PARP-1 selectively impairs KRAS-driven phenotypic and molecular features in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined. DESIGN: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA. RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients. CONCLUSION: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.

The Co-mutational Spectrum Determines the Therapeutic Response in Murine FGFR2 Fusion-Driven Cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and a highly lethal malignancy. Chemotherapeutic options are limited, but a considerable subset of patients harbors genetic lesions for which targeted agents exist. Fibroblast growth factor receptor 2 (FGFR2) fusions belong to the most frequent and therapeutically relevant alterations in ICC, and the first FGFR inhibitor was recently approved for the treatment of patients with progressed, fusion-positive ICC. Response rates of up to 35% indicate that FGFR-targeted therapies are beneficial in many but not all patients. Thus far, no established biomarkers exist that predict resistance or response to FGFR-targeted therapies in patients with ICC. APPROACH AND RESULTS: In this study, we use an autochthonous murine model of ICC to demonstrate that FGFR2 fusions are potent drivers of malignant transformation. Furthermore, we provide preclinical evidence that the co-mutational spectrum acts not only as an accelerator of tumor development, but also modifies the response to targeted FGFR inhibitors. Using pharmacologic approaches and RNA-interference technology, we delineate that Kirsten rat sarcoma oncogene (KRAS)-activated mitogen-activated protein kinase signaling causes primary resistance to FGFR inhibitors in FGFR2 fusion-positive ICC. The translational relevance is supported by the observation that a subset of human FGFR2 fusion patients exhibits transcriptome profiles reminiscent of KRAS mutant ICC. Moreover, we demonstrate that combination therapy has the potential to overcome primary resistance and to sensitize tumors to FGFR inhibition. CONCLUSIONS: Our work highlights the importance of the co-mutational spectrum as a significant modifier of response in tumors that harbor potent oncogenic drivers. A better understanding of the genetic underpinnings of resistance will be pivotal to improve biomarker-guided patient selection and to design clinically relevant combination strategies.

CD44 Contributes to the Regulation of MDR1 Protein and Doxorubicin Chemoresistance in Osteosarcoma.

Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.

In the Zoom Where It Happened: Telepractice and the Voice Clinic in 2020.

Telemedicine-conveying medical information virtually for planning, diagnosis, or treatment-has been a part of the American medical system for over 100 years. A constantly evolving modality, telepractice was a supplemental care delivery system for most speech-language pathologists (SLPs) until March 2020 when the COVID-19 pandemic forced clinical operations to halt in-person activities and convert as much as possible to virtual platforms. The purpose of this article is to provide an overview of the need for telepractice prior to and beyond the current pandemic, the efficacy of telepractice for the voice-specialized SLP, limitations of telepractice, and best practices for providing care over telepractice with a specific focus on voice disorder diagnosis and treatment.

Cluster of differentiation 44 promotes osteosarcoma progression in mice lacking the tumor suppressor Merlin.

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.

Application of patient-derived liver cancer cells for phenotypic characterization and therapeutic target identification.

Primary liver cancer (PLC) ranks among the most lethal solid cancers worldwide due to lack of effective biomarkers for early detection and limited treatment options in advanced stages. Development of primary culture models that closely recapitulate phenotypic and molecular diversities of PLC is urgently needed to improve the patient outcome. Long-term cultures of 7 primary liver cancer cell lines of hepatocellular and cholangiocellular origin were established using defined culture conditions. Morphological and histological characteristics of obtained cell lines and xenograft tumors were analyzed and compared to original tumors. Time course analyses of transcriptomic and genomic changes were performed using next-generation sequencing (NGS). Key oncogenic alterations were identified by targeted NGS and cell lines carrying potentially actionable mutations were treated with corresponding specific inhibitors. PDCL fully resembled morphological features of the primary cancers in vitro and in vivo over extended period in culture. Genomic alterations as well as transcriptome profiles showed high similarity with primary tumors and remained stable during long-term culturing. Targeted-NGS confirmed that key oncogenic mutations such as TP53, KRAS, CTNNB1 as well as actionable mutations (e.g. MET, cKIT, KDR) were highly conserved in PDCL and amenable for individualized therapeutic approaches. Integrative genomic and transcriptomic approaches further demonstrated that PDCL more closely resemble molecular and prognostic features of PLC than established cell lines and are valuable tool for direct target evaluation. Our integrative analysis demonstrates that PDCL represents refined model for discovery of relevant molecular subgroups and exploration of precision medicine approaches for the treatment of this deadly disease.

