SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck.

Cancers often display immune escape, but the mechanisms are incompletely understood. Herein, we identify SMYD3 as a mediator of immune escape in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor response to immunotherapy with pembrolizumab. SMYD3 depletion induces upregulation of multiple type I interferon (IFN) response and antigen presentation machinery genes in HNSCC cells. Mechanistically, SMYD3 binds to and regulates the transcription of UHRF1, encoding for a reader of H3K9me3, which binds to H3K9me3-enriched promoters of key immune-related genes, recruits DNMT1, and silences their expression. SMYD3 further maintains the repression of immune-related genes through intragenic deposition of H4K20me3. In vivo, Smyd3 depletion induces influx of CD8+ T cells and increases sensitivity to anti-programmed death 1 (PD-1) therapy. SMYD3 overexpression is associated with decreased CD8 T cell infiltration and poor response to neoadjuvant pembrolizumab. These data support combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in HPV-negative HNSCC.

SMYD3: a regulator of epigenetic and signaling pathways in cancer.

Chromatin modifiers and their implications in oncogenesis have been an exciting area of cancer research. These are enzymes that modify chromatin via post-translational modifications such as methylation, acetylation, sumoylation, phosphorylation, in addition to others. Depending on the modification, chromatin modifiers can either promote or repress transcription. SET and MYN-domain containing 3 (SMYD3) is a chromatin modifier that has been implicated in the development and progression of various cancer types. It was first reported to tri-methylate Histone 3 Lysine 4 (H3K4), a methylation mark known to promote transcription. However, since this discovery, other histone (H4K5 and H4K20, for example) and non-histone (VEGFR, HER2, MAP3K2, ER, and others) substrates of SMYD3 have been described, primarily in the context of cancer. This review aims to provide a background on basic characteristics of SMYD3, such as its protein structure and tissue expression profiles, discuss reported histone and non-histone substrates of SMYD3, and underscore prognostic and functional implications of SMYD3 in cancer. Finally, we briefly discuss ongoing efforts to develop inhibitors of SMYD3 for future therapeutic use. It is our hope that this review will help synthesize existing research on SMYD3 in an effort to propel future discovery.

Clinicopathologic significance of protein lysine methyltransferases in cancer.

Protein lysine methyltransferases (PKMTs) constitute a large family of approximately 50 chromatin modifiers that mono-, di- and/or tri-methylate lysine residues on histone and non-histone substrates. With the advent of The Cancer Genome Atlas, it became apparent that this family of chromatin modifiers harbors frequent genetic and expression alterations in multiple types of cancer. In this regard, past and ongoing preclinical studies have provided insight into the mechanisms of action of some of these enzymes, laying the ground for the ongoing development of PKMT inhibitors as novel anticancer therapeutics. The purpose of this review is to summarize existing data obtained by different research groups through immunohistochemical analysis of the protein expression levels of PKMTs, and their respective clinicopathologic associations. We focused on studies that used immunohistochemistry to associate protein expression levels of specific PKMTs, as well as several established histone methylation marks, with clinicopathologic features and survival outcomes in various cancer types. We also review ongoing clinical trials of PKMT inhibitors in cancer treatment. This review underscores the clinical relevance and potential of targeting the family of PKMT enzymes as the next generation of cancer therapy.

WHSC1 monomethylates histone H1 and induces stem-cell like features in squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is a malignancy with poor outcomes, thus novel therapies are urgently needed. We recently showed that WHSC1 is necessary for the viability of SCCHN cells through H3K36 di-methylation. Here, we report the identification of its novel substrate, histone H1, and that WHSC1-mediated H1.4K85 mono-methylation may enhance stemness features in SCCHN cells. To identify proteins interacting with WHSC1 in SCCHN cells, WHSC1 immunoprecipitation and mass spectrometry identified H1 as a WHSC1-interacting candidate. In vitro methyltransferase assays showed that WHSC1 mono-methylates H1 at K85. We generated an H1K85 mono-methylation-specific antibody and confirmed that this methylation occurs in vivo. Sphere formation assays using SCC-35 cells stably expressing either wild-type (FLAG-H1.4-WT) or mutated (FLAG-H1.4K85A) vector with lysine 85 to alanine substitution which is not methylated, indicated a higher number of spheres in SCC-35 cells expressing the wild type than those with the mutant vector. SCC-35 cells expressing the wild type H1.4 proliferated faster than those expressing the mutated vector. RNA sequencing, RT-PCR and Western blotting of the FLAG-H1.4-WT or FLAG-H1.4K85A SCC-35 cells revealed that OCT4 levels were higher in wild type compared to mutant cells. These results were reproduced in SCC-35 cells genetically modified with CRISPR to express H1.4K85R. Chromatin immunoprecipitation showed that FLAG-H1.4K85A had decreased occupancy in the OCT4 gene compared to FLAG-H1.4-WT. This study supports that WHSC1 mono-methylates H1.4 at K85, it induces transcriptional activation of OCT4 and stemness features in SCCHN cells, providing rationale to target H1.4K85 mono-methylation through WHSC1 in SCCHN.

