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Background: Filamin A (FLNA) is a member of the filamin family and has been found to be critical for the progression of several cancers. However, its biological function in papillary thyroid cancer (PTC) remains largely unexplored. Methods: Data from The Cancer Genome Atlas (TCGA) databases were utilized to analyze the FLNA expression level and its influence on the clinical implications of patients with PTC. Gene Expression Omnibus (GEO) and qRT-PCR was used to verify the expression levels of FLNA in PTC. Kaplan–Meier survival analysis was conducted to evaluate the prognostic value of FLNA in PTC. Transwell assays and wound healing were performed to examine the biological function of FLNA knockdown in PTC cells. Gene set enrichment analysis (GSEA) and Western blotting were conducted to investigate the potential mechanisms underlying the role of FLNA in PTC progression. In addition, the relationship between FLNA expression and the tumor immune microenvironment (TME) in PTC was explored. Results: FLNA was significantly upregulated in PTC tissues. High expression levels of FLNA was correlated with advanced TNM stage, T stage, and N stage, as well as poor disease-free interval (DFI) and progression-free interval (PFI) time in PTC patients. Moreover, we found that FLNA knockdown inhibited the migration and invasion of PTC cells. Mechanistically, FLNA knockdown inhibited epithelial–mesenchymal transition (EMT) in PTC and affected the activation of the FAK/AKT signaling pathway. In addition, FLNA expression was associated with TME in PTC. Conclusion: FLNA may be regarded as a new therapeutic target for PTC patients.

ABSTRACTOur study investigated the role of WTAP in colon cancer. We employed experiments including m6A dot blot hybridization, methylated RNA immunoprecipitation, dual-luciferase, and RNA immunoprecipitation to investigate the regulatory mechanism of WTAP. Western blot was performed to analyze the expression of WTAP, FLNA and autophagy-related proteins in cells. Our results confirmed the up-regulation of WTAP in colon cancer and its promoting effect on proliferation and inhibiting effect on apoptosis. FLNA was the downstream gene of WTAP and WTAP-regulated m6A modification led to post-transcriptional repression of FLNA. The rescue experiments showed that WTAP/FLNA could inhibit autophagy. WTAP-mediated m6A modification was confirmed to be crucial in colon cancer development, providing new insights into colon cancer therapy.

Abstract Background Periventricular nodular heterotopia (PNH), associated with FLNA mutations, is a rare clinical condition potentially associated with multiple systemic conditions, including cardiac, pulmonary, skeletal, and cutaneous diseases. However, due to a paucity of information in the literature, accurate prognostic advice cannot be provided to patients with the disease. Case presentation We report a 2-year-old female whose PNH was associated with a nonsense mutation in the q28 region of the X chromosome, in exon 31 of FLNA (c.5159dupA). The patient is currently seizure-free and has no congenital heart disease, lung disease or skeletal or joint issues, and her development is normal. Conclusions FLNA-associated PNH is a genetically-heterogeneous disease, and the FLNA mutation, c.5159dupA (p.Tyr1720*) is a newly identified pathogenic variant. FLNA characterization will help the clinical diagnosis and treatment of PNH and provide individualized genetic counseling for patients.

Mutations in FLNA, which encodes the cytoskeletal protein FLNA, cause a spectrum of sclerosing skeletal dysplasias. Although many of these genetic variants are recurrent and cluster within the gene, the pathogenic mechanism that underpins the development of these skeletal phenotypes is unknown. To determine if the skeletal dysplasia in FLNA-related conditions is due to a cell-autonomous loss-of-function localising to osteoblasts and/or osteocytes, we utilised mouse models to conditionally remove Flna from this cellular lineage. Flna was conditionally knocked out from mature osteocytes using the Dmp1-promoter driven Cre-recombinase expressing mouse, as well as the committed osteoblast lineage using the Osx-Cre or Col1a1-Cre expressing lines. We measured skeletal parameters with μCT and histological methods, as well as gene expression in the mineralised skeleton. We found no measureable differences between the conditional Flna knockout mice, and their control littermate counterparts. Moreover, all of the conditional Flna knockout mice, developed and aged normally. From this we concluded that the skeletal dysplasia phenotype associated with pathogenic variants in FLNA is not caused by a cell-autonomous loss-of-function in the osteoblast-osteocyte lineage, adding more evidence to the hypothesis that these phenotypes are due to gain-of-function in FLNA.

