Keloid Disease Can Be Inhibited by Antagonizing Excessive mTOR Signaling With a Novel Dual TORC1/2 Inhibitor

Keloid disease (KD) is a fibroproliferative lesion of unknown etiopathogenesis that possibly targets the PI3K/Akt/mTOR pathway. We investigated whether PI3K/Akt/mTOR inhibitor, Palomid 529 (P529), which targets both mammalian target of rapamycin complex 1 (mTORC-1) and mTORC-2 signaling, could exert anti-KD effects in a novel KD organ culture assay and in keloid fibroblasts (KF). Treatment of KF with P529 significantly (P < 0.05) inhibited cell spreading, attachment, proliferation, migration, and invasive properties at a low concentration (5 ng/mL) and induced substantial KF apoptosis when compared with normal dermal fibroblasts. P529 also inhibited hypoxia-inducible factor-1α expression and completely suppressed Akt, GSK3β, mTOR, eukaryotic initiation factor 4E-binding protein 1, and S6 phosphorylation. P529 significantly (P < 0.05) inhibited proliferating cell nuclear antigen and cyclin D and caused considerable apoptosis. Compared with rapamycin and wortmannin, P529 also significantly (P < 0.05) reduced keloid-associated phenotypic markers in KF. P529 caused tissue shrinkage, growth arrest, and apoptosis in keloid organ cultures and substantially inhibited angiogenesis. pS6, pAkt-Ser473, and mTOR phosphorylation were also suppressed in situ. P529 reduced cellularity and expression of collagen, fibronectin, and α-smooth muscle actin (substantially more than rapamycin). These pre-clinical in vitro and ex vivo observations are evidence that the mTOR pathway is a promising target for future KD therapy and that the dual PI3K/Akt/mTOR inhibitor P529 deserves systematic exploration as a candidate agent for the future treatment of KD

Combined BRAFV600E- and SRC-inhibition induces apoptosis, evokes an immune response and reduces tumor growth in an immunocompetent orthotopic mouse model of anaplastic thyroid cancer

Anaplastic (ATC) and refractory papillary thyroid cancer (PTC) lack effective treatments. Inhibition of either oncogenic BRAF or SRC has marked anti-tumor effects in mouse models of thyroid cancer, however, neither drug induces notable apoptosis. Here we report that the SRC-inhibitor dasatinib further sensitizes BRAFV600E-positive thyroid cancer cells to the BRAFV600E-inhibitor PLX4720. Combined treatment with PLX4720 and dasatinib synergistically inhibited proliferation and reduced migration in PTC and ATC cells. Whereas PLX4720 did not induce robust apoptosis in thyroid cancer cells, combined treatment with dasatinib induced apoptosis in 4 of 6 lines. In an immunocompetent orthotopic mouse model of ATC, combined PLX4720 and dasatinib treatment significantly reduced tumor volume relative to PLX4720 treatment alone. Immune cell infiltration was increased by PLX4720 treatment and this effect was maintained in mice treated with both PLX4720 and dasatinib. Further, combined treatment significantly increased caspase 3 cleavage in vivo relative to control or either treatment alone. In conclusion, combined PLX4720 and dasatinib treatment induces apoptosis, increases immune cell infiltration and reduces tumor volume in a preclinical model of ATC, suggesting that the combination of these FDA-approved drugs may have potential for the treatment of patients with ATC or refractory PTC

The Next Generation of Orthotopic Thyroid Cancer Models: Immunocompetent Orthotopic Mouse Models of BRAF