Deletion of organic cation transporter Oct3 promotes hepatic fibrosis via upregulation of TGFß.

Organic cation transporters (OCT) are responsible for the intracellular uptake and detoxification of a broad spectrum of endogenous and exogenous substrates. OCTs are downregulated in cholestasis, fibrosis, and hepatocellular carcinoma, but the underlying molecular mechanisms and downstream effects of OCT deletion are unknown. Oct3-knockout (Oct3-/-; FVB.Slc22a3tm10pb) and wild-type (WT; FVB) mice were subject to escalating doses of carbon tetrachloride (CCl4) or thioacetamide (TAA) for 6 wk to induce advanced parenchymal liver fibrosis. Secondary biliary fibrosis was generated by bile duct ligation. Liver fibrosis was assessed by hydroxyproline determination, quantitative Sirius red morphometry, and quantitative real-time PCR for fibrosis and inflammation-related genes. Ductular reaction was assessed by bile duct count per field of view in hematoxylin and eosin staining. General gene expression analyses were performed in liver tissue from untreated Oct3-/- and WT mice. Finally, primary murine hepatocytes were treated with the nonselective OCT inhibitor quinine, and transforming growth factor-ß1 (Tgfß1) protein expression was quantified by quantitative real-time PCR and Western blot. Oct3-/- mice developed significantly more fibrosis after bile duct ligation and CCl4 treatment compared with WT mice. Ductular reaction was enhanced in the long-term model. Concomitantly, Oct1 mRNA expression was downregulated during cholestatic and chemically (TAA and CCl4) induced fibrogenesis. The downregulation of Oct1 mRNA in fibrotic liver tissue reversed within 4 wk after TAA cessation. Gene expression analysis by next-generation sequencing revealed an enrichment of Tgfß1 target genes in Oct3-/- mice. Tgfß1 mRNA expression was significantly upregulated after chemically induced fibrosis (P < 0.001) in Oct3-/- compared with WT mice. Accordingly, in primary murine hepatocytes functional inhibition of OCT led to an upregulation of Tgfß1 mRNA expression. Loss of Oct3 promotes fibrogenesis by affecting Tgfß-mediated homeostasis in mice with chronic biliary and parenchymal liver damage and fibrosis.NEW & NOTEWORTHY We show for the first time that organic cation transporter 3 (Oct3) is not only downregulated in fibrosis but loss of Oct3 also leads to an upregulation of transforming growth factor-ß contributing to fibrosis progression.

Induction of Chromosome Instability by Activation of Yes-Associated Protein and Forkhead Box M1 in Liver Cancer.

