IL)-6 were correlated with primary subcutaneous growth; TGF-ß, IL-6, and interferon-γ were related to number of metastatic lung nodules. Cytotoxicity against melanoma cells in splenocytes and in tumor-draining lymph node cells were not defective by the absence of adhesion molecules, suggesting that the enhancement of tumor growth and metastasis caused by the loss of selectins results from an impaired migration of effector cells into the tissue. CONCLUSIONS: The results indicate the complexity of anti-tumor responses mediated by adhesion molecules in primary subcutaneous tumors and pulmonary metastasis of murine experimental melanoma.