Matthew A. Bill

The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity

BackgroundWe characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells.ResultsFLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-γ-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-γ, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-γ production when cultured with K562 targets as compared to vehicle (DMSO).ConclusionsThese data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.

Curcumin induces proapoptotic effects against human melanoma cells and modulates the cellular response to immunotherapeutic cytokines

Curcumin has potential as a chemopreventative and chemotherapeutic agent, but its interactions with clinically relevant cytokines are poorly characterized. Because cytokine immunotherapy is a mainstay of treatment for malignant melanoma, we hypothesized that curcumin could modulate the cellular responsiveness to interferons and interleukins. As a single agent, curcumin induced a dose-dependent increase in apoptosis of human melanoma cell lines, which was most prominent at doses >10 μmol/L. Immunoblot analysis confirmed that curcumin induced apoptosis and revealed caspase-3 processing, poly ADP ribose polymerase cleavage, reduced Bcl-2, and decreased basal phosphorylated signal transducers and activators of transcription 3 (STAT3). Despite its proapoptotic effects, curcumin pretreatment of human melanoma cell lines inhibited the phosphorylation of STAT1 protein and downstream gene transcription following IFN-α and IFN-γ as determined by immunoblot analysis and real time PCR, respectively. Pretreatment of peripheral blood mononuclear cells from healthy donors with curcumin also inhibited the ability of IFN-α, IFN-γ, and interleukin-2 to phosphorylate STAT proteins critical for their antitumor activity (STAT1 and STAT5, respectively) and their respective downstream gene expression as measured by real time PCR. Finally, stimulation of natural killer (NK) cells with curcumin reduced the level of interleukin-12–induced IFN-γ secretion, and production of granzyme b or IFN-γ upon coculture with A375 melanoma cells or NK-sensitive K562 cells as targets. These data show that although curcumin can induce apoptosis of melanoma cells, it can also adversely affect the responsiveness of immune effector cells to clinically relevant cytokines that possess antitumor properties. [Mol Cancer Ther 2009;8(9):2726–35]

Structurally modified curcumin analogs inhibit STAT3 phosphorylation and promote apoptosis of human renal cell carcinoma and melanoma cell lines.

The Janus kinase-2 (Jak2)-signal transducer and activator of transcription-3 (STAT3) pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC) and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3), and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma.

PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27Kip1

Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27Kip1 expression in human melanoma cells.

UV Light B–Mediated Inhibition of Skin Catalase Activity Promotes Gr-1+CD11b+ Myeloid Cell Expansion

Skin cancer incidence and mortality are higher in men compared to women, but the causes of this sex discrepancy remain largely unknown. Ultraviolet light exposure induces cutaneous inflammation and neutralizes cutaneous antioxidants. Gr-1+CD11b+ myeloid cells are heterogeneous bone marrow-derived cells that promote inflammation-associated carcinogenesis. Reduced activity of catalase, an antioxidant present within skin, has been associated with skin carcinogenesis. We utilized the outbred, immune competent Skh-1 hairless mouse model of ultraviolet light B (UVB)-induced inflammation and non-melanoma skin cancer to further define sex discrepancies in UVB-induced inflammation. Our results demonstrated that male skin had relatively lower baseline catalase activity, which was inhibited following acute UVB exposure in both sexes. Further analysis revealed that skin catalase activity inversely correlated with splenic Gr-1+CD11b+ myeloid cell percentage. Acute UVB exposure induced Gr-1+CD11b+ myeloid cell skin infiltration, which was inhibited to a greater extent in males by topical catalase treatment. In chronic UVB studies, we demonstrated that the percentage of splenic Gr-1+CD11b+ myeloid cells was 55% higher in male tumor-bearing mice compared to their female counterparts. Together, our findings indicate that lower skin catalase activity in male mice may at least in part contribute to increased UVB-induced Gr-1+CD11b+ myeloid cells and subsequent skin carcinogenesis.

Modulation of SOCS protein expression influences the interferon responsiveness of human melanoma cells

BackgroundEndogenously produced interferons can regulate the growth of melanoma cells and are administered exogenously as therapeutic agents to patients with advanced cancer. We investigated the role of negative regulators of interferon signaling known as suppressors of cytokine signaling (SOCS) in mediating interferon-resistance in human melanoma cells.MethodsBasal and interferon-alpha (IFN-α) or interferon-gamma (IFN-γ)-induced expression of SOCS1 and SOCS3 proteins was evaluated by immunoblot analysis in a panel of n = 10 metastatic human melanoma cell lines, in human embryonic melanocytes (HEM), and radial or vertical growth phase melanoma cells. Over-expression of SOCS1 and SOCS3 proteins in melanoma cells was achieved using the PINCO retroviral vector, while siRNA were used to inhibit SOCS1 and SOCS3 expression. Tyr701-phosphorylated STAT1 (P-STAT1) was measured by intracellular flow cytometry and IFN-stimulated gene expression was measured by Real Time PCR.ResultsSOCS1 and SOCS3 proteins were expressed at basal levels in melanocytes and in all melanoma cell lines examined. Expression of the SOCS1 and SOCS3 proteins was also enhanced following stimulation of a subset of cell lines with IFN-α or IFN-γ. Over-expression of SOCS proteins in melanoma cell lines led to significant inhibition of Tyr701-phosphorylated STAT1 (P-STAT1) and gene expression following stimulation with IFN-α (IFIT2, OAS-1, ISG-15) or IFN-γ (IRF1). Conversely, siRNA inhibition of SOCS1 and SOCS3 expression in melanoma cells enhanced their responsiveness to interferon stimulation.ConclusionsThese data demonstrate that SOCS proteins are expressed in human melanoma cell lines and their modulation can influence the responsiveness of melanoma cells to IFN-α and IFN-γ.