Abstract Background Filamin A, encoded by the X‐linked gene FLNA, links the cell membrane with the cytoskeleton and acts as a regulator of the actin cytoskeleton. Mutations in FLNA cause a large spectrum of congenital malformations during embryonic development, including Melnick–Needles syndrome (MNS). However, reports of MNS, especially in males, are rare, and the pathogenesis molecular mechanisms are not well understood. Methods We found a family with two consecutive miscarriages of similar fetuses with multiple malformations. DNA was extracted from peripheral blood and tissues, and whole exome sequencing was performed for genetic analysis. Then, we created a C57BL/6 mouse with a point mutation by CRISPR/Cas‐mediated genome engineering. The migration of primary abdominal muscle cell was detected by wound healing assay. Results The first fetus showed congenital hygroma colli and omphalocele identified by ultrasound at 12 wks; the second fetus showed hygroma colli and thoraco abdominoschisis at 12 wks, with a new hemizygous mutation c.4420G>A in exon 26 of the FLNA gene, which is predicted to cause an amino acid substitution (p.Asp1474Asn). The mother and grandmother were both present in the c.4420G>A heterozygous state, and the mother's healthy brother had wild‐type FLNA. These FLNA‐mutated mice exhibited a broader central gap between the rectus abdominis than the wild type (WT), similar to the midline structure dysplasia of the abdominal wall in the two fetuses. Wound healing assays showed the attenuated migration capacity of abdominal muscle cells in mice with mutated FLNA. Finally, we summarized the cases of MNS with FLNA mutation from the accessible published literature thus far. Conclusion Our research revealed a mutation site of the FLNA for MNS and explored the mechanism of midline structure dysplasia in the abdominal wall of male patients, which could provide more evidence for the clinical diagnosis and genetic counseling of families with these disorders.

Background: Periventricular nodular heterotopia (PVNH) is caused by abnormal neuronal migration, resulting in the neurons accumulate as nodules along the surface of the lateral ventricles. PVNH often cause epilepsy, psychomotor development or cognition problem. Mutations in FLNA (Filamin A) is the most common underlying genetic etiology. Our purpose is to delineate the clinical and imaging spectrum that differentiates FLNA-positive and FLNA-negative PVNH patients. Methods: We included 21 patients with confirmed PVNH. The detailed clinical information, electroencephalography, and other clinical findings were recorded. Detailed brain MR imaging was assessed. Mutation analysis of the FLNA gene was used Sanger sequencing or a next generation sequencing based assay. Results: FLNA mutations were identified in 9 patients (7 females and 2 males), including two nonsense, two splice site, three frameshift, and two missense mutations. In FLNA-positive group, 8 patients had anterior predominant bilateral symmetric presentation and only one had asymmetrical distribution and dilated ventricles. Extra-cerebral features were more often observed in FLNA-positive group than FLNA-negative group. Conclusion: Genetics of PVNH is heterogenous, and mutations in FLNA gene account for less than half of the patients in our cohort. Our finding between FLNA-positive and FLNA-negative patients could guide the clinicians to select relevant genetic testing.

Cell cytoskeleton proteins are involved in tumor pathogenesis, progression and pharmacological resistance. Filamin A (FLNA) is a large actin-binding protein with both structural and scaffold functions implicated in a variety of cellular processes, including migration, cell adhesion, differentiation, proliferation and transcription. The role of FLNA in cancers has been studied in multiple types of tumors. FLNA plays a dual role in tumors, depending on its subcellular localization, post-translational modification (as phosphorylation at Ser2125) and interaction with binding partners. This review summarizes the experimental evidence showing the critical involvement of FLNA in the complex biology of endocrine tumors. Particularly, the role of FLNA in regulating expression and signaling of the main pharmacological targets in pituitary neuroendocrine tumors, pancreatic neuroendocrine tumors, pulmonary neuroendocrine tumors and adrenocortical carcinomas, with implications on responsiveness to currently used drugs in the treatment of these tumors, will be discussed.