Background: While the development of new treatments for aggressive thyroid cancer has advanced in the last 10 years, progress has trailed headways made with other malignancies. A lack of reliable authenticated human cell lines and reproducible animal models is one major roadblock to preclinical testing of novel therapeutics. Existing xenograft and orthotopic mouse models of aggressive thyroid cancer rely on the implantation of highly passaged human thyroid carcinoma lines in immunodeficient mice. Genetically engineered models of papillary and undifferentiated (anaplastic) thyroid carcinoma (PTC and ATC) are immunocompetent; however, slow and stochastic tumor development hinders high-throughput testing. Novel models of PTC and ATC in which tumors arise rapidly and synchronously in immunocompetent mice would facilitate the investigation of novel therapeutics and approaches. Methods: We characterized and utilized mouse cell lines derived from PTC and ATC tumors arising in genetically engineered mice with thyroid-specific expression of endogenous BrafV600E/WTand deletion of either Trp53 (p53) or Pten. These murine thyroid cancer cells were transduced with luciferase- and GFP-expressing lentivirus and implanted into the thyroid glands of immunocompetent syngeneic B6129SF1/J mice in which the growth characteristics were assessed. Results: Large locally aggressive thyroid tumors form within one week of implantation. Tumors recapitulate their histologic subtype, including well-differentiated PTC and ATC, and exhibit CD3+, CD8+, B220+, and CD163+ immune cell infiltration. Tumor progression can be followed in vivo using luciferase and ex vivo using GFP. Metastatic spread is not detected at early time points. Conclusions: We describe the development of the next generation of murine orthotopic thyroid cancer models. The implantation of genetically defined murine BRAF-mutated PTC and ATC cell lines into syngeneic mice results in rapid and synchronous tumor formation. This model allows for preclinical investigation of novel therapeutics and/or therapeutic combinations in the context of a functional immune system

A novel type of cellular senescence that can be enhanced in mouse models and human tumor xenografts to suppress prostate tumorigenesis

Irreversible cell growth arrest, a process termed cellular senescence, is emerging as an intrinsic tumor suppressive mechanism. Oncogene-induced senescence is thought to be invariably preceded by hyperproliferation, aberrant replication, and activation of a DNA damage checkpoint response (DDR), rendering therapeutic enhancement of this process unsuitable for cancer treatment. We previously demonstrated in a mouse model of prostate cancer that inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (Pten) elicits a senescence response that opposes tumorigenesis. Here, we show that Pten-loss-induced cellular senescence (PICS) represents a senescence response that is distinct from oncogene-induced senescence and can be targeted for cancer therapy. Using mouse embryonic fibroblasts, we determined that PICS occurs rapidly after Pten inactivation, in the absence of cellular proliferation and DDR. Further, we found that PICS is associated with enhanced p53 translation. Consistent with these data, we showed that in mice p53-stabilizing drugs potentiated PICS and its tumor suppressive potential. Importantly, we demonstrated that pharmacological inhibition of PTEN drives senescence and inhibits tumorigenesis in vivo in a human xenograft model of prostate cancer. Taken together, our data identify a type of cellular senescence that can be triggered in nonproliferating cells in the absence of DNA damage, which we believe will be useful for developing a 'pro-senescence' approach for cancer prevention and therapy

Cloning and characterization of a p-53-inducible gene, WIG-1

Elucidating the mechanisms by which p53 inhibits tumor growth is a central issue in cancer research, as the TP53 gene is mutated in over half of all human cancers. Lack of functional p53 also contributes to the resistance of many tumor types to chemotherapy and radiotherapy. p53 is a transcription factor, which regulates the expression of a large number of genes. In fact, most of the knowledge about the molecular mechanisms by which p53 exerts its biological functions has been acquired by the identification and characterization of genes whose expression is regulated by p53. This thesis reports on the identification and characterization of a novel p53-inducble gene, designated WIG-1 (Wild type p53 Induced Gene 1). The mouse homologue was first identified by an improved differential display technique. A 7661 bp wig-1 transcript was cloned from the p53negative mouse T lymphoma cell line (J3D), transfected with a temperature-sensitive mouse mutant p53 (Val- 135), using the 5 Marathon cDNA amplification technique. Two mouse wig-1 transcripts of 7.6 kb and 2.2 kb were detected in Northern analysis. The 2.2 kb mouse wig-1 transcript was suggested to arise due to alternative polyadenylation and encodes the same zinc finger protein as the 7.6 kb transcript. Mouse wig-1 induction in response to DNA damage was shown to be p53 dependent. Induction of mouse wig-1 was demonstrated to occur in vivo, in a tissue-specific manner by Northern analysis. The coding region of human WIG-1 was cloned from a brain cDNA library. The human and mouse WIG-1 coding sequences are 84% identical at the nucleotide level and 86.9% identical at the amino acid level, with the three conserved zinc finger motifs. Two human WIG-1 transcripts of 8 kb and 6 kb were detected by Northern analysis. Their induction was demonstrated to occur in human cell lines containing wild type p53 but not in cell lines with mutant p53. A p53-specific binding site with perfect match to the p53 consensus-binding site was found in intron 1 of human WIG-1. In vitro binding of wild type p53 to this site was shown by electrophoretic mobility gel shift assay. Tissue- and species-specific basal expression of both human and mouse WIG-1 were demonstrated by Northern analysis. The abundance of both human and mouse WIG-1 mRNAs and the ratio between the transcripts were shown to vary among different tissues. Human and mouse WIG-1 were mapped to 3q26.3 and mouse chromosome 3B, respectively. Human WIG-1 was demonstrated to be amplified with increased expression in primary squamous cell carcinomas of the lung, using Southern blot and RT-PCR analysis. Polyclonal antibodies against the mouse wig-1 were raised and purified. Further investigation into the function of WIG-1 should contribute to a better understanding of the p53-regulated pathways. This in turn will provide further information for logical, interventional therapeutic strategies against p53-deficient tumors

