Distinct promoter methylation patterns of LKB1 in the hamartomatous polyps of Peutz-Jeghers syndrome and its potential in gastrointestinal malignancy prediction.

BACKGROUND: Peutz-Jeghers Syndrome (PJS) is known as a rare inherited polyposis due to the malfunction of serine/threonine kinase gene LKB1. However, not all of PJS patients carry LKB1 germline mutation. Previous researches have observed the elevated DNA methylation level in PJS polyps. Nevertheless, the mechanism of such abnormal and its impact on PJS patients remains to be fully described. RESULTS: The results proved a significant increase on the methylation level of LKB1 promoter in PJS polyps compared with normal colon biopsies through bisulfite PCR followed by Sanger sequencing. Moreover, the methylation pattern in PJS polyps could be further categorized as three different scenarios: hypermethylated, hemimethylated and hypomethylated pattern. Furthermore, immunohistochemistry of DNMT1/3a/3b suggested the up-regulation of DNMT1 and 3a might participate the epigenetic alternation of LKB1 in PJS polyps. Logistic regression suggested hypomethylated LKB1 promoter in PJS polyps as a risk factor for gastrointestinal malignancies in PJS patients. CONCLUSIONS: The promoter methylation level of LKB1 gene in PJS polyps is generally elevated compared with normal colon mucosa. Yet not all of PJS polyps carry hypermethylated LKB1 promoter. Hypomethylation in this region has linked to malignant tumors in PJS patients. Given the rarity of PJS, this work together with previous researches, have proved the importance of LKB1 promoter methylation in PJS development and prognosis.

Formononetin attenuates monocrotaline­induced pulmonary arterial hypertension via inhibiting pulmonary vascular remodeling in rats.

Pulmonary arterial hypertension (PAH) is a life­threatening disease induced by the excessive proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Formononetin (FMN) is a natural isoflavone with numerous cardioprotective properties, which can inhibit the proliferation and induce the apoptosis of tumor cells; however, whether FMN has a therapeutic effect on PAH remains unclear. In the present study, PAH was induced in rats with monocrotaline (MCT, 60 mg/kg); rats were then administered FMN (10, 30 or 60 mg/kg/day). At the end of the experiment, hemodynamic changes, right ventricular hypertrophy and lung morphological characteristics were evaluated. α­smooth muscle actin (α­SMA), proliferating cell nuclear antigen (PCNA), and TUNEL were detected by immunohistochemical staining. The expression of PCNA, Bcl­2­associated X protein (Bax), Bcl­2 and, cleaved caspase­3, and activation of AKT and ERK were examined by western blot analysis. The results demonstrated that FMN significantly ameliorated the right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling induced by MCT. FMN also attenuated MCT­induced increased expression of α­SMA and PCNA. The ratio of Bax/Bcl­2 and cleaved caspase­3 expression increased in rat lung tissue in response to FMN treatment. Furthermore, reduced phosphorylation of AKT and ERK was also observed in FMN­treated rats. Therefore, FMN may provide protection against MCT­induced PAH by preventing pulmonary vascular remodeling, potentially by suppressing the PI3K/AKT and ERK pathways in rats.

Dual role of the integrated stress response in medulloblastoma tumorigenesis.

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). Phosphorylated eIF2α is quickly dephosphorylated by the protein phosphatase 1 and growth arrest and DNA damage 34 (GADD34) complex. Data indicate that the ISR can either promote or suppress tumor development. Our previous studies showed that the ISR is activated in medulloblastoma in both human patients and animal models, and that the decreased ISR via PERK heterozygous deficiency attenuates medulloblastoma formation in Patched1 heterozygous deficient (Ptch1+/-) mice by enhancing apoptosis of pre-malignant granule cell precursors (GCPs) during cell transformation. We showed here that GADD34 heterozygous mutation moderately enhanced the ISR and noticeably increased the incidence of medulloblastoma in adult Ptch1+/- mice. Surprisingly, GADD34 homozygous mutation strongly enhanced the ISR, but significantly decreased the incidence of medulloblastoma in adult Ptch1+/- mice. Intriguingly, GADD34 homozygous mutation significantly enhanced pre-malignant GCP apoptosis in cerebellar hyperplastic lesions and reduced the lesion numbers in young Ptch1+/- mice. Nevertheless, neither GADD34 heterozygous mutation nor GADD34 homozygous mutation had a significant effect on medulloblastoma cells in adult Ptch1+/- mice. Collectively, these data imply the dual role of the ISR, promoting and inhibiting, in medulloblastoma tumorigenesis by regulating apoptosis of pre-malignant GCPs during the course of malignant transformation.

