A novel selective ERK1/2 inhibitor, Laxiflorin B, targets EGFR mutation subtypes in non-small-cell lung cancer.

Extracellular regulated protein kinases 1/2 (ERK1/2) are key members of multiple signaling pathways, including the ErbB axis. Ectopic ERK1/2 activation contributes to various types of cancer, especially drug resistance to inhibitors of RTK, RAF and MEK, and specific ERK1/2 inhibitors are scarce. In this study, we identified a potential novel covalent ERK inhibitor, Laxiflorin B, which is a herbal compound with anticancer activity. However, Laxiflorin B is present at low levels in herbs; therefore, we adopted a semi-synthetic process for the efficient production of Laxiflorin B to improve the yield. Laxiflorin B induced mitochondria-mediated apoptosis via BAD activation in non-small-cell lung cancer (NSCLC) cells, especially in EGFR mutant subtypes. Transcriptomic analysis suggested that Laxiflorin B inhibits amphiregulin (AREG) and epiregulin (EREG) expression through ERK inhibition, and suppressed the activation of their receptors, ErbBs, via a positive feedback loop. Moreover, mass spectrometry analysis combined with computer simulation revealed that Laxiflorin B binds covalently to Cys-183 in the ATP-binding pocket of ERK1 via the D-ring, and Cys-178 of ERK1 through non-inhibitory binding of the A-ring. In a NSCLC tumor xenograft model in nude mice, Laxiflorin B also exhibited strong tumor suppressive effects with low toxicity and AREG and EREG were identified as biomarkers of Laxiflorin B efficacy. Finally, Laxiflorin B-4, a C-6 analog of Laxiflorin B, exhibited higher binding affinity for ERK1/2 and stronger tumor suppression. These findings provide a new approach to tumor inhibition using natural anticancer compounds.

RXXPEG motif of MERIT40 is required to maintain spindle structure and function through its interaction with Tankyrase1.

Deubiquitinase BRISC complex plays important role in the maintenance of spindle structure and function; however, the underlying mechanism remains largely undefined. Here we demonstrated that MERIT40, a core component of BRISC complex, directly interacts with the RXXPEG motif in the ARC-V domain of Tankyrase1(TNKS1). Mutation of the RXXPEG motif in the MERIT40 (R28A) disrupted its interaction with TNKS1. Consistent with these data, R28A mutant cells displayed multiple mitotic defects including aberrant spindle assembly and chromosome misalignment. These results support a critical role of RXXPEG motif of MERIT40 in BRISC-mediated regulation of TNKS1 function during spindle assembly.

Laxiflorin B covalently binds the tubulin colchicine-binding site to inhibit triple negative breast cancer proliferation and induce apoptosis.

Laxiflorin B is a natural ent-kaurane diterpenoid that can be isolated from the leaves of the Isodon eriocalyx var. laxiflora, a perennial shrub native to parts of China. While this compound has potent cytotoxic activity against various tumor cells, the anti-tumor targets and molecular mechanisms of Laxiflorin B are unclear. Here, we show that Laxiflorin B exhibits strong antiproliferative and proapoptotic effects on triple-negative breast cancer (TNBC) cells. At the mechanistic level, we show that ß-tubulin (TUBB) is a cellular target of Laxiflorin B. By covalently binding the Cys239 and C354 residues of the TUBB colchicine-binding site, Laxiflorin B disturbs microtubule integrity and structure in vitro and in vivo. Cytotoxicity analyses also showed that the α, ß-unsaturated carbonyl in the D ring of Laxiflorin B is responsible for mediating its covalent binding and anti-tumor activity. To assess the therapeutic effects of Laxiflorin B, we synthesized a Laxiflorin B-ALA pro-drug and delivered it by intraperitoneal injection (10 mg/kg) into a 4T1 orthotopic tumor mouse model. Drug treatment had anti-tumor effects without inducing notable weight loss or organ dysfunction. We conclude that Laxiflorin B is a promising colchicine binding site inhibitor that might be exploited in the context of TNBC treatment in the future.

Methylation of KRAS by SETD7 promotes KRAS degradation in non-small cell lung cancer.