Cellular Senescence as a Possible Mechanism for Halting Progression of Keloid Lesions

Keloid scarring is a consequence of aberrant wound healing that leads to expansion of the scar beyond the confines of the skin injury. Keloid scars are characterized by excessive extracellular matrix disposition, prolonged proliferation of fibroblasts, increased angiogenesis, and inflammatory cell infiltration. There is no single satisfactory treatment for keloid, and it can lead to severe disfigurements and bodily dysfunction. Thus, clarification of the mechanisms underlying keloid formation, as well as those that prevent it from behaving as a malignant tumor, has significant consequences not only for treatment of keloid but also for the prevention of malignant tumor formation. Senescence is an irreversible form of growth arrest that has been shown to play a role, both in vitro and in vivo, in preventing malignant tumorigenesis upon oncogenic stress. In this study it is shown that fibroblasts embedded inside keloid scars proliferate at a slower rate compared with either those residing at the proliferative edges of the scar or normal fibroblasts. Likewise it is demonstrated that keloid fibroblasts exhibit a cell-cycle arrest with a G2/M DNA content and a higher rate of senescence. The results also indicate that levels of the tumor suppressor protein PML are higher in the active regions of keloid. The study therefore suggests that senescence is one possible mechanism by which keloid is maintained in a benign state. On this basis, “pro-senescence therapy” should be taken into consideration when designing treatment strategies for keloid

VISAGE Reveals a Targetable Mitotic Spindle Vulnerability in Cancer Cells

There is an unmet need for new antimitotic drug combinations that target cancer-specific vulnerabilities. Based on our finding of elevated biomolecule oxidation in mitotically arrested cancer cells, we combined Plk1 inhibitors with TH588, an MTH1 inhibitor that prevents detoxification of oxidized nucleotide triphosphates. This combination showed robust synergistic killing of cancer, but not normal, cells that, surprisingly, was MTH1-independent. To dissect the underlying synergistic mechanism, we developed VISAGE, a strategy integrating experimental synergy quantification with computational-pathway-based gene expression analysis. VISAGE predicted, and we experimentally confirmed, that this synergistic combination treatment targeted the mitotic spindle. Specifically, TH588 binding to β-tubulin impaired microtubule assembly, which when combined with Plk1 blockade, synergistically disrupted mitotic chromosome positioning to the spindle midzone. These findings identify a cancer-specific mitotic vulnerability that is targetable using Plk1 inhibitors with microtubule-destabilizing agents and highlight the general utility of the VISAGE approach to elucidate molecular mechanisms of drug synergy.National Institutes of Health (U.S.) (Grant R35-ES028374)National Institutes of Health (U.S.) (Grant R01-GM104047)National Institutes of Health (U.S.) (Grant R01-ES015339)National Institutes of Health (U.S.) (Grant U54-CA112967)National Institutes of Health (U.S.) (Grant U54-CA217377)American Cancer Society. Post-Doctoral Fellowship (PF-13-355-01-TBE)National Cancer Institute (U.S.) (Support Grant P30-ES002109

Transite: A Computational Motif-Based Analysis Platform That Identifies RNA-Binding Proteins Modulating Changes in Gene Expression

© 2020 The Author(s) Krismer et al. present a computational approach to identify RNA-binding proteins (RBPs) that modulate post-transcriptional control of gene expression using RNA expression data as inputs. By applying this approach to publicly available patient datasets, they identify and experimentally confirm that the RBP hnRNPC contributes to chemotherapy resistance in lung cancer