SHARPIN stabilizes ß-catenin through a linear ubiquitination-independent manner to support gastric tumorigenesis.

BACKGROUND: Aberrant activation of Wnt/ß-catenin signaling by dysregulated post-translational protein modifications, especially ubiquitination is causally linked to cancer development and progression. Although Lys48-linked ubiquitination is known to regulate Wnt/ß-catenin signaling, it remains largely obscure how other types of ubiquitination, such as linear ubiquitination governs its signaling activity. METHODS: The expression and regulatory mechanism of linear ubiquitin chain assembly complex (LUBAC) on Wnt/ß-catenin signaling was examined by immunoprecipitation, western blot and immunohistochemical staining. The ubiquitination status of ß-catenin was detected by ubiquitination assay. The impacts of SHARPIN, a core component of LUBAC on malignant behaviors of gastric cancer cells were determined by various functional assays in vitro and in vivo. RESULTS: Unlike a canonical role in promoting linear ubiquitination, SHARPIN specifically interacts with ß-catenin to maintain its protein stability. Mechanistically, SHARPIN competes with the E3 ubiquitin ligase ß-Trcp1 for ß-catenin binding, thereby decreasing ß-catenin ubiquitination levels to abolish its proteasomal degradation. Importantly, SHARPIN is required for invasiveness and malignant growth of gastric cancer cells in vitro and in vivo, a function that is largely dependent on its binding partner ß-catenin. In line with these findings, elevated expression of SHARPIN in gastric cancer tissues is associated with disease malignancy and correlates with ß-catenin expression levels. CONCLUSIONS: Our findings reveal a novel molecular link connecting linear ubiquitination machinery and Wnt/ß-catenin signaling via SHARPIN-mediated stabilization of ß-catenin. Targeting the linear ubiquitination-independent function of SHARPIN could be exploited to inhibit the hyperactive ß-catenin signaling in a subset of human gastric cancers.

Microvascular fractal dimension predicts prognosis and response to chemotherapy in glioblastoma: an automatic image analysis study.

The microvascular profile has been included in the WHO glioma grading criteria. Nevertheless, microvessels in gliomas of the same WHO grade, e.g., WHO IV glioblastoma (GBM), exhibit heterogeneous and polymorphic morphology, whose possible clinical significance remains to be determined. In this study, we employed a fractal geometry-derived parameter, microvascular fractal dimension (mvFD), to quantify microvessel complexity and developed a home-made macro in Image J software to automatically determine mvFD from the microvessel-stained immunohistochemical images of GBM. We found that mvFD effectively quantified the morphological complexity of GBM microvasculature. Furthermore, high mvFD favored the survival of GBM patients as an independent prognostic indicator and predicted a better response to chemotherapy of GBM patients. When investigating the underlying relations between mvFD and tumor growth by deploying Ki67/mvFD as an index for microvasculature-normalized tumor proliferation, we discovered an inverse correlation between mvFD and Ki67/mvFD. Furthermore, mvFD inversely correlated with the expressions of a glycolytic marker, LDHA, which indicated poor prognosis of GBM patients. Conclusively, we developed an automatic approach for mvFD measurement, and demonstrated that mvFD could predict the prognosis and response to chemotherapy of GBM patients.

Central nervous system progression in advanced non-small cell lung cancer patients with EGFR mutations in response to first-line treatment with two EGFR-TKIs, gefitinib and erlotinib: a comparative study.