Oncogenic KRAS mutations are a key driver for initiation and progression in non-small cell lung cancer (NSCLC). However, how post-translational modifications (PTMs) of KRAS, especially methylation, modify KRAS activity remain largely unclear. Here, we show that SET domain containing histone lysine methyltransferase 7 (SETD7) interacts with KRAS and methylates KRAS at lysines 182 and 184. SETD7-mediated methylation of KRAS leads to degradation of KRAS and attenuation of the RAS/MEK/ERK signaling cascade, endowing SETD7 with a potent tumor-suppressive role in NSCLC, both in vitro and in vivo. Mechanistically, RABGEF1, a ubiquitin E3 ligase of KRAS, is recruited and promotes KRAS degradation in a K182/K184 methylation-dependent manner. Notably, SETD7 is inversely correlated with KRAS at the protein level in clinical NSCLC tissues. Low SETD7 or RABGEF1 expression is associated with poor prognosis in lung adenocarcinoma patients. Altogether, our results define a tumor-suppressive function of SETD7 that operates via modulating KRAS methylation and degradation.

Role of the Tumor Microenvironment in Regulating Pancreatic Cancer Therapy Resistance.

Pancreatic cancer has a notoriously poor prognosis, exhibits persistent drug resistance, and lacks a cure. Unique features of the pancreatic tumor microenvironment exacerbate tumorigenesis, metastasis, and therapy resistance. Recent studies emphasize the importance of exploiting cells in the tumor microenvironment to thwart cancers. In this review, we summarize the hallmarks of the multifaceted pancreatic tumor microenvironment, notably pancreatic stellate cells, tumor-associated fibroblasts, macrophages, and neutrophils, in the regulation of chemo-, radio-, immuno-, and targeted therapy resistance in pancreatic cancer. The molecular insight will facilitate the development of novel therapeutics against pancreatic cancer.

The Epigenetic Regulation of Nonhistone Proteins by SETD7: New Targets in Cancer.

Epigenetic modifications are essential mechanism by which to ensure cell homeostasis. One such modification is lysine methylation of nonhistone proteins by SETD7, a mono-methyltransferase containing SET domains. SETD7 methylates over 30 proteins and is thus involved in various classical pathways. As such, SETD7 has been implicated in both the basic functions of normal tissues but also in several pathologies, such as cancers. In this review, we summarize the current knowledge of SETD7 substrates, especially transcriptional-related proteins and enzymes, and their putative roles upon SETD7-mediated methylation. We focus on the role of SETD7 in cancers, and speculate on the possible points of intervention and areas for future research.

YT521-B homology domain family proteins as N6-methyladenosine readers in tumors.

N6-methyladenosine (m6A) is the most abundant internal chemical modification of eukaryotic mRNA and plays diverse roles in gene regulation. The m6A modification plays a significant role in numerous cancer types, including kidney, stomach, lung, bladder tumors, and melanoma, through varied mechanisms. As direct m6A readers, the YT521-B homology domain family proteins (YTHDFs) play a key role in tumor transcription, translation, protein synthesis, tumor stemness, epithelial-mesenchymal transition (EMT), immune escape, and chemotherapy resistance. An in-depth understanding of the molecular mechanism of YTHDFs is expected to provide new strategies for tumor treatment. In this review, we provide a systematic description of YTHDF protein structure and its function in tumor progression.

Dezocine induces apoptosis in human cervical carcinoma Hela cells via the endoplasmic reticulum stress pathway.

Dezocine, a dual agonist and antagonist of the µ-opioid receptor and κ-opioid receptor, is widely used as an analgesic in China. At present, there are few studies on anti-tumor effects of dezocine, most of which are used to treat cancer pain. However, it has recently been reported that dezocine can induce apoptosis of triple negative breast cancer cells. Dezocine may have some anti-tumor activity, but the effect and potential mechanism of dezocine in the treatment of other types of cancer remain to be fully studied. The purpose of the present study was to investigate the effect of dezocine on human Hela cervical carcinoma cells, and to elucidate the underlying molecular mechanisms. We performed CCK-8 assays, clone formation assays, xenograft, flow cytometry analysis, western blot and RNA-seq analysis to evaluate the effects of dezocine on Hela cells. In addition, the role of endoplasmic reticulum (ER) stress in dezocine-induced apoptosis was investigated using qPCR and western blot analysis. Dezocine inhibited Hela cell viability in dose-dependent and time-dependent manners, and notably did not achieve this effect by targeting the opioid receptors. Further mechanistic studies demonstrated that dezocine activated ER stress by upregulating the expression of GRP78, IRE1 and p-JNK, and that dezocine-induced apoptosis was attenuated when the ER stress pathway was blocked. Our results provide a foundation to support the redefinition of dezocine as a novel, adjuvant treatment for patients with cervical cancer, although further research will be required to support its application in clinical practice.