Novel Small Molecule XPO1/CRM1 Inhibitors Induce Nuclear Accumulation of TP53, Phosphorylated MAPK and Apoptosis in Human Melanoma Cells

XPO1/CRM1 is a key nuclear exporter protein that mediates translocation of numerous cellular regulatory proteins. We investigated whether XPO1 is a potential therapeutic target in melanoma using novel selective inhibitors of nuclear export (SINE). In vitro effects of SINE on cell growth and apoptosis were measured by MTS assay and flow cytometry [Annexin V/propidium iodide (PI)], respectively in human metastatic melanoma cell lines. Immunoblot analysis was used to measure nuclear localization of key cellular proteins. The in vivo activity of oral SINE was evaluated in NOD/SCID mice bearing A375 or CHL-1 human melanoma xenografts. SINE compounds induced cytostatic and pro-apoptotic effects in both BRAF wild type and mutant (V600E) cell lines at nanomolar concentrations. The cytostatic and pro-apoptotic effects of XPO1 inhibition were associated with nuclear accumulation of TP53, and CDKN1A induction in the A375 cell line with wild type TP53, while pMAPK accumulated in the nucleus regardless of TP53 status. The orally bioavailable KPT-276 and KPT-330 compounds significantly inhibited growth of A375 (p<0.0001) and CHL-1 (p = 0.0087) human melanoma cell lines in vivo at well tolerated doses. Inhibition of XPO1 using SINE represents a potential therapeutic approach for melanoma across cells with diverse molecular phenotypes by promoting growth inhibition and apoptosis.

Ultraviolet Light B-Mediated Inhibition of Skin Catalase Activity Promotes Gr-1+CD11b+ Myeloid Cell Expansion

Skin cancer incidence and mortality are higher in men compared to women, but the causes of this sex discrepancy remain largely unknown. Ultraviolet light exposure induces cutaneous inflammation and neutralizes cutaneous antioxidants. Gr-1+CD11b+ myeloid cells are heterogeneous bone marrow-derived cells that promote inflammation-associated carcinogenesis. Reduced activity of catalase, an antioxidant present within skin, has been associated with skin carcinogenesis. We utilized the outbred, immune competent Skh-1 hairless mouse model of ultraviolet light B (UVB)-induced inflammation and non-melanoma skin cancer to further define sex discrepancies in UVB-induced inflammation. Our results demonstrated that male skin had relatively lower baseline catalase activity, which was inhibited following acute UVB exposure in both sexes. Further analysis revealed that skin catalase activity inversely correlated with splenic Gr-1+CD11b+ myeloid cell percentage. Acute UVB exposure induced Gr-1+CD11b+ myeloid cell skin infiltration, which was inhibited to a greater extent in males by topical catalase treatment. In chronic UVB studies, we demonstrated that the percentage of splenic Gr-1+CD11b+ myeloid cells was 55% higher in male tumor-bearing mice compared to their female counterparts. Together, our findings indicate that lower skin catalase activity in male mice may at least in part contribute to increased UVB-induced Gr-1+CD11b+ myeloid cells and subsequent skin carcinogenesis.

Abstract 933: The expression of PRMT5 methyltransferase mediates cell survival and metastatic phenotype in malignant melanoma