Regulation of PERK-eIF2α signalling by tuberous sclerosis complex-1 controls homoeostasis and survival of myelinating oligodendrocytes.

Tuberous sclerosis complex-1 or 2 (TSC1/2) mutations cause white matter abnormalities, including myelin deficits in the CNS; however, underlying mechanisms are not fully understood. TSC1/2 negatively regulate the function of mTOR, which is required for oligodendrocyte differentiation. Here we report that, unexpectedly, constitutive activation of mTOR signalling by Tsc1 deletion in the oligodendrocyte lineage results in severe myelination defects and oligodendrocyte cell death in mice, despite an initial increase of oligodendrocyte precursors during early development. Expression profiling analysis reveals that Tsc1 ablation induces prominent endoplasmic reticulum (ER) stress responses by activating a PERK-eIF2α signalling axis and Fas-JNK apoptotic pathways. Enhancement of the phospho-eIF2α adaptation pathway by inhibition of Gadd34-PP1 phosphatase with guanabenz protects oligodendrocytes and partially rescues myelination defects in Tsc1 mutants. Thus, TSC1-mTOR signalling acts as an important checkpoint for maintaining oligodendrocyte homoeostasis, pointing to a previously uncharacterized ER stress mechanism that contributes to hypomyelination in tuberous sclerosis.

Inhibition of Vascular Endothelial Growth Factor Receptor 2 Exacerbates Loss of Lower Motor Neurons and Axons during Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are inflammatory demyelinating and neurodegenerative diseases in the central nervous system (CNS). It is believed that MS and EAE are initiated by autoreactive T lymphocytes that recognize myelin antigens; however, the mechanisms responsible for neurodegeneration in these diseases remain elusive. Data indicate that vascular endothelial growth factor A (VEGF-A) plays a role in the development of MS and EAE. Interestingly, VEGF-A is regarded as a neurotrophic factor in the CNS that promotes neuron survival and neurogenesis in various neurodegenerative diseases by activating VEGF receptor 2 (VEGFR2). In this study, we sought to explore the role of the VEGF-A/VEGFR2 signaling in neurodegeneration in MS and EAE. We showed that the expression of VEGF-A was decreased in the spinal cord during EAE and that VEGFR2 was activated in lower motor neurons in the spinal cord of EAE mice. Interestingly, we found that treatment with SU5416, a selective VEGFR2 inhibitor, starting after the onset of EAE clinical symptoms exacerbated lower motor neuron loss and axon loss in the lumbar spinal cord of mice undergoing EAE, but did not alter Purkinje neuron loss in the cerebellum or upper motor neuron loss in the cerebral cortex. Moreover, SU5416 treatment had a minimal effect on EAE clinical symptoms as well as inflammation, demyelination, and oligodendrocyte loss in the lumbar spinal cord. These results imply the protective effects of the VEGF-A/VEGFR2 signaling on lower motor neurons and axons in the spinal cord in MS and EAE.

Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α.