BACKGROUND: Central nervous system (CNS) brain metastasis of advanced non-small cell lung cancer (NSCLC) patients confers a worse quality of life and prognosis. The efficacy comparison of two first-generation epidermal growth factor receptor (EGFR) inhibitors erlotinib or gefitinib as first-line treatment for CNS metastasis NSCLC patients with EGFR-sensitizing mutations is yet to be elucidated. METHODS: A retrospective analysis was done on cerebral metastasis rate after erlotinib or gefitinib as first-line treatment for advanced NSCLC patients with EGFR-sensitizing mutations. Time to neurological progression (nTTP) and median progression-free survival (mPFS) were calculated. RESULTS: The study involved 279 patients (erlotinib group: 108, gefitinib group: 171). After a median follow-up of 22 months, 27 patients (25%) in the erlotinib group and 60 patients (35.1%) in the gefitinib group showed CNS progression. The HR of CNS progression for erlotinib versus gefitinib was 0.695 [95% confidence interval (CI), 0.406-1.190], suggesting a risk reduction of 30.5% although not achieving statistical significance. The 6-, 12- and 18-month cumulative CNS progression rates were 0.9, 3.7 and 12% for erlotinib compared with corresponding rates of 5.8, 9.4 and 17% for gefitinib (P = 0.181). However, for those patients with preexisting brain metastases prior to EGFR-TKI treatment, erlotinib as first line treatment significantly extended the median nTTP in comparison to gefitinib (30 months vs 15.8 months, p = 0.024). CONCLUSIONS: Our data show that nTTP can be effectively extended in preexisting brain metastases patients with EGFR-sensitizing mutations initially treated with erlotinib compared with gefitinib. If confirmed, our results indicate that erlotinib may play an important role in controlling CNS progression from EGFR mutation-positive NSCLC.

Monitoring dynamic alterations in calcium homeostasis by T1-mapping manganese-enhanced MRI (MEMRI) in the early stage of small intestinal ischemia-reperfusion injury.

Manganese-enhanced MRI studies have proven to be useful in monitoring physiological activities associated with calcium ions (Ca(2+)) due to the paramagnetic property of the manganese ion (Mn(2+)), which makes it an excellent probe of Ca(2+) . In this study, we developed a method in which a Mn(2+)-enhanced T1 -map MRI could enable the monitoring of Ca(2+) influx during the early stages of intestinal ischemia-reperfusion (I/R) injury. The Mn(2+) infusion protocol was optimized by obtaining dose-dependent and time-course wash-out curves using a Mn(2+)-enhanced T1-map MRI of rabbit abdomens following an intravenous infusion of 50 mmol/l MnCl2 (5-10 nmol/g body weight (BW)). In the rabbit model of intestinal I/R injury, T1 values were derived from the T1 maps in the intestinal wall region and revealed a relationship between the dose of the infused MnCl2 and the intestinal wall relaxation time. Significant Mn(2+) clearance was also observed over time in control animals after the infusion of Mn(2+) at a dose of 10 nmol/g BW. This technique was also shown to be sensitive enough to monitor variations in calcium ion homeostasis in vivo after small intestinal I/R injury. The T1 values of the intestinal I/R group were significantly lower (P < 0.05) than that of the control group at 5, 10, and 15 min after Mn(2+) infusion. Our data suggest that MnCl2 has the potential to be an MRI contrast agent that can be effectively used to monitor changes in intracellular Ca(2+) homeostasis during the early stages of intestinal I/R injury.

ALDH1A1 defines invasive cancer stem-like cells and predicts poor prognosis in patients with esophageal squamous cell carcinoma.

Invasion and metastasis are the major cause of deaths in patients with esophageal cancer. In this study, we isolated cancer stem-like cells from an esophageal squamous cell carcinoma cell line EC109 based on aldehyde dehydrogenase 1A1 (ALDH1A1), and found that ALDH1A1(high) cells possessed the capacities of self-renewal, differentiation and tumor initiation, indications of stem cell properties. To support their stemness, ALDH1A1(high) cells exhibited increased potential of invasion and metastasis as compared with ALDH1A1(low) cells. ALDH1A1(high) esophageal squamous cell carcinoma cells expressed increased levels of mRNA for vimentin, matrix metalloproteinase 2, 7 and 9 (MMP2, MMP7 and MMP9), but decreased the level of E-cadherin mRNA, suggesting that epithelial-mesenchymal transition and secretary MMPs may be attributed to the high invasive and metastatic capabilities of ALDH1A1(high) cells. Furthermore, we examined esophageal squamous cell carcinoma specimens from 165 patients and found that ALDH1A1(high) cells were associated with esophageal squamous dysplasia and the grades, differentiation and invasion depth, lymph node metastasis and UICC stage of esophageal squamous cell carcinoma, as well as poor prognosis of patients. Our results provide the strong evidence that ALDH1A1(high) cancer stem-like cells contribute to the invasion, metastasis and poor outcome of human esophageal squamous cell carcinoma.