Abstract Background FLNA Loss-of-Function (LoF) causes periventricular nodular heterotopia type 1 (PVNH1), an acknowledged cause of seizures of various types. Neurological symptoms are inconstant, and cardiovascular (CV) defects or connective tissue disorders (CTD) have regularly been associated. We aimed at refining the description of CV and CTD features in patients with FLNA LoF and depicting the multisystemic nature of this condition. Methods We retrospectively evaluated FLNA variants and clinical presentations in FLNA LoF patient with at least one CV or CTD feature, from three cohorts: ten patients from the French Reference Center for Rare Vascular Diseases, 23 patients from the national reference diagnostic lab for filaminopathies-A, and 59 patients from literature review. Results Half of patients did not present neurological symptoms. Most patients presented a syndromic association combining CV and CTD features. CV anomalies, mostly aortic aneurysm and/or dilation were present in 75% of patients. CTD features were present in 75%. Variants analysis demonstrated an enrichment of coding variants in the CH1 domain of FLNA protein. Conclusion In FLNA LoF patients, the absence of seizures should not be overlooked. When considering a diagnosis of PVNH1, the assessment for CV and CTD anomalies is of major interest as they represent interlinked features. We recommend systematic study of FLNA within CTD genes panels, regardless of the presence of neurological symptoms.

We present a family with 3 generations of FLNA gene-associated periventricular nodular heterotopia (PVNH), with a unique presentation in a fetus with multiple neurologic malformations. Neurologic abnormalities were noted on routine fetal imaging for a 33-year-old G1P0 woman; absence of the corpus callosum and PVNH was confirmed on follow-up MRI. This prompted genetic evaluation, revealing a nonsense mutation in the FLNA gene. Familial genetic analysis and neuroimaging revealed the same variant and MRI evidence of PVNH in the fetus’s asymptomatic mother, and maternal grandmother, who had a long history of seizure disorder. Such phenotypic variability within a single family demonstrates the spectrum of PVNH and the importance of genetic counseling for patients with PVNH. This case also adds to existing literature on the rare but not unique presentation of FLNA-associated fetal malformations.

The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST2) and 5 (SST5) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)–based approach for the in situ visualization and quantification of SST2/SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2. SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P < 0.05 vs. A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P < 0.05 vs. basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2/SST5 hetero-dimers. Moreover, we showed that FLNA regulates SST2 and SST5 intracellular trafficking induced by octreotide and pasireotide.

Colorectal cancer (CRC) has a high incidence rate and poor prognosis, and the available treatment approaches have limited therapeutic benefits. Therefore, understanding the underlying mechanisms of occurrence and development is particularly crucial. Increasing attention has been paid to the pathophysiological role of cancer-associated fibroblasts (CAFs) in the heterogeneous tumour microenvironment. CAFs play a crucial role in tumorigenesis, tumour progression and treatment response. However, routine tissue sequencing cannot adequately reflect the heterogeneity of tumours. In this study, single-cell sequencing was used to examine the fibroblast population in CRC. After cluster analysis, the fibroblast population was divided into four subgroups. The distribution and role of these four subgroups in CRC were found to be different. Based on differential gene expression and lasso regression analysis of the main marker genes in these subgroups, four representative genes were obtained, namely, TCF7L1, FLNA, GPX3 and MMP11. Patients with CRC were divided into the low- and high-risk groups using the prognostic risk model established based on the expression of these four genes. The prognosis of patients in different risk groups varied significantly; patients with low-risk scores had a greater response to PDL1 inhibitors, significant clinical benefits and significantly prolonged overall survival. These effects may be attributed to inhibition of the function of T cells in the immune microenvironment and promotion of the function of tumour-associated macrophages.

