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Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout of the nuclear retinoid X receptor alpha (RXRαep−/−) results in increased melanoma formation after chronic ultraviolet-B (UVB) irradiation compared with control mice with functional RXRα. Melanomas from both groups of bigenic RXRαep−/− mice are larger in size with higher proliferative capacity, and exhibit enhanced angiogenic properties and increased expression of malignant melanoma markers. Analysis of tumor adjacent normal skin from these mice revealed altered expression of several biomarkers indicative of enhanced melanoma susceptibility, including reduced expression of tumor suppressor p53 and loss of PTEN, with concomitant increase in activated AKT. Loss of epidermal RXRα in combination with UVB significantly enhances invasion of melanocytic cells to draining lymph nodes in bigenic mice expressing oncogenic NRASQ61K compared with controls with functional RXRα. These results suggest a crucial role of keratinocytic RXRα to suppress formation of UVB-induced melanomas and their progression to malignant cancers in the context of driver mutations such as activated CDK4R24C/R24C or oncogenic NRASQ61K.Implications: These findings suggest that RXRα may serve as a clinical diagnostic marker and therapeutic target in melanoma progression and metastasis. Mol Cancer Res; 13(1); 186–96. ©2014 AACR.

Antibodies used for experiments. Supplementary Table S2: PCR primers used for genotyping of mouse lines.

Supplementary Figure S5: No change in infiltrating mast cells as determined by CEM staining is observed in melanocytic lesions from either Cdk4R24c or Tyr-NRASQ61K mice with epidermis-specific knockout of Rxra. Assay was performed on bleached 5 J.lm paraffin sections. Infiltrating mast cells are labeled with light blue staining. E=Epidermis, 0= Dermis. Scale bars= 50 micron

Supplementary Figure S8: Chronically UVB-irradiated tumor-adjacent normal skin from Tyr-NRASQBtK or Cdk4R24c mice with epidermis-specific knockout of Rxra show modest increase in CDK4 expression; downregulation of phosphorylated ERK, as well as increased E2F1 expression specifically in Rxraep-/- 1 Cdk4R24c mice. (A-D) Modest increase of CDK4 expression in both epidermis and dermis of Rxraep-/- I Tyr- NRASQ61K and Rxraep-/- 1 Cdk4R24c mice as compared to RxraL2/L2 controls, as determined by immunohistochemistry (5 IJm bleached paraffin sections). E=Epidermis, D= Dermis. Scale bars= 50 IJm. # = no statistical significance , *= p s 0.05, *** = p s 0.001 (E, F) Down regulation in phosphorylated ERK in tumor-adjacent normal skin of Rxraep-/- 1 Tyr-NRASQotK and Rxraep-/- 1 Cdk4R24cmice as compared to RxraL2/L2 controls; as shown by Western Blot (Two biological replicates for each group are shown). (G, H) Analysis of E2F1 protein levels in tumor-adjacent normal skin of Rxraep-/- 1 Tyr-NRASQotK and Rxraep-/- I Cdk4R24c mice as compared to RxraL2/L2 controls; as shown by Western Blot (Two biological replicates for each group are shown). No difference was observed in the Tyr-NRASQotK group, while increased E2F1 levels were observed in Rxraep-/- 1 Cdk4R'

Supplementary methods

Supplementary Figure S2: Chronic-UVB treated and parallel non-treated controls used in this study. (A,B) H&E staining of non-UVB treated skin from parallel age-matched mice (A) and TAN skin from chronic-UVB treated mice (B). In all groups, TAN skin has similar morphology to non-UVB treated skin, except for increased epidermal thickening; and dermal pigmentation which is enhanced further in the RXRaep-/- mice. Mice carrying Cdk4R24c in all groups show sebaceous gland hyperplasia within hair follicles (yellow boxes). E=Epidermis, D=Dermis, scale bar =100 IJm. (C) Epidermis measurements taken from H&E stained samples. In non-UVB treated mice, Tyr-NRAS0 61K or Cdk4R24c combined with Rxraep-/- mutation exhibited slightly thicker epidermis than RxraL2/L2 counterparts [7]. This is attenuated by chronic-UVB treatment, which results in a similarly thickened epidermis across all groups. ** = p