Should stage T2 esophageal squamous cell carcinoma be subclassified?

BACKGROUND: In patients with esophageal squamous cell carcinoma (ESCC), pathologic examination allows T2 tumors to be further subclassified according to whether the circular or longitudinal muscle layers are invaded. Therefore, we aimed to investigate whether subclassifying the T2 stages can aid in determining the prognosis for patients with ESCC. METHODS: The clinical and pathologic characteristics of 85 ESCC patients with T2 tumors who underwent thoracoscopic esophagectomy between 2008 and 2013 were retrospectively analyzed. Univariate and multivariate analyses were performed to identify prognostic factors. The Kaplan-Meier method was used to compare survival differences with respect to each prognostic factor. RESULTS: Thirty-nine patients had tumors invading the circular muscle layer and were designated as having T2a disease. The remaining 46 patients had T2b disease, with tumors invading the longitudinal muscle layer. The overall 1-, 3-, and 5-year survival rates were 96.1, 53.8, and 36.4 %, respectively, with a median survival of 39.0 months. Univariate analysis indicated that sex, smoking history, grade, location, and tumor length did not significantly influence on survival. Only T stage (P = 0.017) and N stage (P = 0.003) were associated with survival. The results of multivariate Cox proportional hazard regression analysis showed that T stage (P = 0.045) and N stage (P = 0.003) were independent prognostic factors. CONCLUSIONS: N stage and subclassified T stage are independent prognostic factors in patients with T2 tumors. Therefore, we concluded that T2 tumors can be subclassified further into T2a and T2b stages, and patients with different T2 stages may have different prognoses.

Tumor-associated microglia/macrophages enhance the invasion of glioma stem-like cells via TGF-ß1 signaling pathway.

The invasion of malignant glioma cells into the surrounding normal brain tissues is crucial for causing the poor outcome of this tumor type. Recent studies suggest that glioma stem-like cells (GSLCs) mediate tumor invasion. However, it is not clear whether microenvironment factors, such as tumor-associated microglia/macrophages (TAM/Ms), also play important roles in promoting GSLC invasion. In this study, we found that in primary human gliomas and orthotopical transplanted syngeneic glioma, the number of TAM/Ms at the invasive front was correlated with the presence of CD133(+) GSLCs, and these TAM/Ms produced high levels of TGF-ß1. CD133(+) GSLCs isolated from murine transplanted gliomas exhibited higher invasive potential after being cocultured with TAM/Ms, and the invasiveness was inhibited by neutralization of TGF-ß1. We also found that human glioma-derived CD133(+) GSLCs became more invasive upon treatment with TGF-ß1. In addition, compared with CD133(-) committed tumor cells, CD133(+) GSLCs expressed higher levels of type II TGF-ß receptor (TGFBR2) mRNA and protein, and downregulation of TGFBR2 with short hairpin RNA inhibited the invasiveness of GSLCs. Mechanism studies revealed that TGF-ß1 released by TAM/Ms promoted the expression of MMP-9 by GSLCs, and TGFBR2 knockdown reduced the invasiveness of these cells in vivo. These results demonstrate that TAM/Ms enhance the invasiveness of CD133(+) GSLCs via the release of TGF-ß1, which increases the production of MMP-9 by GSLCs. Therefore, the TGF-ß1 signaling pathway is a potential therapeutic target for limiting the invasiveness of GSLCs.

Connexin 43 reverses malignant phenotypes of glioma stem cells by modulating E-cadherin.

Malfunctioned gap junctional intercellular communication (GJIC) has been thought associated with malignant transformation of normal cells. However, the role of GJIC-related proteins such as connexins in sustaining the malignant behavior of cancer stem cells remains unclear. In this study, we obtained tumorspheres formed by glioma stem cells (GSCs) and adherent GSCs and then examined their GJIC. All GSCs showed reduced GJIC, and differentiated glioma cells had more gap junction-like structures than GSCs. GSCs expressed very low level of connexins, Cx43 in particular, which are key components of gap junction. We observed hypermethylation in the promoter of gap junction protein α1, which encodes Cx43 in GSCs. Reconstitution of Cx43 in GSCs inhibited their capacity of self-renewal, invasiveness, and tumorigenicity via influencing E-cadherin and its coding protein, which leads to changes in the expression of Wnt/ß-catenin targeting genes. Our results suggest that GSCs require the low expression of Cx43 for maintaining their malignant phenotype, and upregulation of Cx43 might be a potential strategy for treatment of malignant glioma.