Abstract Background Melnick-Needles syndrome (MNS) is an extremely rare osteochondrodysplasia caused by a mutation of FLNA, the gene encoding filamin A. MNS is inherited in an X-linked dominant manner. In this study, we describe three members of the same family with MNS, who exhibited different phenotypic severity despite having an identical FLNA gene mutation. Case presentation The patient was 16 months old, with a history of delayed physical development, multiple upper respiratory infections and otitis media episodes. She was referred to our orthopedic clinic because of bowed legs and an abnormal plain chest radiograph. Both upper and lower extremities were bowed. Plain X-rays showed thoracolumbar kyphoscoliosis, with anterior and posterior vertebral scalloping, and thin, wavy ribs. Hypoplasia of the pubis and ischium, with bilateral coxa valga, were also noted. Target exome sequencing revealed a heterozygous mutation of FLNA, c.3578 T > C, p.Lys1193Pro, which confirmed the diagnosis of MNS. Her older sister and mother had minimal deformities of the axial and extremity skeleton, but genetic analyses revealed the same FLNA mutation as the patient. The mutation identified in this family has not been previously reported. Conclusion This report illustrates the potential inherited nature of MNS and the phenotypic variability of clinicoradiologic characteristics. In patients with traits suggestive of MNS, a careful medical and family history should be obtained, and genetic testing should be performed for the patient, as well as all family members.

OBJECTIVE: Colorectal cancer (CRC) is the third most common type of cancer observed in cancer-related mortality because it has a high metastasis ratio. This study aims to investigate the expression levels of several genes, including metastasis-related colon cancer 1 (MACC1), Filamin A (FLNA), F-box/WD repeat-containing protein 7 (FBXW7), which has an important role in cell signaling, migration and adhesion through the remodeling of the cell skeleton. METHODS: In this study, 21 patients with a precise diagnosis of CRC and 21 controls were included. Gene expressions were examined using the RT-PCR technique. To define the relationship of the genes with metastasis, blood samples were collected from all patients with colon/rectal cancer diagnosis without metastasis at six months before and after the medication with Xelox. RESULTS: Our findings showed that no significant difference was observed in the pre-treatment values compared to the control group, whereas FLNA (p=0.001) expression was observed to be significantly increased following treatment with Xelox. CONCLUSION: To our knowledge, our study is the first study to investigate the effects of Xelox treatment on the expression levels of MACC1, FBXW7 and FLNA genes in non-metastatic colorectal cancer patients in Turkey.

Abstract Background Prune belly syndrome (PBS) is a rare, multi-system congenital myopathy primarily affecting males that is poorly described genetically. Phenotypically, its morbidity spans from mild to lethal, however, all isolated PBS cases manifest three cardinal pathological features: 1) wrinkled flaccid ventral abdominal wall with skeletal muscle deficiency, 2) urinary tract dilation with poorly contractile smooth muscle, and 3) intra-abdominal undescended testes. Despite evidence for a genetic basis, previously reported PBS autosomal candidate genes only account for one consanguineous family and single cases. Methods We performed whole exome sequencing (WES) of two maternal adult half-brothers with syndromic PBS (PBS + Otopalatodigital spectrum disorder [OPDSD]) and two unrelated sporadic individuals with isolated PBS and further functionally validated the identified mutations. Results We identified three unreported hemizygous missense point mutations in the X-chromosome gene Filamin A (FLNA) (c.4952 C > T (p.A1448V), c.6727C > T (p.C2160R), c.5966 G > A (p.G2236E)) in two related cases and two unrelated sporadic individuals. Two of the three PBS mutations map to the highly regulatory, stretch-sensing Ig19–21 region of FLNA and enhance binding to intracellular tails of the transmembrane receptor β-integrin 1 (ITGβ1). Conclusions FLNA is a regulatory actin-crosslinking protein that functions in smooth muscle cells as a mechanosensing molecular scaffold, transmitting force signals from the actin-myosin motor units and cytoskeleton via binding partners to the extracellular matrix. This is the first evidence for an X-linked cause of PBS in multiple unrelated individuals and expands the phenotypic spectrum associated with FLNA in males surviving even into adulthood.