B7-H3 is a member of B7 family of immunoregulatory transmembrane glycoproteins expressed by T cells. While B7-H3 overexpression is associated with poor outcomes in multiple cancers, it also has immune-independent roles outside T cells and its precise mechanistic contributions to cancer are unclear. In this study, we investigated the role of B7-H3 in metabolic reprogramming of cancer cells in vitro and in vivo We found that B7-H3 promoted the Warburg effect, evidenced by increased glucose uptake and lactate production in B7-H3-expressing cells. B7-H3 also increased the protein levels of HIF1α and its downstream targets, LDHA and PDK1, key enzymes in the glycolytic pathway. Furthermore, B7-H3 promoted reactive oxygen species-dependent stabilization of HIF1α by suppressing the activity of the stress-activated transcription factor Nrf2 and its target genes, including the antioxidants SOD1, SOD2, and PRX3. Metabolic imaging of human breast cancer xenografts in mice confirmed that B7-H3 enhanced tumor glucose uptake and tumor growth. Together, our results illuminate the critical immune-independent contributions of B7-H3 to cancer metabolism, presenting a radically new perspective on B7 family immunoregulatory proteins in malignant progression. Cancer Res; 76(8); 2231-42. ©2016 AACR.

PERK Activation Promotes Medulloblastoma Tumorigenesis by Attenuating Premalignant Granule Cell Precursor Apoptosis.

Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum. Here, we determined the role of PERK signaling on medulloblastoma tumorigenesis by assessing its effects on premalignant GCPs and tumor cells. We found that PERK signaling was activated in both premalignant GCPs in young Ptch1(+/-) mice and medulloblastoma cells in adult mice. We demonstrated that PERK haploinsufficiency reduced the incidence of medulloblastomas in Ptch1(+/-) mice. Interestingly, PERK haploinsufficiency enhanced apoptosis of premalignant GCPs in young Ptch1(+/-) mice but had no significant effect on medulloblastoma cells in adult mice. Moreover, we showed that the PERK pathway was activated in medulloblastomas in humans. These results suggest that PERK signaling promotes medulloblastoma tumorigenesis by attenuating apoptosis of premalignant GCPs during the course of malignant transformation.

Pancreatic endoplasmic reticulum kinase activation promotes medulloblastoma cell migration and invasion through induction of vascular endothelial growth factor A.

Evidence is accumulating that activation of the pancreatic endoplasmic reticulum kinase (PERK) in response to endoplasmic reticulum (ER) stress adapts tumor cells to the tumor microenvironment and enhances tumor angiogenesis by inducing vascular endothelial growth factor A (VEGF-A). Recent studies suggest that VEGF-A can act directly on certain tumor cell types in an autocrine manner, via binding to VEGF receptor 2 (VEGFR2), to promote tumor cell migration and invasion. Although several reports show that PERK activation increases VEGF-A expression in medulloblastoma, the most common solid malignancy of childhood, the role that either PERK or VEGF-A plays in medulloblastoma remains elusive. In this study, we mimicked the moderate enhancement of PERK activity observed in tumor patients using a genetic approach and a pharmacologic approach, and found that moderate activation of PERK signaling facilitated medulloblastoma cell migration and invasion and increased the production of VEGF-A. Moreover, using the VEGFR2 inhibitor SU5416 and the VEGF-A neutralizing antibody to block VEGF-A/VEGFR2 signaling, our results suggested that tumor cell-derived VEGF-A promoted medulloblastoma cell migration and invasion through VEGFR2 signaling, and that both VEGF-A and VEGFR2 were required for the promoting effects of PERK activation on medulloblastoma cell migration and invasion. Thus, these findings suggest that moderate PERK activation promotes medulloblastoma cell migration and invasion through enhancement of VEGF-A/VEGFR2 signaling.

A deregulated integrated stress response promotes interferon-γ-induced medulloblastoma.

Endoplasmic reticulum (ER) stress activates pancreatic ER kinase (PERK), which coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). There is evidence that the ISR is involved in tumor development. Recent studies also show that the ISR stimulates the expression of vascular endothelial growth factor A (VEGF-A), a master regulator of angiogenesis. Our previous studies have demonstrated that enforced expression of interferon-γ (IFNγ) in the central nervous system during development induces sonic hedgehog expression and leads to cerebellar dysplasia or medulloblastoma. Here we report that PERK was activated in cerebellar dysplasia and medulloblastoma in IFNγ-expressing mice. We found that inactivation of the growth arrest and DNA damage 34 (GADD34) gene, encoding the stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2α, enhanced ISR signaling and facilitated medulloblastoma formation in IFNγ-expressing mice. Moreover, we found that the induction of VEGF-A and enhanced angiogenesis were associated with medulloblastoma formation in IFNγ-expressing mice on the GADD34 mutation background. Thus, our data provide genetic evidence that the ISR facilitates medulloblastoma development.