Pulmonary adenofibroma in a middle-aged man: report of a case.

We report a case of pulmonary adenofibroma, a rare benign soft-tissue tumor characterized by a combination of glandular and spindle stromal elements, in a 55-year-old man. This case is interesting because of the unusual X-ray and computed tomography (CT) imaging findings of a well-defined, subpleural soft-tissue nodule in the inferior lobe of the left lung, which showed no enhancement after contrast scan. The tumor was resected completely via video-assisted thoracoscopy. Microscopic and immunohistochemical examinations supported the diagnosis of a benign pulmonary adenofibroma. The patient remains well with no evidence of recurrence 16 months after surgery. Although the imaging findings were nonspecific, adenofibroma may be one of diagnostic inclusions of soft-tissue nodules of the lung in middle-aged patients.

Decreased expression of C-erbB-2 and CXCR4 in breast cancer after primary chemotherapy.

BACKGROUND: Biological molecular markers such as proto-oncogene erbB-2 (HER-2/neu, c-erbB-2), the CXC chemokine receptor 4 (CXCR4), estrogen receptor (ER), Proliferating Cell Nuclear Antigen (PCNA), DNA topoisomerase II (topo II), P-glycoprotein (P-gp) and glutathione S-transferase (GST) were observed for changes after administration of neochemotherapy and whether these protein expression changes were correlated with response to chemotherapy. METHODS: Sixty-four patients with primary breast cancer who had undergone neo-adjuvant chemotherapy were enrolled in the present study. The expressions of C-erbB-2, CXCR4 and ER-α were measured by immunohistochemistry (IHC) on full tissue sections and on tissue microarrays (TMAs). PCNA, TopoII, P-gp and GST were measured by IHC on TMAs. On the other hand, CXCR4, C-erbB-2 and ER-α expressions were detected using western blot analysis to 16 pairs of fresh preoperative core biopsies. The final surgical specimens were obtained from patients with breast carcinoma who received neo-adjuvant chemotherapy and obtained a partial response (PR). RESULTS: Our data demonstrated that the levels of C-erbB-2, CXCR4 and ER-α in patients decreased after they received neo-adjuvant chemotherapy on full tissue sections and on TMAs. The PCNA level was down-regulated after receiving neo-adjuvant chemotherapy, and no significant change was observed for TopoII, P-gp and GST. The levels of C-erbB-2, CXCR4 and ER-α were also down-regulated after neo-adjuvant chemotherapy was administered, as detected by western blot. In addition, the change expressions of C-erbB-2 and CXCR4 in specimens tended to be correlated with pathological change to neo-adjuvant chemotherapy on full tissue sections and on TMAs in a Pearson chi-square analysis. CONCLUSIONS: As demonstrated in our study, after breast cancer patients were treated with neo-adjuvant systemic therapy, decreased expressions of C-erbB2, ER-α and CXCR4 were observed. Down-regulated expressions of c-erbB-2 and CXCR4 may be a novel mechanism of chemotherapy; the changes of these objective markers may be useful in evaluating the clinical response of neo-adjuvant chemotherapy in breast cancer.

Retrospective analysis for thirty-nine patients with solitary fibrous tumor of pleura and review of the literature.

BACKGROUND: Solitary fibrous tumor of the pleura (SFTP) is an uncommon neoplasm arising from mesenchymal cells. The aim of this study is to summarize the experience and the outcome of the surgical treatment for 39 cases of SFTP. METHODS: From January 2004 to December 2008, 39 patients underwent surgical resection of SFTP in our department. All patients had clinical follow-up by the same team of surgeons. The mean follow-up was 40.3 months. RESULTS: A local removal of the neoplasm was accomplished by video-assisted thoracic surgery (VATS) in 9 patients (group A) and by thoracotomy in 30 patients (group B) respectively. Comparing with group B, operations in group A took significantly less operative time, blood loss and spent less time in the intensive care unit and hospital. All specimens were positive for CD34 and Bcl-2. One patient developed recurrence, and the remaining 38 patients are alive and disease free at the end of follow-up. CONCLUSIONS: Malignant SFTP still had the potential recurrence. VATS represents the more acceptable choice for the selected patients with SFTP.