Recurrent metastasis is a major fatal cause of breast cancer. Regretfully, the driving force and the molecular beneath have not been fully illustrated yet. In this study, a cohort of breast cancer patients with locoregional metastasis was recruited. For them, we collected the matched samples of the primary tumor and metastatic tumor, and then we determined the mutation profiles with whole-exome sequencing (WES). On basis of the profiles, we identified a list of deleterious variants in eight susceptible genes. Of them, filamin A (FLNA) was considered a potential driver gene of metastasis, and its low expression could enhance 5 years’ relapse survival rate by 15%. To prove the finding, we constructed a stable FLNA knockout tumor cell line, which manifested that the cell abilities of proliferation, migration, and invasion were significantly weakened in response to the gene knockout. Subsequently, xenograft mouse experiments further proved that FLNA knockout could inhibit local or distal metastasis. Putting all the results together, we consolidated that FLNA could be a potential driver gene to metastasis of breast cancer, in particular triple-negative breast cancer. Additional experiments also suggested that FLNA might intervene in metastasis via the regulation of MMP-1 expression. In summary, this study demonstrates that FLNA may play as a positive regulator in cancer proliferation and recurrence. It provides new insight into breast cancer metastasis and suggests a potential new therapeutic target for breast cancer therapy.

Background: Emery–Dreifuss muscular dystrophy (EDMD) is a rare disease characterized by early joint contractures, slowly progressive muscular dystrophy, and cardiac involvement, which includes arrhythmia, dilated cardiomyopathy, hypertrophic cardiomyopathy, heart failure, and sudden death.Methods: Clinical data of the proband and family members were collected. The next-generation sequencing technology was used to analyze the pathogenic variants and copy number variations. Polymerase chain reaction was used to sequence the breakpoints of gene locus rearrangements.Results: Here, we report two siblings with EDMD in a family. The proband, a 17-year-old boy, manifested a dilated right heart, bradycardia, mild muscle weakness, and joint contractures. His younger brother only showed a mild bowing limitation with elevated creatine kinase. Next-generation sequencing revealed the complete deletion of EMD and a rearrangement in FLNA (exon29_48dup) in these two patients. The EMD deletion and partial FLNA duplication were accompanied by a 5 bp overlap (GTCCC) on the background of the FLNA-EMD inversion. These findings support the pathogenic mechanism of microhomology-mediated nonhomologous end joining.Conclusion: We report two siblings with complete EMD deletion and FLNA duplication in a family. A microhomology-mediated nonhomologous end joining event involving EMD and FLNA acts as the underlying mechanism.

Filamins (FLNs) are actin cross-linking proteins, and as scaffolding proteins, FLNs are closely associated with the stabilization of the cytoskeleton. Nevertheless, the biological importance of FLNs in aortic dissection (AD) has not been well-elucidated. In this study, we first reanalyzed datasets downloaded from the Gene Expression Omnibus (GEO) database, and we found that in addition to the extracellular matrix, the actin cytoskeleton is a key structure associated with AD. Given that FLNs are involved in remodeling the cytoskeleton to affect cellular functions, we measured their expression levels in the aortas of patients with Stanford type A AD (TAAD). Our results showed that the mRNA and protein levels of FLNA were consistently decreased in dissected aortas of both humans and mice, while the FLNB protein level was upregulated despite decreased FLNB mRNA levels, and comparable expression levels of FLNC were observed between groups. Furthermore, the immunohistochemistry results demonstrated that FLNA was highly expressed in smooth muscle cells (SMCs) of aorta in non-AD samples, and downregulated in the medial layer of the dissected aortas of humans and mice. Moreover, we revealed that FOS and JUN, forming a dimeric transcription factor called AP-1 (activating protein-1), were positively correlated with the expression of FLNA in aorta. Either overexpression of FOS or JUN alone, or overexpression of FOS and JUN together, facilitated the expression of FLNA in primary cultured human aortic SMCs. In the present study, we not only detected the expression pattern of FLNs in aortas of humans and mice with or without AD, but we also found that the expression of FLNA in the AD samples was significantly reduced and that AP-1 might regulate the expression of FLNA. Our findings will contribute to the elucidation of the pathological mechanisms of AD and provide potential therapeutic targets for AD.