Modulation of Diacylglycerol-Induced Melanogenesis in Human Melanoma and Primary Melanocytes: Role of Stress Chaperone Mortalin.

Skin color/pigmentation is regulated through melanogenesis process in specialized melanin-producing cells, melanocytes, involving multiple signaling pathways. It is highly influenced by intrinsic and extrinsic factors such as oxidative, ultraviolet radiations and other environmental stress conditions. Besides determining the color, it governs response and tolerance of skin to a variety of environmental stresses and pathological conditions including photodamage, hyperpigmentation, and skin cancer. Depigmenting reagents have been deemed useful not only for cosmetics but also for pigmentation-related pathologies. In the present study, we attempted modulation of 1-oleoyl-2-acetyl-glycerol- (OAG-) induced melanogenesis in human melanoma and primary melanocytes. In both cell types, OAG-induced melanogenesis was associated with increase in enhanced expression of melanin, tyrosinase, as well as stress chaperones (mortalin and HSP60) and Reactive Oxygen Species (ROS). Treatment with TXC (trans-4-(Aminomethyl) cyclohexanecarboxylic acid hexadecyl ester hydrochloride) and 5/40 natural compounds resulted in their reduction. The data proposed an important role of mortalin and oxidative stress in skin pigmentation and the use of TXC and natural extracts for modulation of pigmentation pathways in normal and pathological conditions.

Anticancer activity of the supercritical extract of Brazilian green propolis and its active component, artepillin C: Bioinformatics and experimental analyses of its mechanisms of action.

Propolis, a resinous substance collected by honeybees by mixing their saliva with plant sources, including tree bark and leaves and then mixed with secreted beeswax, possesses a variety of bioactivities. Whereas caffeic acid phenethyl ester (CAPE) has been recognized as a major bioactive ingredient in New Zealand propolis, Brazilian green propolis, on the other hand, possesses artepillin C (ARC). In this study, we report that, similar to CAPE, ARC docks into and abrogates mortalin-p53 complexes, causing the activation of p53 and the growth arrest of cancer cells. Cell viability assays using ARC and green propolis-supercritical extract (GPSE) revealed higher cytotoxicity in the latter, supported by nuclear translocation and the activation of p53. Furthermore, in vivo tumor suppression assays using nude mice, we found that GPSE and its conjugate with γ cyclodextrin (γCD) possessed more potent anticancer activity than purified ARC. GPSE­Î³CD may thus be recommended as a natural, effective and economic anticancer amalgam.

Molecular Characterization and Enhancement of Anticancer Activity of Caffeic Acid Phenethyl Ester by γ Cyclodextrin.

Caffeic Acid Phenethyl Ester (CAPE) is a key component in New Zealand propolis, known for a variety of health promoting and therapeutic potentials. We investigated the molecular mechanism of anticancer and anti-metastasis activities of CAPE. cDNA array performed on the control and CAPE-treated breast cancer cells revealed activation of DNA damage signaling involving upregulation of GADD45α and p53 tumor suppressor proteins. Molecular docking analysis revealed that CAPE is capable of disrupting mortalin-p53 complexes. We provide experimental evidence and demonstrate that CAPE induced disruption of mortalin-p53 complexes led to nuclear translocation and activation of p53 resulting in growth arrest in cancer cells. Furthermore, CAPE-treated cells exhibited downregulation of mortalin and several other key regulators of cell migration accountable for its anti-metastasis activity. Of note, we found that whereas CAPE was unstable in the culture medium (as it gets degraded into caffeic acid by secreted esterases), its complex with gamma cyclodextrin (γCD) showed high efficacy in anti-tumor and anti-metastasis assays in vitro and in vivo (when administered through either intraperitoneal or oral route). The data proposes that CAPE-γCD complex is a potent anti-cancer and anti-metastasis reagent.