BACKGROUND & AIMS: Many different types of cancer cells have chromosome instability. The hippo pathway leads to phosphorylation of the transcriptional activator yes-associated protein 1 (YAP1, YAP), which regulates proliferation and has been associated with the development of liver cancer. We investigated the effects of hippo signaling via YAP on chromosome stability and hepatocarcinogenesis in humans and mice. METHODS: We analyzed transcriptome data from 242 patients with hepatocellular carcinoma (HCC) to search for gene signatures associated with chromosomal instability (CIN); we investigated associations with overall survival time and cancer recurrence using Kaplan-Meier curves. We analyzed changes in expression of these signature genes, at mRNA and protein levels, after small interfering RNA-mediated silencing of YAP in Sk-Hep1, SNU182, HepG2, or pancreatic cancer cells, as well as incubation with thiostrepton (an inhibitor of forkhead box M1 [FOXM1]) or verteporfin (inhibitor of the interaction between YAP and TEA domain transcription factor 4 [TEAD4]). We performed co-immunoprecipitation and chromatin immunoprecipitation experiments. We collected liver tissues from mice that express a constitutively active form of YAP (YAPS127A) and analyzed gene expression signatures and histomorphologic parameters associated with chromosomal instability. Mice were given injections of thiostrepton and livers were collected and analyzed by immunoblotting, immunohistochemistry, histology, and real-time polymerase chain reaction. We performed immunohistochemical analyses on tissue microarrays of 105 HCCs and 7 nontumor liver tissues. RESULTS: Gene expression patterns associated with chromosome instability, called CIN25 and CIN70, were detected in HCCs from patients with shorter survival time or early cancer recurrence. TEAD4 and YAP were required for CIN25 and CIN70 signature expression via induction and binding of FOXM1. Disrupting the interaction between YAP and TEAD4 with verteporfin, or inhibiting FOXM1 with thiostrepton, reduced the chromosome instability gene expression patterns. Hyperplastic livers and tumors from YAPS127A mice had increased CIN25 and CIN70 gene expression patterns, aneuploidy, and defects in mitosis. Injection of YAPS127A mice with thiostrepton reduced liver overgrowth and signs of chromosomal instability. In human HCC tissues, high levels of nuclear YAP correlated with increased chromosome instability gene expression patterns and aneuploidy. CONCLUSIONS: By analyzing cell lines, genetically modified mice, and HCC tissues, we found that YAP cooperates with FOXM1 to contribute to chromosome instability. Agents that disrupt this pathway might be developed as treatments for liver cancer. Transcriptome data are available in the Gene Expression Omnibus public database (accession numbers: GSE32597 and GSE73396).

Translating bioinformatics in oncology: guilt-by-profiling analysis and identification of KIF18B and CDCA3 as novel driver genes in carcinogenesis.

MOTIVATION: Co-regulated genes are not identified in traditional microarray analyses, but may theoretically be closely functionally linked [guilt-by-association (GBA), guilt-by-profiling]. Thus, bioinformatics procedures for guilt-by-profiling/association analysis have yet to be applied to large-scale cancer biology. We analyzed 2158 full cancer transcriptomes from 163 diverse cancer entities in regard of their similarity of gene expression, using Pearson's correlation coefficient (CC). Subsequently, 428 highly co-regulated genes (|CC| ≥ 0.8) were clustered unsupervised to obtain small co-regulated networks. A major subnetwork containing 61 closely co-regulated genes showed highly significant enrichment of cancer bio-functions. All genes except kinesin family member 18B (KIF18B) and cell division cycle associated 3 (CDCA3) were of confirmed relevance for tumor biology. Therefore, we independently analyzed their differential regulation in multiple tumors and found severe deregulation in liver, breast, lung, ovarian and kidney cancers, thus proving our GBA hypothesis. Overexpression of KIF18B and CDCA3 in hepatoma cells and subsequent microarray analysis revealed significant deregulation of central cell cycle regulatory genes. Consistently, RT-PCR and proliferation assay confirmed the role of both genes in cell cycle progression. Finally, the prognostic significance of the identified KIF18B- and CDCA3-dependent predictors (P = 0.01, P = 0.04) was demonstrated in three independent HCC cohorts and several other tumors. In summary, we proved the efficacy of large-scale guilt-by-profiling/association strategies in oncology. We identified two novel oncogenes and functionally characterized them. The strong prognostic importance of downstream predictors for HCC and many other tumors indicates the clinical relevance of our findings. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Control of mitogenic and motogenic pathways by miR-198, diminishing hepatoma cell growth and migration.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths, worldwide. MicroRNAs, inhibiting gene expression by targeting various transcripts, are involved in genomic dysregulation during hepatocellular tumorigenesis. In previous studies, microRNA-198 (miR-198) was shown to be significantly downregulated in HCV-positive hepatocellular carcinoma (HCC). Herein, the function of miR-198 in hepatocellular carcinoma cell growth and gene expression was studied. In hepatoma cell-types with low levels of liver-specific transcription factor HNF1α indicating a low differentiation grade, miR-198 expression was most downregulated. However, miR-198 treatment did not restore the expression of the liver-specific transcription factors HNF1α or HNF4α. Importantly, overexpression of miR-198 in Pop10 hepatoma cells markedly reduced cell growth. In agreement, comprehensive gene expression profiling by microarray hybridisation and real-time quantification revealed that central signal transducers of proliferation pathways were downregulated by miR-198. In contrast, genes mediating cellular adherence were highly upregulated by miR-198. Thus, the low expression of E-cadherin and claudin-1, involved in cell adhesion and cell-cell contacts, was abolished in hepatoma cells after miR-198 overexpression. This definite induction of both proteins by miR-198 was shown to be accompanied by a significantly impaired migration activity of hepatoma Pop10 cells. In conclusion, miR-198 acts as a tumor suppressor by repression of mitogenic and motogenic pathways diminishing cell growth and migration.