Abstract Background Accumulating evidence shows that circular RNAs (circRNAs) plays vital roles in tumor progression. However, the biological functions of circRNAs in laryngeal squamous cell carcinoma (LSCC) metastasis is still unclear. Methods qRT-PCR was used to detect circFLNA, miRNAs and FLNA mRNA expression. Transwell assay and western blot were performed to evaluate cell migration ability and to detect FLNA, MMP2 and MLK1 protein expression, respectively. RNA pull-down analysis was used to find the binding-miRNAs of circFLNA. Luciferase reporter assay was used to examine the effect of circFLNA on miRNAs and miR-486-3p on FLNA expression. Results In this study, we confirmed that a Filamin A (FLNA)-derived hsa_circ_0092012 known as circFLNA, was upregulated in LSCC, and the higher expression of circFLNA was correlated with LSCC lymph node metastasis. Increased circFLNA facilitates LSCC cell migration ability through upregulating FLNA and MMP2 protein expression. Mechanistically, we find that circFLNA sponges miR-486-3p in LSCC cells, relieving miR-486-3p-induced repression of FLNA which promotes LSCC cell migration. Accordingly, FLNA mRNA is overexpressed in LSCC tissues and a higher FLNA level is correlated with poor survival. Dysregulation of the circFLNA/miR-486-3p/FLNA regulatory pathway contributes to LSCC migration. Conclusions In summary, our study sheds light on the regulatory mechanism of circFLNA in LSCC migration via sponging miR‐486-3p, which downregulates the FLNA protein expression. Targeting circFLNA/miR-486-3p/FLAN axis provides a potential therapeutic target for aggressive LSCC.

Abstract Background Septo-optic dysplasia (SOD), also known as de-Morsier syndrome, is a rare disorder characterized by any combination of optic nerve hypoplasia, pituitary gland hypoplasia, and midline abnormalities of the brain including absence of the septum pellucidum and corpus callosum dysgenesis. The variable presentation of SOD includes visual, neurologic, and/or hypothalamic-pituitary endocrine defects. The unclear aetiology of a large proportion of SOD cases underscores the importance of identifying novel SOD-associated genes. Case presentation To identify the disease-causing gene in a male infant with neonatal hypoglycaemia, dysmorphic features, and hypoplasia of the optic nerve and corpus callosum, we designed a targeted next-generation sequencing panel for brain morphogenesis defects. We identified a novel hemizygous deletion, c.6355 + 4_6355 + 5delAG, in intron 38 of the FLNA gene that the patient had inherited from his mother. cDNA studies showed that this variant results in the production of 3 aberrant FLNA transcripts, the most abundant of which results in retention of intron 38 of FLNA. Conclusions We report for the first time a case of early-onset SOD associated with a mutation in the FLNA gene. This finding broadens the spectrum of genetic causes of this rare disorder and expands the phenotypic spectrum of the FLNA gene.

Background: Androgen receptor (AR) has been described to play a prominent role in male breast cancer (MBC). It maps on chromosome X, and recent reports indicate that X-chromosome polysomy is frequent in MBC. Since the response to anti-androgen therapy may depend on AR polysomy and on its overexpression similarly to prostate cancer, the aim of the present study was to investigate the DNA methylation level of AR and its coregulators, especially those mapped on the X-chromosome, that may influence the activity of AR in MBC.Methods: The DNA methylation level of AR, MAGEA2, MAGEA11, MAGEC1, MAGEC2, FLNA, HDAC6, and UXT, mapped on the X-chromosome, was evaluated by quantitative bisulfite-NGS. Bioinformatic analysis was performed in a Galaxy Project environment using BWA-METH, MethylDackel, and Methylation Plotter tools. The study population consisted of MBC (41 cases) compared with gynecomastia (17 cases).Results:MAGEA family members, especially MAGEA2, MAGEA11, MAGEC, and UXT and HDAC6 showed hypomethylation of several CpGs, reaching statistical significance by the Kruskal–Wallis test (p < 0.01) in MBC when compared to gynecomastia. AR showed almost no methylation at all.Conclusions: Our study demonstrated for the first time that MAGEA family members mapped on the X-chromosome and coregulators of AR are hypomethylated in MBC. This may lead to their overexpression, enhancing AR activity.