Overcoming trastuzumab resistance in breast cancer by targeting dysregulated glucose metabolism.

Trastuzumab shows remarkable efficacy in treatment of ErbB2-positive breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. In this study, we investigated the antitumor effects of trastuzumab in combination with glycolysis inhibitors in ErbB2-positive breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of heat shock factor 1 (HSF1) and lactate dehydrogenase A (LDH-A) in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab resistance. Importantly, we found that combining trastuzumab with glycolysis inhibition synergistically inhibited trastuzumab-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. Taken together, our findings show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance.

Amyloidosis cutis dyschromica in two female siblings: cases report.

BACKGROUND: Cutaneous amyloidosis has been classified into primary cutaneous amyloidosis (PCA, OMIM #105250), secondary cutaneous amyloidosis and systemic cutaneous amyloidosis. PCA is the deposition of amyloid in previously apparent normal skin without systemic involvement. Amyloidosis cutis dyschromica (ACD) is a rare distinct type of PCA. Here, the unique clinical and histological findings of two Chinese female siblings with ACD were described. CASES PRESENTATIONS: Patient 1 was a 34-year-old female, presented with mildly pruritic, diffuse mottled hyperpigmentation and hypopigmentation. The lesions involved all over the body since she was 10 years old. There were a few itchy blisters appearing on her arms, lower legs and truck, especially on the sun-exposed areas in summer. Some hypopigmented macules presented with slight atrophy. Patient 2 was 39-year-old, the elder sister of patient 1. She had similar skin lesions since the same age as the former. The atrophy and blisters on the skin of the patient with amyloidosis cutis dyschromica have not been described in previous literature. Histological examinations of the skin biopsies taken from both patients revealed amyloid deposits in the whole papillary dermis. Depending on the histological assessment, the two cases were diagnosed as amyloidosis cutis dyschromica. CONCLUSION: The two cases suggest that the atrophy and blisters may be the uncommon manifestations of amyloidosis cutis dyschromica. It alerts clinicians to consider the possibility of ACD when meeting patients with cutaneous dyschromia. Skin biopsy is essential and family consultation of genetic investigation is very important in such cases.

Enhanced integrated stress response promotes myelinating oligodendrocyte survival in response to interferon-gamma.

The T-cell-derived, pleiotropic cytokine interferon (IFN)-gamma is believed to play a key regulatory role in immune-mediated demyelinating disorders of the central nervous system, including multiple sclerosis and experimental autoimmune encephalomyelitis. Our previous work has demonstrated that the endoplasmic reticulum (ER) stress response modulates the response of oligodendrocytes to this cytokine. The ER stress response activates the pancreatic ER kinase, which coordinates an adaptive program known as the integrated stress response by phosphorylating translation initiation factor 2alpha (eIF2alpha). In this study, we found that growth arrest and DNA damage 34 (GADD34), a stress-inducible regulatory subunit of a phosphatase complex that dephosphorylates eIF2alpha, was selectively up-regulated in myelinating oligodendrocytes in mice that ectopically expressed IFN-gamma in the central nervous system. We also found that a GADD34 mutant strain of mice displayed increased levels of phosphorylated eIF2alpha (p-eIF2alpha) in myelinating oligodendrocytes when exposure to IFN-gamma, as well as diminished oligodendrocyte loss and hypomyelination. Furthermore, treatment with salubrinal, a small chemical compound that specifically inhibits protein phosphatase 1(PP1)-GADD34 phosphatase activity, increased the levels of p-eIF2alpha and ameliorated hypomyelination and oligodendrocyte loss in cultured hippocampal slices exposed to IFN-gamma. Thus, our data provide evidence that an enhanced integrated stress response could promote oligodendrocyte survival in immune-mediated demyelination diseases.