Post-translational modification of proteins is involved at all levels of cellular regulation. The PRMT5 enzyme is a type II arginine methyltransferase that catalyzes the transfer of a methyl group to two of three guanidino nitrogen atoms within the arginine molecule. This enzyme has been shown to methylate histone H3 at arginine 8 (H3R8) and H4R3 to trigger silencing of tumor suppressor genes. In addition, PRMT5 has been shown to interact with p53, TRAIL receptor, and the CDK4 complex to regulate the cell cycle or apoptosis. Although prior studies have provided insight into these mechanistic features of the PRMT enzymes, most data are derived from a limited panel of cell lines. PRMT5 over-expression has recently been shown to influence progression of leukemia and lymphoma. However, few, if any publications exist which document the role of PRMT enzymes in melanoma. We hypothesized that PRMT5 expression promotes metastasis and contributes to reduced immunologic recognition of melanoma cells. Immunoblot analysis and confocal microscopy revealed that PRMT5 is expressed in a panel of melanoma cell lines regardless of the mutational status of B-Raf, NRas, or p53. siRNA-mediated inhibition of PRMT5 led to apoptosis and restored CXCL10 chemokine expression. PRMT5 expression was next determined by immunohistochemical (IHC) analysis of formalin-fixed samples from patients with melanoma (n=56 primary; n=20 metastases) or benign nevi (n=24). The specimens were obtained as a commercially-available tissue microarray, or procured as de-identified primary patient samples (IRB #20100071; P.I. Lesinski; OSU IRB # 2002H0089; co-PI: Peters). PRMT5 expression was significantly increased in the nucleus of melanoma cells as compared to normal epidermis or benign nevi (p=0.001). The level of cytoplasmic PRMT5 expression was also elevated in melanoma lesions, however its distribution in benign tissues was bimodal, and expressed in a subset of benign samples. Notably, nuclear PRMT5 expression increased as melanoma cells invaded the dermis or in melanoma lesions exhibiting pagetoid spread. Finally, we have identified a specific inhibitor of PRMT5 arginine methyltransferase activity from a small molecule library. This lead compound (BLL-1) blocked the dimethylation of arginine 3 on histone H4 (H4R3me2s). A series of titration experiments revealed reduction of both H4R3me2s and proliferation rate. Treatment of multiple melanoma cell lines with BLL-1 (24 hr) led to significantly increased apoptosis. These data represent the first report of PRMT5 and its inhibition in melanoma. Together, this suggests PRMT5 inhibition as a potential therapeutic strategy by virtue of its ability to promote apoptosis and restore immunomodulatory gene expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 933. doi:10.1158/1538-7445.AM2011-933

Abstract 162: Reduced pro-inflammatory cytokines and immunosuppressive cells in patients with prostate cancer following consumption of soy isoflavone enriched bread.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Soybeans are a rich source of bioactive phytochemcials. Among the phytochemicals, the isoflavones (primarily genistein and daidzein) have received the greatest attention regarding cancer prevention. Indeed, we and others have reported dietary soy has measureable benefit in experimental tumor models of prostate cancer (PCa). Thus, we and others are actively investigating the potential of diets rich in soy components to prevent or slow human PCa progression, particularly in the early hormone sensitive state. Isoflavones are hypothesized to act through various pathways to impact PCa progression, such as inhibition of tumor growth factor signaling, anti-angiogenesis, and cell cycle inhibition. However, one area that has received very little focus is the impact of soy components on the immune system. We hypothesized that dietary soy may benefit prostate cancer (PCa) patients by modulating the balance of pro-inflammatory cytokines and immunosuppressive cells. A randomized, phase II trial was conducted in 32 PCa patients with asymptomatic biochemical failure (rising prostate specific antigen/PSA). Patients were randomized to 3 slices of soy bread (33 mg isoflavones/slice) or soy bread containing almond extract daily as a source of β-glucosidase. Flow cytometry was used to study cellular phenotype and bioplex assay to measure cytokines in peripheral blood on days 0 (baseline) and 56. Soy isoflavone metabolites in patient urine were assessed by LC-MS/MS. Adequate blood samples were available from 25 of 32 men completing the >56 day protocol and evaluated. Phenotypic analysis of Day 0 and 56 blood revealed no change in percentage of CD8+ T cells, but showed increased CD56+ NK cells (p=0.038) and reduced CD4+ T cells (p=0.04). Cells with a natural T regulatory cell phenotype (CD4+CD25+FoxP3+) were significantly decreased after 56 days consuming either type of soy bread (p=0.035). A significant decrease in cells with a monocytic (CD33+HLADRlowCD14+) and granulocytic (CD33+HLADR-CD11b+) MDSC phenotype was observed in patients consuming both types of soy bread (p=0.0064 and p=0.049). Plasma cytokine and chemokine levels were significantly decreased on Day 56 compared to baseline. Subgroup analysis indicated reduction in both Th1 type cytokines (p=0.0349) and MDSC associated cytokines (p=0.0284). Th2 and Th17 cytokines were not significantly altered as compared to baseline. Exploratory analyses using a K-means clustering approach revealed patients who metabolized soy isoflavones into dihyrdodaidzein and equol had significantly lower MDSC after soy intervention (ps<0.02). Finally, patients with prolonged or no change in PSA doubling time had fewer T regulatory cells or MDSC on day 56 versus baseline. Our data suggests that soy bread modulates immune suppressor cell biology in humans and may enhance the efficacy of anti-cancer immune responses. Citation Format: Gregory Brian Lesinski, Thomas A. Mace, Patrick Reville, Jennifer Ahn-Jarvis, Matthew Bill, Courtney Nicholas, Yael Vodovotz, Elizabeth Grainger, Kenneth Riedl, Gregory Young, Steven Schwartz, Steven Clinton. Reduced pro-inflammatory cytokines and immunosuppressive cells in patients with prostate cancer following consumption of soy isoflavone enriched bread. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 162. doi:10.1158/1538-7445.AM2013-162