Stress chaperone mortalin regulates human melanogenesis.

In order to identify the cellular factors involved in human melanogenesis, we carried out shRNA-mediated loss-of-function screening in conjunction with induction of melanogenesis by 1-oleoyl-2-acetyl-glycerol (OAG) in human melanoma cells using biochemical and visual assays. Gene targets of the shRNAs (that caused loss of OAG-induced melanogenesis) and their pathways, as determined by bioinformatics, revealed involvement of proteins that regulate cell stress response, mitochondrial functions, proliferation, and apoptosis. We demonstrate, for the first time, that the mitochondrial stress chaperone mortalin is crucial for melanogenesis. Upregulation of mortalin was closely associated with melanogenesis in in vitro cell-based assays and clinical samples of keloids with hyperpigmentation. Furthermore, its knockdown resulted in compromised melanogenesis. The data proposed mortalin as an important protein that may be targeted to manipulate pigmentation for cosmetic and related disease therapeutics.

Targeting Mortalin by Embelin Causes Activation of Tumor Suppressor p53 and Deactivation of Metastatic Signaling in Human Breast Cancer Cells.

Embelin, a natural quinone found in the fruits of Embelia ribes, is commonly used in Ayurvedic home medicine for a variety of therapeutic potentials including anti-inflammation, anti-fever, anti-bacteria and anti-cancer. Molecular mechanisms of these activities and cellular targets have not been clarified to-date. We demonstrate that the embelin inhibits mortalin-p53 interactions, and activates p53 protein in tumor cells. We provide bioinformatics, molecular docking and experimental evidence to the binding affinity of embelin with mortalin and p53. Binding of embelin with mortalin/p53 abrogates their complex resulted in nuclear translocation and transcriptional activation function of p53 causing growth arrest in cancer cells. Furthermore, analyses of growth factors and metastatic signaling using antibody membrane array revealed their downregulation in embelin-treated cells. We also found that the embelin causes transcriptional attenuation of mortalin and several other proteins involved in metastatic signaling in cancer cells. Based on these molecular dynamics and experimental data, it is concluded that the anticancer activity of embelin involves targeting of mortalin, activation of p53 and inactivation of metastatic signaling.

Embelin inhibits TNF-α converting enzyme and cancer cell metastasis: molecular dynamics and experimental evidence.

BACKGROUND: Embelin, a quinone derivative, is found in the fruits of Embelia ribes Burm (Myrsinaceae). It has been shown to have a variety of therapeutic potentials including anthelmintic, anti-tumor, anti-diabetic, anti-bacterial and anti-inflammation. Inflammation is an immunological response to external harmful stimuli and is regulated by an endogenous pyrogen and pleiotropic pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α). TNF-α production has been implicated in a variety of other human pathologies including neurodegeneration and cancer. Several studies have shown that the anti-inflammatory activity of embelin is mediated by reduction in TNF-α. The latter is synthesized as a membrane anchored protein (pro-TNF-α); the soluble component of pro-TNF-α is then released into the extracellular space by the action of a protease called TNF-α converting enzyme (TACE). TACE, hence, has been proposed as a therapeutic target for inflammation and cancer. METHODS: We used molecular docking and experimental approaches to investigate the docking potential and molecular effects of embelin to TACE and human cancer cell characteristics, respectively. RESULTS: We demonstrate that embelin is a potential inhibitor of TACE. Furthermore, in vitro studies revealed that it inhibits malignant properties of cancer cells through inactivation of metastatic signaling molecules including MMPs, VEGF and hnRNP-K in breast cancer cells. CONCLUSION: Based on the molecular dynamics and experimental data, embelin is proposed as a natural anti-inflammatory and anticancer drug.