Intraparenchymal myofibromatosis of the brain in an adult: report of an unusual case.

An unusual case of intraparenchymal myofibromatosis of the brain occurring in a 29-year-old woman is described. Preoperative CT and MRI examinations revealed two well-circumscribed nodular masses localized in the wall of the left lateral ventricle and right temporal lobe, respectively. Both masses were completely resected, and the patient remains disease-free 2 years post-surgery. Histopathologically, the lesions were characterized by stratification. From outer to inner, there was a reactive glial component, lamellated well-differentiated muscle-like cells, densely compact collagen fibers and cellular tumor with nodular and hemangiopericytoma-like patterns, respectively. The myofibroblastic nature of this tumor was verified by immunohistochemical staining and ultrastructural analysis. Intraparenchymal myofibromatosis may be confused with, and should be distinguished from, meningioma, myopericytoma, solitary fibrous tumor, leiomyoma and inflammatory myofibroblastic tumor for accurate diagnosis and optimal treatment.

RBBP6 induces non-small cell lung cancer cell proliferation and high expression is associated with poor prognosis.

Lung cancer is the most common cause of cancer-associated mortality in China with 85% of patients having non-small cell lung cancer (NSCLC). Identifying NSCLC driver genes and prognostic markers is critical to reducing these numbers. The studies of retinoblastoma binding protein 6 (RBBP6) performed on NSCLC is limited. The present study aimed to investigate the molecular function and the prognostic potential of RBBP6 in NSCLC using the A549 cell line and patient samples, respectively. The functional effect on cancer cell proliferation and prognostic value of RBBP6 were examined in vitro and in vivo using reverse transcription-quantitative PCR, immunofluorescence, immunohistochemistry (IHC) and xenograft implantation. The results demonstrated that RBBP6 mRNA expression was significantly higher in NSCLC tissues compared with in adjacent normal samples. When RBBP6 mRNA expression was interfered with using short hairpin RNA, A549 cell proliferation and xenograft tumor growth were reduced. Additionally, IHC and survival analysis demonstrated that patients with NSCLC with high expression levels of RBBP6 had a shorter median overall survival time compared with patients with low RBBP6 expression (31 vs. 51.5 months), and this was more prominent in stage I-II patients (43 vs. >67 months). High expression levels of RBBP6 indicated poor prognosis in patients with NSCLC. This may be due to the ability of RBBP6 to promote cancer cell proliferation. RBBP6 may be a potential prognostic biomarker and a therapeutic target for NSCLC.

Increased ski expression levels are associated with a higher risk and poor prognosis in patients with gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most commonly diagnosed primary mesenchymal tumors of the gastrointestinal tract and 30% of GISTs are associated with a high recurrence risk or metastasis. The current risk classification criteria of the National Comprehensive Cancer Network are based on tumor size, mitotic activity and localization. Investigating additional biomarkers associated with clinical risk may aid in the diagnosis of GIST and improves prediction of patient prognosis. In the present study, the value of using the expression levels of the oncoprotein ski as a prognostic predictor for GISTs was investigated. The results demonstrated that high ski expression levels were correlated with high risk and recurrence rates and indicated poor prognosis regarding median disease-free survival. Overall, the present study suggests that ski expression levels may serve as a predictor for clinical risk and prognosis of patients with GISTs.

Value of SATB2, ISL1, and TTF1 to differentiate rectal from other gastrointestinal and lung well-differentiated neuroendocrine tumors.

