PLT Counts as a Predictive Marker after Plasma Exchange in Patients with Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

BACKGROUND AND OBJECTIVES: The platelet (PLT) value in hepatitis B-related acute-on-chronic liver failure (HBV-ACLF) is not sufficiently understood. The present study aimed to evaluate the prognostic effect of PLT on the prediction of HBV-ACLF outcomes after plasma exchange (PE). METHODS: HBV-ACLF patients treated with PE between January 2017 and August 2021 were followed up for at least 6 months. Cox regression was performed to develop the predictive model, and the model's performance was analyzed using the receiver operating characteristic curve (ROC). RESULTS: A total of 170 patients were included. The overall survival rate within 180 days was 75.88%. Age, PLT, total bilirubin (TBil), and the iMELD scores were independent risk factors affecting the prognosis of HBV-ACLF patients after PE. According to the Cox regression results, the new model was calculated: R = 0.142 × iMELD-0.009 × PLT. The area under the curve (AUC) of the receiver operating characteristic curve (ROC) was 0.758 (95% CI 0.678-0.838), and patients with lower PLT-iMELD scores (<4.50) had a better prognosis (p < 0.001). CONCLUSION: PLT is a valuable prognostic biomarker for HBV-ACLF patients after PE. The modified iMELD model incorporating PLT has a better sensitivity and efficacy in predicting the prognosis of patients.

SOX9 Transcriptionally Regulates mTOR-Induced Proliferation of Basal Cell Carcinomas.

Currently available smoothened targeted therapies in patients with basal cell nevus syndrome are associated with substantial tumor recurrence and clinical resistance. Strategies bypassing smoothened and/or identifying additional downstream components of the Hedgehog pathway could provide novel antitumor targets with a better therapeutic index. Sry-related high mobility group box 9 (SOX9) is a Hedgehog/glioma-associated oncogene homolog-regulated transcription factor known to be overexpressed in basal cell carcinomas (BCCs). A sequence motif search for SOX9-responsive elements identified three motifs in the promoter region of mammalian target of rapamycin (mTOR). In murine BCC cells, SOX9 occupies the mTOR promoter and induces its transcriptional activity. Short hairpin RNA (shRNA)-mediated knockdown of SOX9, as well as smoothened inhibition by itraconazole and vismodegib, reduces mTOR expression and the phosphorylation of known downstream mTOR targets. These effects culminate in diminishing the proliferative capacity of BCC cells, demonstrating a direct mechanistic link between the Hedgehog and mTOR pathways capable of driving BCC growth. Furthermore, rapamycin, a pharmacologic mTOR inhibitor, suppressed the growth of UV-induced BCCs in Ptch1+/-/SKH-1 mice, a model that closely mimics the accelerated BCC growth pattern of patients with basal cell nevus syndrome. Our data demonstrate that Hedgehog signaling converges on mTOR via SOX9, and highlight the SOX9-mTOR axis as a viable additional target downstream of smoothened that could enhance tumor elimination in patients with BCC.

Correction: Inhibition of p38 MAPK Signaling Augments Skin Tumorigenesis via NOX2 Driven ROS Generation.

[This corrects the article DOI: 10.1371/journal.pone.0097245.].

AKT1 Activation is Obligatory for Spontaneous BCC Tumor Growth in a Murine Model that Mimics Some Features of Basal Cell Nevus Syndrome.