The Impact of TRPV1 on Cancer Pathogenesis and Therapy: A Systematic Review.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a transmembrane protein that can be activated by various physical and chemical stimuli and is associated with pain transduction. In recent years, TRPV1 was discovered to play essential roles in cancer tumorigenesis and development, as TRPV1 expression levels are altered in numerous cancer cell types. Several investigations have discovered direct associations between TRPV1 and cancer cell proliferation, cell death, and metastasis. Furthermore, about two dozen TRPV1 agonists/antagonists are under clinical trial, as TRPV1 is a potential drug target for treating various diseases. Hence, more researchers are focusing on the effects of TRPV1 agonists or antagonists on cancer tumorigenesis and development. However, both agonists and antagonists may reveal anti-cancer effects, and the effect may function via or be independent of TRPV1. In this review, we provide an overview of the impact of TRPV1 on cancer cell proliferation, cell death, and metastasis, as well as on cancer therapy and the tumor microenvironment, and consider the implications of using TRPV1 agonists and antagonists for future research and potential therapeutic approaches.

The significance of SMARCB1 in the pathogenesis of renal cell carcinoma with rhabdoid features.

BACKGROUND: Renal cell carcinoma with rhabdoid features (RCC-RF) is an aggressive histologic variant in the adults and is usually unresponsive to standard chemotherapy. METHODS: Expression of SMARCB1/INI1 was examined in primary RCC-RF (n = 5). Stable INI1 with/without prostaglandin E2 receptor 1 (EP1) knockdown cell lines were created in the ACHN and 786-O RCC cell lines and measured for epidermal growth factor receptor (EGFR)-related signaling pathways. Chemosensitivity to targeted drugs in vitro was tested after knocking down of INI1 in both cell lines. The outcome of co-targeting of INI1 and EP1 in RCC was examined using a tumorigenicity assay. RESULTS: Expression of INI1 was markedly reduced at both transcriptional and translational levels in primary RCC-RF. Immunohistochemical expression of INI1 protein was lost in the nuclei of rhabdoid cells compared with conventional RCC (n = 8). Using two cell lines with different genetic background, we showed that knocking down of INI1 activates the EGFR signaling with up-regulated AKT and ERK pathways and sensitizes cancer cells to Erlotinib treatment in vitro. However, cell-line dependent effects were also demonstrated with reference to impact of INI1 or EP1 on cell growth, migration and response to Gefitinib or Everolimus treatment in vitro. CONCLUSION: Inactivation of INI1 may play a role in the pathogenesis of RCC-RF. Erlotinib is recommended in the management of patients with INI1-related RCC.

Tacrolimus and ascomycin inhibit melanoma cell growth, migration and invasion via targeting nuclear factor of activated T-cell 3.

Melanoma is the most malignant form of skin cancer with high metastatic potential. Nuclear factor of activated T-cells (NFATs) are discovered as transcription factors that regulate the expression of proinflammatory cytokines and other genes during the immune response. Among five NFAT members, NFAT3 is exclusively not expressed in immune cells and its role in progression of different types of cancer remains controversial. Our previous study showed that NFAT3 was highly expressed in skin cancer compared with normal skin tissues and critical for melanoma cell survival and tumor growth. Here, we reported that knockdown of NFAT3 expression, as well as treatment with the calcineurin (CaN) inhibitors, tacrolimus (FK506) or ascomycin (FK520) inhibits melanoma cell migration and invasion, and also proliferation and colony formation. Mechanistic studies revealed that FK506 or FK520 blocked the nuclear translocation and reduced the transcriptional activity of NFAT3. These data support that the antimelanoma effect of FK506 and FK520 is partially mediated by inhibiting the oncogenic factor NFAT3, suggesting that therapeutics based on NFAT3 inhibition may be effective in clinical melanoma treatment.