Inhibition of PRMT5-mediated regulation of DKK1 sensitizes colorectal cancer cells to chemotherapy.

The Dickkopf family proteins (DKKs) are strong Wnt signaling antagonists that play a significant role in colorectal cancer (CRC) development and progression. Recent work has shown that DKKs, mainly DKK1, are associated with the induction of chemoresistance in CRC and that DKK1 expression in cancer cells correlates with that of protein arginine N-methyltransferase 5 (PRMT5). This points to the presence of a regulatory loop between DKK1 and PRMT5. Herein, we addressed the question of whether PRMT5 contributes to DKK1 expression in CRC and hence CRC chemoresistance. Both in silico and in vitro approaches were used to explore the relationship between PRMT5 and different DKK members. Our data demonstrated that DKK1 expression is significantly upregulated in CRC clinical samples, KRAS-mutated CRC in particular and that the levels of DKK1 positively correlate with PRMT5 activation. Chromatin immunoprecipitation (ChIP) data indicated a possible epigenetic role of PRMT5 in regulating DKK1, possibly through the symmetric dimethylation of H3R8. Knockdown of DKK1 or treatment with the PRMT5 inhibitor CMP5 in combination with doxorubicin yielded a synergistic anti-tumor effect in KRAS mutant, but not KRAS wild-type, CRC cells. These findings suggest that PRMT5 regulates DKK1 expression in CRC and that inhibition of PRMT5 modulates DKK1 expression in such a way that reduces CRC cell growth.

Next generation sequencing of the Ago2 interacting transcriptome identified chemokine family members as novel targets of neuronal microRNAs in hepatic stellate cells.

BACKGROUND & AIMS: After myofibroblastic transdifferentiation, hepatic stellate cells (HSC), mainly involved in liver fibrosis by extracellular matrix production, exhibit an altered growth factor profile including increased expression of neuronal mediators. Here, we analyzed putative targets of neuronal microRNAs miR-9, miR-125b, and miR-128 by deep sequencing of the transcript population, interacting with the miRNA/Argonaute 2 (Ago2) complex in myofibroblastic HSC. METHODS: MicroRNA expression was quantified by real-time PCR in primary HSC, isolated from the rat or human liver. Myofibroblastic HSC were transfected either with mimics or inhibitors of miR-9, miR-125b, and miR-128. RNA from immunoprecipitated Ago2-miRNA/transcript complexes was purified and used for next generation sequencing. Additionally, gene expression was investigated in quiescent and activated primary HSC, treated with the miR-128 mimic or inhibitor, by microarray analysis. RESULTS: During myofibroblastic transdifferentiation of HSC, miR-9, miR-125b, and miR-128 expression was markedly increased. Transcriptome analysis of Ago2 bound mRNA by deep sequencing identified a broad spectrum of transcripts that interact with neuronal miRNAs in myofibroblastic HSC. In particular, in HSC overexpressing miR-128, many members of the chemokine family were bound to the Ago2 repression complex. Furthermore, a comprehensive profiling of gene expression demonstrates the high impact of neuronal miRNAs on the chemokine network. CONCLUSIONS: Ago2 immunoprecipitation followed by deep sequencing is a useful tool to identify novel miRNA targets. Upregulation of neuronal miR-9, miR-125b, and miR-128 during myofibroblastic transition and the identified interaction with a wide range of chemokines and chemokine receptors suggest a prominent role of neuronal miRNAs in the inflammatory response of HSC during fibrosis.