Patients with basal cell nevus syndrome (BCNS), also known as Gorlin syndrome, develop numerous basal cell carcinomas (BCC) due to germline mutations in the tumor suppressor PTCH1 and aberrant activation of Hedgehog (Hh) signaling. Therapies targeted at components of the Hh pathway, including the smoothened (SMO) inhibitor vismodegib, can ablate these tumors clinically, but tumors recur upon drug discontinuation. Using SKH1-Ptch1+/- as a model that closely mimics the spontaneous and accelerated growth pattern of BCCs in patients with BCNS, we show that AKT1, a serine/threonine protein kinase, is intrinsically activated in keratinocytes derived from the skin of newborn Ptch1+/- mice in the absence of carcinogenic stimuli. Introducing Akt1 haplodeficiency in Ptch1+/- mice (Akt1+/- Ptch1+/-) significantly abrogated BCC growth. Similarly, pharmacological inhibition of AKT with perifosine, an alkyl phospholipid AKT inhibitor, diminished the growth of spontaneous and UV-induced BCCs. Our data demonstrate an obligatory role for AKT1 in BCC growth, and targeting AKT may help reduce BCC tumor burden in BCNS patients. Cancer Prev Res; 9(10); 794-802. ©2016 AACR.

Inhibition of p38 MAPK signaling augments skin tumorigenesis via NOX2 driven ROS generation.

p38 mitogen-activated protein kinases (MAPKs) respond to a wide range of extracellular stimuli. While the inhibition of p38 signaling is implicated in the impaired capacity to repair ultraviolet (UV)-induced DNA damage-a primary risk factor for human skin cancers-its mechanism of action in skin carcinogenesis remains unclear, as both anti-proliferative and survival functions have been previously described. In this study, we utilized cultured keratinocytes, murine tumorigenesis models, and human cutaneous squamous cell carcinoma (SCC) specimens to assess the effect of p38 in this regard. UV irradiation of normal human keratinocytes increased the expression of all four p38 isoforms (α/ß/γ/δ); whereas irradiation of p53-deficient A431 keratinocytes derived from a human SCC selectively decreased p38α, without affecting other isoforms. p38α levels are decreased in the majority of human cutaneous SCCs assessed by tissue microarray, suggesting a tumor-suppressive effect of p38α in SCC pathogenesis. Genetic and pharmacological inhibition of p38α and in A431 cells increased cell proliferation, which was in turn associated with increases in NAPDH oxidase (NOX2) activity as well as intracellular reactive oxygen species (ROS). These changes led to enhanced invasiveness of A431 cells as assessed by the matrigel invasion assay. Chronic treatment of p53-/-/SKH-1 mice with the p38 inhibitor SB203580 accelerated UV-induced SCC carcinogenesis and increased the expression of NOX2. NOX2 knockdown suppressed the augmented growth of A431 xenografts treated with SB203580. These findings indicate that in the absence of p53, p38α deficiency drives SCC growth and progression that is associated with enhanced NOX2 expression and ROS formation.

Resveratrol-mediated downregulation of Rictor attenuates autophagic process and suppresses UV-induced skin carcinogenesis.

Macroautophagy is a cellular response to various environmental stresses that ensures lysosomal degradation of long-lived and damaged proteins and cellular organelles. It occurs through the formation of an autophagosome, which then fuses with a lysosome to form an autolysosome. Depending on the cellular context, autophagy may promote cancer cell survival or it may serve as a mechanism of tumor suppression. Herein, we show that resveratrol, a natural phytoalexin, induces premature senescence in human A431 SCC cells, and that resveratrol-induced premature senescence is associated with a blockade of autolysosome formation, as assessed by the absence of colocalization of LC3 and Lamp-2, markers for autophagosomes and lysosomes, respectively. Further, we show that resveratrol downregulates the level of Rictor, a component of mTORC2, leading to decreased RhoA-GTPase and altered actin cytoskeleton organization. Exogenous overexpression of Rictor restores RhoA-GTPase activity and actin cytoskeleton network, and decreases resveratrol-induced senescence-associated ß-gal activity, indicating a direct role of Rictor in senescence induction. Rictor is overexpressed in UV-induced murine SCCs, whereas its expression is diminished by oral administration of resveratrol. These data indicate that resveratrol attenuates autophagic process via Rictor, and suggest that downregulation of Rictor may be a mechanism of tumor suppression associated with premature senescence.