Therapeutic potential of targeting MKK3-p38 axis with Capsaicin for Nasopharyngeal Carcinoma.

Background: Capsaicin is an active compound found in plants of the Capsicum genus; it has a range of therapeutic benefits, including anti-tumor effects. Here we aimed to delineate the inhibitory effects of capsaicin on nasopharyngeal carcinoma (NPC). Methods: The anti-cancer effects of capsaicin were confirmed in NPC cell lines and xenograft mouse models, using CCK-8, clonogenic, wound-healing, transwell migration and invasion assays. Co-immunoprecipitation, western blotting and pull-down assays were used to determine the effects of capsaicin on the MKK3-p38 axis. Cell proliferation and EMT marker expression were monitored in MKK3 knockdown (KD) or over-expression NPC cell lines treated with or without capsaicin. Finally, immunohistochemistry was performed on NPC specimens from NPC patients (n = 132) and the clinical relevance was analyzed. Results: Capsaicin inhibited cell proliferation, mobility and promoted apoptosis in NPC cells. Then we found that capsaicin directly targets p38 for dephosphorylation. As such, MKK3-induced p38 activation was inhibited by capsaicin. Furthermore, we found that capsaicin-induced inhibition of cell motility was mediated by fucokinase. Xenograft models demonstrated the inhibitory effects of capsaicin treatment on NPC tumor growth in vivo, and analysis of clinical NPC samples confirmed that MKK3 phosphorylation was associated with NPC tumor growth and lymphoid node metastasis. Conclusions: The MKK3-p38 axis represents a potential therapeutic target for capsaicin. MKK3 phosphorylation might serve as a biomarker to identify NPC patients most likely to benefit from adjunctive capsaicin treatment.

SH3BGRL3 Protein as a Potential Prognostic Biomarker for Urothelial Carcinoma: A Novel Binding Partner of Epidermal Growth Factor Receptor.

PURPOSE: Mass spectrometry-based biomarker discovery has clinical benefit. To identify novel biomarkers for urothelial carcinoma, we performed quantitative proteomics on pooled urine pairs from patients with and without urothelial carcinoma. EXPERIMENTAL DESIGN: Shot-gun proteomics using liquid chromatography-tandem mass spectrometry and stable isotope dimethyl labeling identified 219 candidate proteins. The potential implication of SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) was examined by immunoblotting of the urine (n = 13) and urothelial tumors (n = 32). Additional immunohistochemistry was performed on bladder cancer array (n = 1145) and correlated with tumor aggressiveness. Then, biologic functions and signaling pathways of SH3BGRL3 were explored using stable cell lines. RESULTS: The detectable urine SH3BGRL3 in patients with urothelial carcinoma was positively associated with higher histologic grading and muscle invasiveness of urothelial carcinoma. SH3BGRL3 is expressed in 13.9% (159/1145) of bladder cancer cohort and is positively associated with muscle invasion (P = 0.0028). SH3BGRL3 expression is associated with increased risk of progression in patients with nonmuscle-invasive bladder cancer (P = 0.032). SH3BGRL3 expression is significantly associated with a high level of epidermal growth factor receptor (EGFR) in bladder cancer (P < 0.0001). SH3BGRL3 promotes the epithelial-mesenchymal transition, cell migration, and proliferation of urothelial carcinoma in vitro. SH3BGRL3 interacts with phosphor-EGFR at Y1068, Y1086, and Y1173 through Grb2 by its proline-rich motif, and activates the Akt-associated signaling pathway. CONCLUSIONS: Evaluation of SH3BGRL3 expression status or urine content may identify a subset of patients with bladder cancer who may require more intensive treatment. SH3BGRL3 deserves further investigation as a cotargeting candidate for designing EGFR-based cancer therapies. Clin Cancer Res; 21(24); 5601-11. ©2015 AACR.