Supplementary Figure S1: Breeding/Genotyping of mouse lines used in this study. (A, B) Breeding crossings used. K14-Cre'g/O I RxraL2/L2 is also known as RXRaep-/-. (C) Semi-quantitative PCR was performed on biopsies containing both epidermal and non-epidermal tissue; to confirm genotypes with regard to presence/absence of K14-Cre recombinase (top), epidermis-specific ere-mediated excision of Rxra exon 4 (middle), and the mutant Cdk4R24c allele (bottom). Presence of K14-Cre recombinase is indicated by a single 349 bp band; animals negative for K14-Cre show no amplification. In K14-Cre positive animals, ere-mediated epidermis-specific excision of Rxra is indicated by a 110 bp (L-) band, while a 203 bp (L2) band is also amplified due to presence of non-epidermal tissue in the biopsy in which excision did not occur. Animals negative for K14-Cre show only the 203 bp (L2) band for Rxra, as no excision occurred in any tissue. Animals homozygous for the mutant Cdk4R24c allele show a single band at 530 bp, while animals homozygous for the WT Cdk4 allele show a single band at 440 bp. Primers used are detailed in the Supplementary Table S2. (D) Tyr-NRAS061K genotype is determined phenotypically 10-12 days postnatal by presence/absence of dark pigmentation of the foot pads.

Supplementary Figure S6: Heat Map and Clustergram of dysregulated genes in melanocytic lesions from Rxraep-/- 1 Tyr-NRASQ61K mice_ Tissue from melanocytic lesions was captured using Laser Capture Microdissection (LCM) (See Figure 4 for details). Isolated RNA was converted to eDNA and gene expression in Rxraep-/- lesions relative to RxraL2/L2 lesions was assayed using an RT-qPCR array (SA Biosciences, PAMM-033Z (Mouse Cancer)). Mean fold change relative to Rxrcf21L2 group is represented by the heat map (A). Three replicate arrays were performed for both RxraL2/L2 and Rxraep-/- groups; magnitude of gene expression for each replicate is shown in the clustergram (B), which correlates genes with similar expression levels.

Supplementary Figure S3: Controls for IHC experiments used in this study. (A) For all IHC studies, a liquid-repellant slide marker pen was used to separate out a single section on each slide that does not receive primary antibody but otherwise carried through the same labeling protocol; for use as a negative control to demonstrate that signals observed were specific. (B) PEP1 antibody (anti-TYRP1) and PEPS antibody (anti-TYRP2) give similar specific staining patterns for melanocytic cells in RxraL2/L2 neonatal skin following irradiation with a single UVB dose. (C) Fluorescent IHC for p-AKT and PTEN on chronically-irradiated TAN skin showing similar staining pattern to chromogenic IHC shown in Figure 5. (D,E) Non-UVB treated skin showed reduced staining intensity of PTEN, p-AKT, and p53 compared to irradiated skin. Unlike skin treated with chronic UVB, no discernable difference in expression pattern between RxraL2/L2 and bi enic Rxraep-/- mice was observed. E=E idermis, D=Dermis, scale bars =1 00 J.Jm.

Supplementary Figure S4: An increase in infiltrating macrophages is observed in melanocytic lesions from Cdk4R24cmice with epidermal-specific knockout of Rxra, but not in Rxraep-/- Tyr-NRASQBtK mice. No change in infiltrating CD4+ T-cells observed in melanocytic lesions from either Cdk4R24c or Tyr-NRASQ61K mice with epidermal-specific knockout of Rxra. (A, B) No change in infiltrating macrophages, as determined by fluorescent immunohistochemistry (I HC) of of 5 J.lm paraffin sections (MAC1 antigen), was observed in Rxraep-/- 1 Tyr-NRAS061K mice as compared to RxraL2/L2 controls. (C, D) An increase in infiltrating macrophages was observed in Rxraep-/- 1 Cdk4R24c mice as compared to RxraL2/L2 controls. (E-H) No change in infiltrating CD4+ T-cells as determined by fluorescent IHC was observed in Rxraep-/- Tyr-NRAS061K or Rxraep-/- Cdk4R24c mice as compared to RxraL 21L2 controls. Scale bars= 50 J.lm. ** = p :5 0.01

PURPOSE IDH mutations occur in about 30% of patients with cholangiocarcinoma. Analysis of mutations in circulating tumor DNA (ctDNA) can be performed by droplet digital polymerase chain reaction (ddPCR). The analysis of ctDNA is a feasible approach to detect IDH mutations. METHODS We isolated ctDNA from the blood of patients with IDH-mutated advanced cholangiocarcinoma collected at baseline, on therapy, and at progression to isocitrate dehydrogenase (IDH) inhibitors. RESULTS Of 31 patients with IDH1R132 (n = 26) or IDH2R172 mutations (n = 5) in the tumor, IDH mutations were detected in 84% of ctDNA samples analyzed by ddPCR and in 83% of ctDNA samples analyzed by next-generation sequencing (NGS). Patients with a low variant allele frequency of ctDNA detected by NGS at baseline had a longer median time to treatment failure compared to patients with high variant allele frequency of ctDNA (3.6 v 1.5 months; P = .008). Patients with a decrease in IDH-mutated ctDNA on therapy by ddPCR compared with no change/increase had a trend to a longer median survival ( P = .07). Most frequent emergent alterations in ctDNA by NGS at progression were ARID1A (n = 3) and TP53 mutations (n = 3). CONCLUSION Detection of IDH mutations in ctDNA in patients with advanced cholangiocarcinoma is feasible, and dynamic changes in ctDNA can correspond with the clinical course and clonal evolution.