Cancer cell survival following DNA damage-mediated premature senescence is regulated by mammalian target of rapamycin (mTOR)-dependent Inhibition of sirtuin 1.

DNA-damaging agents can induce premature senescence in cancer cells, which contributes to the static effects of cancer. However, senescent cancer cells may re-enter the cell cycle and lead to tumor relapse. Understanding the mechanisms that control the viability of senescent cells may be helpful in eliminating these cells before they can regrow. Treating human squamous cell carcinoma (SCC) cells with the anti-cancer compounds, resveratrol and doxorubicin, triggered p53-independent premature senescence by invoking oxidative stress-mediated DNA damage. This process involved the mTOR-dependent phosphorylation of SIRT1 at serine 47, resulting in the inhibition of the deacetylase activity of SIRT1. SIRT1 phosphorylation caused concomitant increases in p65/RelA NF-κB acetylation and the expression of an anti-apoptotic Bfl-1/A1. SIRT1 physically interacts with the mTOR-Raptor complex, and a single amino acid substitution in the TOS (TOR signaling) motif in the SIRT1 prevented Ser-47 phosphorylation and Bfl-1/A1 induction. The pharmacologic and genetic inhibition of mTOR, unphosphorylatable S47A, or F474A TOS mutants restored SIRT1 deacetylase activity, blocked Bfl-1/A1 induction, and sensitized prematurely senescent SCC cells for apoptosis. We further show that the treatment of UVB-induced SCCs with doxorubicin transiently stabilized tumor growth but was followed by tumor regrowth upon drug removal in p53(+/-)/SKH-1 mice. The subsequent treatment of stabilized SCCs with rapamycin decreased tumor size and induced caspase-3 activation. These results demonstrate that the inhibition of SIRT1 by mTOR fosters survival of DNA damage-induced prematurely senescent SCC cells via Bfl-1/A1 in the absence of functional p53.

Resveratrol targets transforming growth factor-ß2 signaling to block UV-induced tumor progression.

Resveratrol (RES) is a potent anti-cancer agent. We have previously reported that RES arrests the growth of invasive human A431 squamous cell carcinoma (SCC) cells. In this study, we show that oral administration of RES to highly tumor-susceptible p53(+/-)/SKH-1 mice markedly delayed UV-induced skin tumorigenesis and reduced the malignant conversion of benign papillomas to SCCs. Transforming growth factor-ß2 (TGF-ß2) was predominantly overexpressed in UV-induced SCCs and its expression was diminished in RES-treated SCCs/skin. In addition to the inhibition of TGF-ß2 expression, RES increased the level of epithelial cadherin. This RES-mediated TGF-ß2 downregulation led to the inhibition of both TGF-ß2/Smad-dependent and -independent pathways, and suppressed the invasiveness of A431 cells. Addition of TGF-ß2, but not TGF-ß1, rescued the RES-mediated downregulation of p-extracellular signal-regulated kinases 1/2, p-Smad3, and α-smooth muscle actin. The protein kinase B (Akt) substrate cAMP response-binding protein (pCREB) transcription factor is known to regulate TGF-ß2 expression, and RES treatment decreased phosphorylation of Akt and pCREB. Expression of constitutively active Akt blocked RES inhibition of CREB and TGF-ß2, and rescued RES inhibition of cellular invasiveness. Our data indicate that RES suppresses UV-induced malignant tumor progression in p53(+/-)/SKH-1 mice and that RES-inhibited invasiveness of human A431 SCC cells appears to occur, in part, through the Akt-mediated downregulation of TGF-ß2.

CP-31398 restores mutant p53 tumor suppressor function and inhibits UVB-induced skin carcinogenesis in mice.