Deletion of Cd44 Inhibits Metastasis Formation of Liver Cancer in Nf2-Mutant Mice.

Primary liver cancer is the third leading cause of cancer-related death worldwide. An increasing body of evidence suggests that the Hippo tumor suppressor pathway plays a critical role in restricting cell proliferation and determining cell fate during physiological and pathological processes in the liver. Merlin (Moesin-Ezrin-Radixin-like protein) encoded by the NF2 (neurofibromatosis type 2) gene is an upstream regulator of the Hippo signaling pathway. Targeting of Merlin to the plasma membrane seems to be crucial for its major tumor-suppressive functions; this is facilitated by interactions with membrane-associated proteins, including CD44 (cluster of differentiation 44). Mutations within the CD44-binding domain of Merlin have been reported in many human cancers. This study evaluated the relative contribution of CD44- and Merlin-dependent processes to the development and progression of liver tumors. To this end, mice with a liver-specific deletion of the Nf2 gene were crossed with Cd44-knockout mice and subjected to extensive histological, biochemical and molecular analyses. In addition, cells were isolated from mutant livers and analyzed by in vitro assays. Deletion of Nf2 in the liver led to substantial liver enlargement and generation of hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (iCCAs), as well as mixed hepatocellular cholangiocarcinomas. Whilst deletion of Cd44 had no influence on liver size or primary liver tumor development, it significantly inhibited metastasis formation in Nf2-mutant mice. CD44 upregulates expression of integrin ß2 and promotes transendothelial migration of liver cancer cells, which may facilitate metastatic spreading. Overall, our results suggest that CD44 may be a promising target for intervening with metastatic spreading of liver cancer.

Genetic signatures shared in embryonic liver development and liver cancer define prognostically relevant subgroups in HCC.

Multiple activations of individual genes during embryonic liver and HCC development have repeatedly prompted speculations about conserved embryonic signatures driving cancer development. Recently, the emerging discussion on cancer stem cells and the appreciation that generally tumors may develop from progenitor cells of diverse stages of cellular differentiation has shed increasing light on the overlapping genetic signatures between embryonic liver development and HCC. However there is still a lack of systematic studies investigating this area. We therefore performed a comprehensive analysis of differentially regulated genetic signaling pathways in embryonic and liver cancer development and investigated their biological relevance.Genetic signaling pathways were investigated on several publically available genome wide microarray experiments on liver development and HCC. Differentially expressed genes were investigated for pathway enrichment or underrepresentation compared to KEGG annotated pathways by Fisher exact evaluation. The comparative analysis of enrichment and under representation of differentially regulated genes in liver development and HCC demonstrated a significant overlap between multiple pathways. Most strikingly we demonstrated a significant overlap not only in pathways expected to be relevant to both conditions such as cell cycle or apoptosis but also metabolic pathways associated with carbohydrate and lipid metabolism. Furthermore, we demonstrated the clinical significance of these findings as unsupervised clustering of HCC patients on the basis of these metabolic pathways displayed significant differences in survival.These results indicate that liver development and liver cancer share similar alterations in multiple genetic signaling pathways. Several pathways with markedly similar patterns of enrichment or underrepresentation of various regulated genes between liver development and HCC are of prognostic relevance in HCC. In particular, the metabolic pathways were identified as novel prognostically relevant players in HCC development.

Investigating Past Trauma in Laryngoresponders Versus Non-Laryngoresponders: Piloting New Methods in an Exploratory Study.