Treatment with vemurafenib, a potent and selective inhibitor of mitogen-activated protein kinase signaling downstream of the BRAFV600E oncogene, elicits dramatic clinical responses in patients with metastatic melanoma. Unfortunately, the clinical utility of this drug is limited by a high incidence of drug resistance. Thus, there is an unmet need for alternative therapeutic strategies to treat vemurafenib-resistant metastatic melanomas. We have conducted high-throughput screening of two bioactive compound libraries (Siga and Spectrum libraries) against a metastatic melanoma cell line (A2058) and identified two structurally analogous compounds, deguelin and rotenone, from a cell viability assay. Vemurafenib-resistant melanoma cell lines, A2058R and A375R (containing the BRAFV600E mutation), also showed reduced proliferation when treated with these two compounds. Deguelin, a mitochondrial complex I inhibitor, was noted to significantly inhibit oxygen consumption in cellular metabolism assays. Mechanistically, deguelin treatment rapidly activates AMPK signaling, which results in inhibition of mTORC1 signaling and differential phosphorylation of mTORC1's downstream effectors, 4E-BP1 and p70S6 kinase. Deguelin also significantly inhibited ERK activation and Ki67 expression without altering Akt activation in the same timeframe in the vemurafenib-resistant melanoma cells. These data posit that treatment with metabolic regulators, such as deguelin, can lead to energy starvation, thereby modulating the intracellular metabolic environment and reducing survival of drug-resistant melanomas harboring BRAF V600E mutations.

Gut dysbiosis is commonly observed in patients with cirrhosis and chronic gastrointestinal disorders, however, its effect on anti-tumor immunity in the liver is largely unknown. Here we studied how the gut microbiome affects anti-tumor immunity in cholangiocarcinoma. Primary sclerosing cholangitis (PSC) or colitis, two known risk factors for cholangiocarcinoma, which promote tumor development in mice caused an accumulation of CXCR2(+) polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). A decrease in gut barrier function observed in mice with PSC and colitis allowed gut derived bacteria and lipopolysaccharide (LPS) to appear in the liver and induced CXCL1 expression in hepatocytes through a TLR4-dependent mechanism and an accumulation of CXCR2(+) PMN-MDSC. On the contrary, neomycin treatment blocked CXCL1 expression, PMN-MDSC accumulation and inhibited tumor growth even in the absence of liver disease or colitis. Our study demonstrates that the gut microbiome controls hepatocytes to form an immunosuppressive environment by increasing PMN-MDSC to promote liver cancer.

The study and comprehension of the molecular mechanisms underlying cancer biology strongly rely on mouse modeling. An ideal mouse model should have molecular, histopathological, and etiological characteristics as close as possible to those of the corresponding human tumors. Among solid tumors, colorectal cancer (CRC) is one of the malignancies that best suits reproduction in an animal model: it evolves through a progressive set of molecular events and is generally associated with a precise histopathology and a neat cataloging of stages and grades. The development of refined CRC mouse models over several decades has seen them recently evolve toward sophisticated systems that ever more closely approximate the human pathology, with different models addressing different human CRC subtypes. In particular, a metastatic CRC model has been seen as a "holy grail" in this field, and we describe in this review the path taken to achieve metastatic models and discuss the path forward.

Table S1. Antibodies used for immuno-histochemical staining and immuno-blotting with details including the host, detection for-, the source, application and the dilution used. Table S2. PCR primers used for genotyping of mouse lines with the forward and reverse primers for K14-Cre, RXRα and CDK4, their sequences and the expected band sizes. Additional Methods. (DOCX 19 kb)