Mutations in the tumor suppressor p53 are detectable in over 50% of all human malignancies. Mutant p53 protein is incapable of transactivating its downstream target genes that are required for DNA repair and apoptosis. Chronic exposure to UVB induces p53 mutations and is carcinogenic in both murine and human skin. CP-31398, a styrylquinazoline compound, restores the tumor suppressor functions of mutant forms of p53 in tumor cells. However, its effectiveness in vivo remains unclear. Here, we demonstrate that CP-31398 blocked UVB-induced skin carcinogenesis and was associated with increases in p53, p21, and BclXs. CP-31398 downregulated Bcl2, proliferating nuclear cell antigen, and cyclin D1. Activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase also occurred in both tumor and perilesional skin following treatment. CP-31398 induced the expression of p53-dependent target proteins, and this was followed by apoptosis in UVB-irradiated wild-type mice but not in their p53-deficient littermates. Similar effects were observed in human skin carcinoma A431 cells expressing mutant p53. In addition, CP-31398 induced mitochondrial translocation of p53, leading to changes in mitochondrial membrane permeability pore transition (MPT) and consequent cytochrome c release in these cells. Blocking MPT diminished p53 translocation and apoptosis. These studies indicate that reconstituting p53 tumor suppressor functions in vivo by small molecular weight compounds may block the pathogenesis and progression of skin cancer.

Resveratrol inhibits proliferation of human epidermoid carcinoma A431 cells by modulating MEK1 and AP-1 signalling pathways.

Resveratrol (trans-3,4',5-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin found in grapes, and has been shown to inhibit the growth of various types of cancer cells. We investigated the mechanism of the antiproliferative effect of resveratrol in A431-transformed keratinocytes harbouring mutant p53, and show that it is accompanied by G1 cell cycle arrest, which coincides with a marked inhibition of G1 cell cycle regulatory proteins, including cyclins A and D1 and cyclin-dependent kinase (CDK)6 and p53-independent induction of p21WAF1. Cell cycle arrest was also associated with the accumulation of hypophosphorylated Rb and p27KIP1. Resveratrol inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1 > extracellular signal-regulated protein kinase (ERK)1/2 signalling, downregulated c-Jun, and suppressed activating protein (AP)-1 DNA-binding and promoter activity. In addition, the inhibition of MEK1 > ERK1/2 signalling appears to be independent of retinoblastoma protein (pRb) hypophosphorylation in A431 cells, as PD098059 did not suppress pRb phosphorylation. Our results demonstrate that resveratrol affects multiple cellular targets in A431 cells, and that the downregulation of both AP-1 and pRb contributes to its antiproliferative activity in these cells.

Role of p38 MAPK in UVB-induced inflammatory responses in the skin of SKH-1 hairless mice.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is activated by numerous inflammatory mediators and environmental stresses. We assessed the effects of ultraviolet B (UVB) on the p38 MAPK pathway and determined whether cyclooxygenase (COX)-2 expression is downstream of this kinase in the skin of UVB-irradiated SKH-1 mice. SKH-1 mice were irradiated with a single dose of UVB (360 mJ per cm2), and activation of the epidermal p38 MAPK pathway was assessed. UVB-induced phosphorylation of p38 MAPK occurred in a time-dependent manner. Phosphorylation of MAPK-activated protein kinase-2 (MAPKAPK-2) also was detected and correlated with an increase in its kinase activity. Phosphorylation of heat shock protein 27 (HSP27), a substrate for MAPKAPK-2, also was detected post-irradiation. Oral administration of the p38 inhibitor, SB242235, prior to UVB irradiation, blocked activation of the p38 MAPK cascade, and abolished MAPKAPK-2 kinase activity and phosphorylation of HSP27. Moreover, SB242235 inhibited expression of the pro-inflammatory cytokines interleukin (IL)-6 and KC (murine IL-8) and COX-2. Our data demonstrate that UVB irradiation of murine skin activates epidermal p38 MAPK signaling and induces a local pro-inflammatory response. Blockade of the p38 MAPK pathway may offer an effective approach to reducing or preventing skin damage resulting from acute solar radiation.