BACKGROUND: Management of neuroendocrine tumors (NETs) depends on the primary site, but the location of many well-differentiated (WD) NETs is elusive. Organ-specific markers are required for pathological diagnosis from biopsy. Transcription factors with good organ specificity include TTF1 (thyroid transcription factor 1; lung), CDX2 (caudal type homeobox transcription factor 2; midgut), and ISL1 (ISL LIM homeobox 1) and PAX8 (paired box 8) for the pancreas and rectum. SATB2 (SATB homeobox 2) has shown high sensitivity and specificity in colorectal adenocarcinoma. This study determined the viability of SATB2 and other transcription factors as markers, single or in combination, for WD-NETs of various sites. METHODS: Immunohistochemical staining of 81 WD-NETs from 8 organ sites was performed to identify SATB2, TTF1, CDX2, ISL1, and PAX8. Receiver operating characteristic (ROC) curves were constructed for different combinations of the 5 markers to determine sensitivity and specificity. RESULTS: Among the WD-NETs, SATB2 was predominantly found in those of the rectum; TTF1 in the lung, larynx, and esophagus; and ISL1 in the duodenum and rectum. PAX8 and CDX2 showed poor organ specificity. ROC profiles showed 50% sensitivity and 96% specificity to lung for TTF1+ ISL1-; and 65% sensitivity and 100% specificity to rectum for SATB2+ ISL1- TTF1-. ISL1+ SATB2- TTF1- showed 83% sensitivity and 85% specificity to the duodenum, and 44% sensitivity and 87% specificity to the pancreas. A literature search showed that there was no significant difference in the expression rates of the five transcription factors (TTF1, CDX2, SATB2, PAX8 and ISL1) between primary and metastatic WD-NETs at the same organ when there was a large sample size. CONCLUSION: Among the 5 transcription factors tested, SATB2 may be a viable marker of WE-NETs of the rectum. The combination of SATB2, ISL1, and TTF1 may help estimate the locations of WD-NETs of unknown origin.

Combination of Metformin and Gefitinib as First-Line Therapy for Nondiabetic Advanced NSCLC Patients with EGFR Mutations: A Randomized, Double-Blind Phase II Trial.

PURPOSE: Preclinical and retrospective studies suggested a role for metformin in sensitizing patients who have diabetes with non-small cell lung cancer (NSCLC) to EGFR tyrosine kinase inhibitors (TKIs). We therefore examined its effects in combination with gefitinib in patients without diabetes harboring EGFR mutations (EGFRm). PATIENTS AND METHODS: A total of 224 patients without diabetes with treatment-naïve stage IIIB-IV EGFRm NSCLC were randomly assigned in a 1:1 ratio to receive gefitinib plus either metformin or placebo. The primary endpoint was progression-free survival (PFS) rate at 1 year and secondary endpoints included overall survival (OS), PFS, objective response rate (ORR), and safety. Serum levels of IL6 were also examined in an exploratory analysis. RESULTS: The median duration of follow-up was 19.15 months. The estimated 1-year PFS rates were 41.2% [95% confidence interval (CI), 30.0-52.2] with gefitinib plus metformin and 42.9% (95% CI, 32.6-52.7) with gefitinib plus placebo (P = 0.6268). Median PFS (10.3 months vs. 11.4 months) and median OS (22.0 months vs. 27.5 months) were numerically lower in the metformin group, while ORRs were similar between the two arms (66% vs. 66.7%). No significant treatment group differences were detected across all subgroups with respect to PFS, including those with elevated levels of IL6. Metformin combined with gefitinib resulted in a remarkably higher incidence of diarrhea compared with the control arm (78.38% vs. 43.24%). CONCLUSIONS: Our study showed that addition of metformin resulted in nonsignificantly worse outcomes and increased toxicity and hence does not support its concurrent use with first-line EGFR-TKI therapy in patients without diabetes with EGFRm NSCLC.

Author Correction: Invasion of white matter tracts by glioma stem cells is regulated by a NOTCH1-SOX2 positive-feedback loop.

The original and corrected figures are shown in the accompanying Author Correction.

Invasion of white matter tracts by glioma stem cells is regulated by a NOTCH1-SOX2 positive-feedback loop.