Metastatic melanoma has a high mortality rate due to lymphatic progression of the disease. Current treatment is surgery followed by radiation and intravenous chemotherapy. However, drawbacks for current chemotherapeutics lie in the fact that they develop resistance and do not achieve therapeutic concentrations in the lymphatic system. We hypothesize that a three-drug nanoscale drug delivery system, tailored for lymphatic uptake, administered subcutaneously, will have decreased drug resistance and therefore offer better therapeutic outcomes. We prepared and characterized nanoparticles (NPs) with docetaxel, everolimus, and LY294002 in polyethyleneglycol-block-poly(ε-caprolactone) (PEG-PCL) polymer with different charge distributions by modifying the ratio of anionic and neutral end groups on the PEG block. These NPs are similarly sized (~48 nm), with neutral, partially charged, or fully charged surface. The NPs are able to load ~2mg/mL of each drug and are stable for 24h. The NPs are assessed for safety and efficacy in two transgenic metastatic melanoma mouse models. All the NPs were safe in both models based on general appearance, weight changes, death, and blood biochemical analyses. The partially charged NPs are most effective in decreasing the number of melanocytes at both the proximal (sentinel) lymph node (LN) and the distal LN from the injection site. The neutral NPs are efficacious at the proximal LN, while the fully charged NPs have no effect on either LNs. Thus, our data indicates that the NP surface charge and lymphatic efficacy are closely tied to each other and the partially charged NPs have the highest potential in treating metastatic melanoma.

Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout of the nuclear retinoid X receptor alpha (RXRαep−/−) results in increased melanoma formation after chronic ultraviolet-B (UVB) irradiation compared with control mice with functional RXRα. Melanomas from both groups of bigenic RXRαep−/− mice are larger in size with higher proliferative capacity, and exhibit enhanced angiogenic properties and increased expression of malignant melanoma markers. Analysis of tumor adjacent normal skin from these mice revealed altered expression of several biomarkers indicative of enhanced melanoma susceptibility, including reduced expression of tumor suppressor p53 and loss of PTEN, with concomitant increase in activated AKT. Loss of epidermal RXRα in combination with UVB significantly enhances invasion of melanocytic cells to draining lymph nodes in bigenic mice expressing oncogenic NRASQ61K compared with controls with functional RXRα. These results suggest a crucial role of keratinocytic RXRα to suppress formation of UVB-induced melanomas and their progression to malignant cancers in the context of driver mutations such as activated CDK4R24C/R24C or oncogenic NRASQ61K. Implications: These findings suggest that RXRα may serve as a clinical diagnostic marker and therapeutic target in melanoma progression and metastasis. Mol Cancer Res; 13(1); 186–96. ©2014 AACR.

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a “non-cell autonomous” manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a “cell autonomous” manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma., Author Summary Melanoma is the deadliest form of skin cancer. It derives from melanocytes, the melanin-producing cells of our skin, which give our skin its tone in addition to protecting it from harmful effects of ultraviolet radiation (UVR). Changes in the skin microenvironment, such as signaling from other cell types, can influence melanoma progression. While several key genes in melanoma development have been identified, the underlying mechanisms are complex; different combinations of mutations can result in melanoma formation and genetic profiles of tumors can vary greatly among patients. Therefore, identification of novel therapeutic targets is crucial. Our present study uses a tissue-specific gene ablation strategy to characterize a novel role of type II nuclear receptors [Retinoid-X-Receptors (RXRs)] in melanocytes to control UVR-induced skin immune responses and cell survival. Several of these observed changes are risk factors for melanoma progression and identify RXRs as potential drug targets for melanoma diagnosis, prevention, and treatment. This newly-discovered role of retinoid receptor signaling in immune surveillance can be studied in different types of cancer and in other diseases including metabolic syndromes and atherosclerosis. The identified pathway is ideal for targeting using specific ligands or small molecule modulators of RXR signaling in different cell types and tissues.

It is appreciated far and wide that increased and regular consumption of fruits and vegetables is linked with noteworthy anticancer benefits. Extensively consumed as a spice in foods and beverages worldwide, ginger (Zingiber officinale Roscoe) is an excellent source of several bioactive phenolics, including non-volatile pungent compounds such as gingerols, paradols, shogaols and gingerones. Ginger has been known to display anti-inflammatory, antioxidant and antiproliferative activities, indicating its promising role as a chemopreventive agent. Here, we show that whole ginger extract (GE) exerts significant growth-inhibitory and death-inductory effects in a spectrum of prostate cancer cells. Comprehensive studies have confirmed that GE perturbed cell-cycle progression, impaired reproductive capacity, modulated cell-cycle and apoptosis regulatory molecules and induced a caspase-driven, mitochondrially mediated apoptosis in human prostate cancer cells. Remarkably, daily oral feeding of 100 mg/kg body weight of GE inhibited growth and progression of PC-3 xenografts by approximately 56 % in nude mice, as shown by measurements of tumour volume. Tumour tissue from GE-treated mice showed reduced proliferation index and widespread apoptosis compared with controls, as determined by immunoblotting and immunohistochemical methods. Most importantly, GE did not exert any detectable toxicity in normal, rapidly dividing tissues such as gut and bone marrow. To the best of our knowledge, this is the first report to demonstrate the in vitro and in vivo anticancer activity of whole GE for the management of prostate cancer.