OBJECTIVES/HYPOTHESIS: This exploratory pilot study aimed to probe the relationship between past experiences of trauma in people who self-identify as "laryngoresponders" compared to those who do not. It also explored the communicative context of past traumatic events in laryngoresponders versus non-laryngoresponders. STUDY DESIGN: Prospective, within-subjects experimental design. METHODS: 29 vocally healthy cisgender women (ages 19 to 56) completed a battery of validated self-report measures relating to their past traumatic experiences. Participants also completed two original self-report measures designed to provide insight about (1) where stress tends to manifest in their body and (2) communicative settings of participants' past trauma. RESULTS: Six participants (21%) self-identified a predictable laryngeal and/or vocal response to acute stress and thus comprise the laryngoresponders group. Laryngoresponders exhibited worse scores on 75% of all trauma-related variables compared to non-laryngoresponders, and Emotional Neglect was disproportionately represented in laryngoresponders. Participants with a reported history of childhood Emotional Neglect (83% of laryngoresponders, 35% of nonlaryngoresponders) reported quantitatively "less ideal" communication experiences in the context of past traumatic experiences. CONCLUSIONS: Other investigators identify the larynx as a "vulnerable body pathway" for some women. This pilot study of adult women without voice complaints revealed several commonalities amongst self-reported laryngoresponders, and compels further exploration of the voice-trauma relationship.

Immediate Effects of Mindfulness Meditation on the Voice.

OBJECTIVES: The benefits of mindfulness meditation are well documented. This study evaluated the immediate effects of mindfulness meditation (MM) on the voice and voice user. STUDY DESIGN: Prospective experimental study. METHODS: Participants: 19 vocally healthy (VH) individuals, and 26 individuals with common voice disorders (CVD; benign lesions and hyperfunctional muscle tension) deemed stimulable for voice therapy. Exclusionary criteria: prior training or regular meditation practice. Participants recorded speech samples before and after a 11.5-minute prerecorded session of MM. PRIMARY OUTCOMES: phonatory aerodynamics and participants' self-reported experience of voice. SECONDARY OUTCOMES: self-reported anxiety, vocal acoustics, speech breathing patterns, and auditory-perceptual outcomes. Baseline self-reported measures of voice (Voice Handicap Index-10 - VHI-10), breathing (Dyspnea Index - DI), stress (Perceived Stress Scale - PSS), and trait mindfulness (Cognitive and Mindfulness Scale - Revised, CAMS-R, Five Facet Mindfulness Questionnaire - FFMQ) were compared between groups. RESULTS: At baseline, CVD had significantly higher VHI-10 (P< 0.001) and DI (P= 0.0014), and lower trait mindfulness (CAMS-R, P= 0.02). No difference between groups for PSS or FFMQ. Changes postMM: decreased CPP for all-voiced sentences for VH (P= 0.003), decreased mean SPL (P= 0.012) on sustained vowel for VH, increased mean phonatory airflow during sustained vowel for CVD (P = 0.012). VH demonstrated a decrease in CPP on the all-voice sentence, and CVD demonstrated an increase, resulting in a significant between group difference (P= 0.013). Participants reported improvements in voice, emotional and physical states. State anxiety decreased for both groups (= < 0.001). No other objective outcomes reached significance. CONCLUSIONS: After a brief MM, participants experienced improvement in physical, emotional, and cognitive states, and in their perceptions of their voice. Results indicate that a brief, single session of MM may be beneficial for some, but not sufficient to override habitual voice and speech patterns. Given the benefits of MM, future work should evaluate MM in a standard voice therapy protocol.

Acquired Resistance to Antiangiogenic Therapies in Hepatocellular Carcinoma Is Mediated by Yes-Associated Protein 1 Activation and Transient Expansion of Stem-Like Cancer Cells.