Early invasive growth along specific anatomical structures, especially the white matter tract, is regarded as one of the main causes of poor therapeutic outcome of people with gliomas. We show that some glioma stem cells (GSCs) are preferentially located along white matter tracts, which exhibit a demyelinated phenotype, at the invasive frontier of glioma tissues. These GSCs are CD133+Notch1+, whereas the nerve fibers express the Notch ligand Jagged1. The Notch-induced transcription factor Sox9 promotes the transcription of SOX2 and the methylation level of the NOTCH1 promoter is attenuated by the upregulation of SOX2 to reinforce NOTCH1 expression in GSCs. This positive-feedback loop in a cohort of glioma subjects is correlated with a poor prognosis. Inhibition of Notch signaling attenuates the white-matter-tract tropism of GSCs. These findings provide evidence indicating that the NOTCH1-SOX2 positive-feedback loop controls GSC invasion along white matter tracts.

BRD4 Promotes Gastric Cancer Progression and Metastasis through Acetylation-Dependent Stabilization of Snail.

Cancer metastasis, a leading cause of death in patients, is associated with aberrant expression of epigenetic modifiers, yet it remains poorly defined how epigenetic readers drive metastatic growth and whether epigenetic readers are targetable to control metastasis. Here, we report that bromodomain-containing protein 4 (BRD4), a histone acetylation reader and emerging anticancer therapeutic target, promotes progression and metastasis of gastric cancer. The abundance of BRD4 in human gastric cancer tissues correlated with shortened metastasis-free gastric cancer patient survival. Consistently, BRD4 maintained invasiveness of cancer cells in vitro and their dissemination at distal organs in vivo. Surprisingly, BRD4 function in this context was independent of its putative transcriptional targets such as MYC or BCL2, but rather through stabilization of Snail at posttranslational levels. In an acetylation-dependent manner, BRD4 recognized acetylated lysine 146 (K146) and K187 on Snail to prevent Snail recognition by its E3 ubiquitin ligases FBXL14 and ß-Trcp1, thereby inhibiting Snail polyubiquitination and proteasomal degradation. Accordingly, genome-wide transcriptome analyses identified that BRD4 and Snail regulate a partially shared metastatic gene signature in gastric cancer cells. These findings reveal a noncanonical posttranscriptional regulatory function of BRD4 in maintaining cancer growth and dissemination, with immediate translational implications for treating gastric metastatic malignancies with clinically available bromodomain inhibitors. SIGNIFICANCE: These findings reveal a novel posttranscriptional regulatory function of the epigenetic reader BRD4 in cancer metastasis via stabilizing Snail, with immediate translational implication for treating metastatic malignancies with clinically available bromodomain inhibitors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/4869/F1.large.jpg.

Epigenetic restriction of Hippo signaling by MORC2 underlies stemness of hepatocellular carcinoma cells.

The evolutionarily conserved Hippo signaling pathway is a key regulator of stem cell self-renewal, differentiation, and organ size. While alterations in Hippo signaling are causally linked to uncontrolled cell growth and a broad range of malignancies, genetic mutations in the Hippo pathway are uncommon and it is unclear how the tumor suppressor function of the Hippo pathway is disrupted in human cancers. Here, we report a novel epigenetic mechanism of Hippo inactivation in the context of hepatocellular carcinoma (HCC). We identify a member of the microrchidia (MORC) protein family, MORC2, as an inhibitor of the Hippo pathway by controlling upstream Hippo regulators, neurofibromatosis 2 (NF2) and kidney and brain protein (KIBRA). Mechanistically, MORC2 forms a complex with DNA methyltransferase 3A (DNMT3A) at the promoters of NF2 and KIBRA, leading to their DNA hyper-methylation and transcriptional repression. As a result, NF2 and KIBRA are crucial targets of MORC2 to regulate confluence-induced activation of Hippo signaling and contact inhibition of cell growth under both physiological and pathological conditions. The MORC2-NF2/KIBRA axis is critical for maintaining self-renewal, sorafenib resistance, and oncogenicity of HCC cells in vitro and in nude mice. Furthermore, MORC2 expression is elevated in HCC tissues, associated with stem-like properties of cancer cells, and disease progression in patients. Collectively, MORC2 promotes cancer stemness and tumorigenesis by facilitating DNA methylation-dependent silencing of Hippo signaling and could be a potential molecular target for cancer therapeutics.