Abstract Background Caveolae are membrane invaginations measuring 50–100 nm. These organelles, composed of caveolin and cavin proteins, are important for cellular signaling and survival. Caveolae play incompletely defined roles in human kidneys. Induction of caveolin-1/CAV1 in diseased tubules has been described previously, but the responsible mechanism remains to be defined. Methods Healthy and atrophying human kidneys were stained for caveolar proteins, (caveolin 1–3 and cavin 1–4) and examined by electron microscopy. Induction of caveolar proteins was studied in isolated proximal tubules and primary renal epithelial cells. These cells were challenged with hypoxia or H2O2. Primary tubular cells were also subjected to viral overexpression of megakaryoblastic leukemia 1 (MKL1) and MKL1 inhibition by the MKL1 inhibitor CCG-1423. Putative coregulators of MKL1 activity were investigated by Western blotting for suppressor of cancer cell invasion (SCAI) and filamin A (FLNA). Finally, correlative bioinformatic studies of mRNA expression of caveolar proteins and MKL1 were performed. Results In healthy kidneys, caveolar proteins were expressed by the parietal epithelial cells (PECs) of Bowman’s capsule, endothelial cells and vascular smooth muscle. Electron microscopy confirmed caveolae in the PECs. No expression was seen in proximal tubules. In contrast, caveolar proteins were expressed in proximal tubules undergoing atrophy. Caveolar proteins were also induced in cultures of primary epithelial tubular cells. Expression was not enhanced by hypoxia or free radical stress (H2O2), but proved sensitive to inhibition of MKL1. Viral overexpression of MKL1 induced caveolin-1/CAV1, caveolin-2/CAV2 and SDPR/CAVIN2. In kidney tissue, the mRNA level of MKL1 correlated with the mRNA levels for caveolin-1/CAV1, caveolin-2/CAV2 and the archetypal MKL1 target tenascin C (TNC), as did the MKL1 coactivator FLNA. Costaining for TNC as readout for MKL1 activity demonstrated overlap with caveolin-1/CAV1 expression in PECs as well as in atrophic segments of proximal tubules. Conclusions Our findings support the view that MKL1 contributes to the expression of caveolar proteins in healthy kidneys and orchestrates the induction of tubular caveolar proteins in renal injury.

Early-stage ovarian serous carcinoma is usually difficult to detect in clinical practice. The profiling of protein expression in high-grade serous carcinoma (HGSC) and low-grade serous carcinoma (LGSC) would provide important information for diagnoses and chemotherapy. Here, we performed proteomic profiling of specimens from 13 HGSC and 7 LGSC patients by iTRAQ. A total of 323 proteins that were differentially expressed were identified. After immunohistochemical confirmation of expressed proteins in 166 clinical tissues, asparagine synthetase (ASNS) and filamin A (FLNA) were selected for further functional study. Cisplatin-sensitive (CS; ASNShigh and FLNAlow) and cisplatin-resistant (CR; ASNSlow and FLNAhigh) SKOV3 and OVCAR3 ovarian cancer cell lines were used for subsequent in vitro and in vivo experiments. Notably, ASNS overexpression (ASNS+) or FLNA knockdown (shFLNA) enabled cisplatin-induced apoptosis and autophagy in CR cells. However, ASNS+ and shFLNA promoted and attenuated tumor growth, respectively. In CS cells, ASNS knockdown (shASNS) attenuated clonogenicity, cell proliferation, and the epithelial–mesenchymal transition, whereas FLNA overexpression (FLNA+) protected cells from cisplatin. In vivo, cisplatin resistance was attenuated in mice xenografted with ASNS+, shFLNA, or ASNS+-shFLNA CR cells, whereas xenografts of shASNS or FLNA+ CS cells exhibited resistance to cisplatin. Clinically, all HGSC patients (83/83) responded to cisplatin, while 6 in 41 LGSC patients exhibited cisplatin resistance. These findings identify ASNS and FLNA as distinct biomarkers for HGSC and LGSC, which may have potential value in the prognosis and clinical treatment of serous carcinoma.