Induction of neoangiogenesis is a hallmark feature during disease progression of hepatocellular carcinoma (HCC). Antiangiogenetic compounds represent a mainstay of therapeutic approaches; however, development of chemoresistance is observed in the majority of patients. Recent findings suggest that tumor-initiating cells (TICs) may play a key role in acquisition of resistance, but the exact relevance for HCC in this process remains to be defined. Primary and established hepatoma cell lines were exposed to long-term sorafenib treatment to model acquisition of resistance. Treatment effects on TICs were estimated by sphere-forming capacity in vitro, tumorigenicity in vivo, and flow cytometry. Adaptive molecular changes were assessed by whole transcriptome analyses. Compensatory mechanisms of resistance were identified and directly evaluated. Sustained antiproliferative effect following sorafenib treatment was observed in three of six HCC cell lines and was followed by rapid regrowth, thereby mimicking responses observed in patients. Resistant cells showed induction in sphere forming in vitro and tumor-initiating capacity in vivo as well as increased number of side population and epithelial cell adhesion molecule-positive cells. Conversely, sensitive cell lines showed consistent reduction of TIC properties. Gene sets associated with resistance and poor prognosis, including Hippo/yes-associated protein (YAP), were identified. Western blot and immunohistochemistry confirmed increased levels of YAP. Combined treatment of sorafenib and specific YAP inhibitor consistently revealed synergistic antioncogenic effects in resistant cell lines. Conclusion: Resistance to antiangiogenic therapy might be driven by transient expansion of TICs and activation of compensatory pro-oncogenic signaling pathways, including YAP. Specific targeting of TICs might be an effective therapeutic strategy to overcome resistance in HCC.

p53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

BACKGROUND & AIMS: A coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury. METHODS: Nitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2. RESULTS: In a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response. CONCLUSIONS: Characteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.

Epigenetic modifications precede molecular alterations and drive human hepatocarcinogenesis.

Development of primary liver cancer is a multistage process. Detailed understanding of sequential epigenetic alterations is largely missing. Here, we performed Infinium Human Methylation 450k BeadChips and RNA-Seq analyses for genome-wide methylome and transcriptome profiling of cirrhotic liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, and early (n = 5) and progressed (n = 3) hepatocellular carcinomas (HCC) synchronously detected in 8 patients with HCC with chronic hepatitis B infection. Integrative analyses of epigenetically driven molecular changes were identified and validated in 2 independent cohorts comprising 887 HCCs. Mitochondrial DNA sequencing was further employed for clonality analyses, indicating multiclonal origin in the majority of investigated HCCs. Alterations in DNA methylation progressively increased from liver cirrhosis (CL) to dysplastic lesions and reached a maximum in early HCCs. Associated early alterations identified by Ingenuity Pathway Analysis (IPA) involved apoptosis, immune regulation, and stemness pathways, while late changes centered on cell survival, proliferation, and invasion. We further validated 23 putative epidrivers with concomitant expression changes and associated with overall survival. Functionally, Striatin 4 (STRN4) was demonstrated to be epigenetically regulated, and inhibition of STRN4 significantly suppressed tumorigenicity of HCC cell lines. Overall, application of integrative genomic analyses defines epigenetic driver alterations and provides promising targets for potentially novel therapeutic approaches.

BAX Redistribution Induces Apoptosis Resistance and Selective Stress Sensitivity in Human HCC.

Cancer therapies induce differential cell responses, ranging from efficient cell death to complete stress resistance. The BCL-2 proteins BAX and BAK govern the cellular decision between survival and mitochondrial apoptosis. Therefore, the status of BAX/BAK regulation can predict the cellular apoptosis predisposition. Relative BAX/BAK localization was analyzed in tumor and corresponding non-tumor samples from 34 hepatocellular carcinoma (HCC) patients. Key transcriptome changes and gene expression profiles related to the status of BAX regulation were applied to two independent cohorts including over 500 HCC patients. The prediction of apoptotic response was tested using cell lines and polyclonal tumor isolates. Cellular protection from BAX was confirmed by challenging cells with mitochondrial BAX. We discovered a subgroup of HCC with selective protection from BAX-dependent apoptosis. BAX-protected tumors showed enrichment of signaling pathways associated with oxidative stress response and DNA repair as well as increased genetic heterogeneity. Gene expression profiles characteristic to BAX-specific protection are enriched in poorly differentiated HCCs and show significant association to the overall survival of HCC patients. Consistently, addiction to DNA repair of BAX-protected cancer cells caused selective sensitivity to PARP inhibition. Molecular characteristics of BAX-protected HCC were enriched in cells challenged with mitochondrial BAX. Our results demonstrate that predisposition to BAX activation impairs tumor biology in HCC. Selective BAX inhibition or lack thereof delineates distinct subgroups of HCC patients with molecular features and differential response pattern to apoptotic stimuli and inhibition of DNA repair mechanisms.