Utilization of acorn fringe for ellagic acid production by Aspergillus oryzae and Endomyces fibuliger.

Conversion of acorn fringe extract into ellagic acid production by Aspergillus oryzae and Endomyces fibuliger were investigated. The results showed that ellagic acid production was maximized when co-fermentation of the two fungi was performed at 30 degrees C and pH 5.0 with 5.7 g/l of initial substrate concentration, which were close to the optimal values for both fungi to yield an appropriate consortium of hydrolytic enzymes. Meanwhile, it was found that the co-fermentation could compensate the deficiencies in the level of polyphenol oxidase activity from pure A. oryzae and the levels of ellagitannin acyl hydrolase and beta-glucosidase activities from pure E. fibuliger, resulting in. 0.91 g/l of biomass concentration containing 1.84 g/l of ellagic acid. The research not only demonstrates that the co-fermentation is an effective approach to utilize forest byproduct for ellagic acid production, but also provides more evidences for understanding evolution of ellagic acid production with enzymes actions, which is important for process control of ellagic acid production in industrial application.

Interferon-gamma induced medulloblastoma in the developing cerebellum.

We have generated a mouse model system with a high incidence of medulloblastoma, a malignant neoplasm believed to arise from immature precursors of cerebellar granule neurons. These animals ectopically express interferon-gamma (IFN-gamma) in astrocytes in the CNS in a controlled manner, exploiting the tetracycline-controllable system. More than 80% of these mice display severe ataxia and develop cerebellar tumors that express synaptophysin, the mouse atonal homolog MATH1, sonic hedgehog (SHH), and Gli1. IFN-gamma-induced tumorigenesis in these mice is associated with increased expression of SHH, and SHH induction and tumorigenesis are dependent on signal transducer and activator of transcription 1 (STAT1). When IFN-gamma expression is shut down with doxycycline at postnatal day 12 (P12), the clinical symptoms dissipate and the mice do not develop tumors, whereas if transgene expression is shut down at P16, the clinical symptoms and tumors progress to lethality, indicating that IFN-gamma is required for tumor induction but not progression. The tumors that occur in the continued presence of IFN-gamma display extensive necrosis and apoptosis as well as macrophage and lymphocytic infiltration, whereas the tumors that develop in mice in which IFN-gamma expression is shut down at P16 do not. Thus, IFN-gamma expression in the perinatal period can induce SHH expression and medulloblastoma in the cerebellum by a STAT1-dependent mechanism, and its continued presence appears to promote a host response to the tumor.

Inducible production of interferon-gamma in the developing brain causes cerebellar dysplasia with activation of the Sonic hedgehog pathway.

Here we examined the role of interferon (IFN)-gamma in regulating the Sonic hedgehog (Shh) pathway and cerebellar development in bigenic mice with temporal control of IFN-gamma gene expression driven by a tetracycline-controllable promoter. In IFN-gamma-expressing but not age-matched non-IFN-gamma-expressing bigenic or control mice, development of the cerebellum was severely affected with the persistence and extensive proliferation of the external granule neuron layer (EGL) and infiltration with modest numbers of T-lymphocytes. Following induction of IFN-gamma transgene expression, both total and tyrosine-phosphorylated signal transducer and activator of transcription (STAT)1 (the major transcriptional factor for IFN-gamma), phosphorylated STAT3 and STAT5, and expression of a number of IFN-gamma-regulated genes were significantly increased in cerebellum. In the cerebellum from IFN-gamma-expressing but not age-matched non-IFN-gamma-expressing mice, the level of Shh and Gli-1 but not Patched (Ptch) 1 RNA was increased as was the 19-kDa signaling product of the Shh precursor protein. In situ localization studies revealed ectopic expression of the Shh gene by the granule neurons. We conclude that IFN-gamma directly affects the proliferation and fate of EGL neurons in the cerebellum by activating the Shh pathway and stimulating an autocrine growth response by these cells.