Targeted inhibition of NRF2 reduces the invasive and metastatic ability of HIP1 depleted lung cancer cells.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) presents as a highly metastatic disease with Kras and P53 as prevalent oncogenic driver mutations. Endocytosis, through its role in receptor recycling and enrichment, is important for cancer cell proliferation and metastasis. Huntingtin Interacting Protein 1 (HIP1) is a clathrin mediated endocytic adapter protein found overexpressed in different cancers. However, conflicting roles both as a tumour promoter and suppressor are reported. HIP1 expression is found repressed at advanced stages and some HIP1-ALK fusions are reported in NSCLC patients. However, the molecular mechanisms and implications of HIP1 depletion are not completely understood. METHODS: HIP1 depletion was performed using siRNA transient transfection and validated using immunoblotting for each experiment. Gene expression dataset from TCGA, GTEX and GEO databases was analysed to explore HIP1 expression in Lung cancer patients. Kaplan-Meier Plotter database was used to analyse the survival correlation between HIP1 mRNA expression in lung cancer patients. HIP1 depleted A549 cells were analysed for deregulated global proteome using label-free LC-MS and this data is available via ProteomeXchange with identifier PXD054307. Various functional assays such as matrigel based invasion, trans-well migration, soft agar colony and angiogenesis tube formation were performed after HIP1 depletion. NRF2 inhibitor was used after HIP1 knockdown to assess its effect on invasion and soft agar colony formation. RESULTS: In silico analysis of HIP1 transcript expression reveals that it is reduced in high-grade and metastatic lung cancer patients correlating with poor survival. Global proteome profiling reveals that HIP1 depleted A549 cells are enriched in pathways associated with metabolism, proliferation and survival. Molecular and functional analysis indicate higher invasive ability of HIP1 depleted cells. The secretome from HIP1 depleted cells also increases the angiogenic potential of HUVEC cells. NRF2 inhibition significantly reverses invasion of HIP1 depleted NSCLC cells with different driver mutations. CONCLUSION: Our study shows that HIP1 depletion leads to activation of various molecular pathways responsible for cell proliferation and survival. Additionally, enhancement of invasion and anchorage-independent growth in HIP1 depleted subsets of NSCLC cells is via upregulation of NRF2 and can be reversed by its inhibitor.

DNA methylation and miR-92a-3p-mediated repression of HIP1R promotes pancreatic cancer progression by activating the PI3K/AKT pathway.

Pancreatic cancer (PAAD) is a highly malignant tumour characterized of high mortality and poor prognosis. Huntingtin-interacting protein 1-related (HIP1R) has been recognized as a tumour suppressor in gastric cancer, while its biological function in PAAD remains to be elucidated. In this study, we reported the downregulation of HIP1R in PAAD tissues and cell lines, and the overexpression of HIP1R suppressed the proliferation, migration and invasion of PAAD cells, while silencing HIP1R showed the opposite effects. DNA methylation analysis revealed that the promoter region of HIP1R was heavily methylated in PAAD cell lines when compared to the normal pancreatic duct epithelial cells. A DNA methylation inhibitor 5-AZA increased the expression of HIP1R in PAAD cells. 5-AZA treatment also inhibited the proliferation, migration and invasion, and induced apoptosis in PAAD cell lines, which could be attenuated by HIP1R silencing. We further demonstrated that HIP1R was negatively regulated by miR-92a-3p, which modulates the malignant phenotype of PAAD cells in vitro and the tumorigenesis in vivo. The miR-92a-3p/HIP1R axis could regulate PI3K/AKT pathway in PAAD cells. Taken together, our data suggest that targeting DNA methylation and miR-92a-3p-mediated repression of HIP1R could serve as novel therapeutic strategies for PAAD treatment.

miR-34a-5p as molecular hub of pathomechanisms in Huntington's disease.

BACKGROUND: Although a pivotal role of microRNA (miRNA, miR) in the pathogenesis of Huntington's disease (HD) is increasingly recognized, the molecular functions of miRNAs in the pathomechanisms of HD await further elucidation. One of the miRNAs that have been associated with HD is miR-34a-5p, which was deregulated in the mouse R6/2 model and in human HD brain tissues. METHODS: The aim of our study was to demonstrate interactions between miR-34a-5p and HD associated genes. By computational means we predicted 12 801 potential target genes of miR-34a-5p. An in-silico pathway analysis revealed 22 potential miR-34a-5p target genes in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway "Huntington's disease". RESULTS: Using our high-throughput miRNA interaction reporter assay (HiTmIR) we identified NDUFA9, TAF4B, NRF1, POLR2J2, DNALI1, HIP1, TGM2 and POLR2G as direct miR-34a-5p target genes. Direct binding of miR-34a-5p to target sites in the 3'UTRs of TAF4B, NDUFA9, HIP1 and NRF1 was verified by a mutagenesis HiTmIR assay and by determining endogenous protein levels for HIP1 and NDUFA9. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis identified protein-protein interaction networks associated with HD like "Glutamine Receptor Signaling Pathway" and "Calcium Ion Transmembrane Import Into Cytosol". CONCLUSION: Our study demonstrates multiple interactions between miR-34a-5p and HD associated target genes and thereby lays the ground for future therapeutic interventions using this miRNA.

Y-box protein-1 modulates circSPECC1 to promote glioma tumorigenesis via miR-615-5p/HIP1/AKT axis.

Y-box binding protein-1 (YB-1) is upregulated in glioma and plays an important role in its occurrence and drug resistance. However, the involved regulatory processes and downstream pathways are still unclear. Since various circular RNAs (circRNAs) and microRNAs (miRNAs) also play roles in the pathogenesis of glioma, we hypothesize that YB-1 may exert its function through a circRNA-miRNA-protein interaction network. In this study, we use the RNA binding protein immunoprecipitation assay and quantitative reverse transcription polymerase chain reaction to determine the circRNAs involved in the regulation of YB-1 and further elucidate their biological functions. The level of circSPECC1 (hsa_circ_0000745) modulated by YB-1 is significantly upregulated in the U251 and U87 glioma cell lines. Downregulation of circSPECC1 markedly inhibits the proliferation and invasiveness of U251 and U87 cells by inducing apoptosis. Bioinformatics analysis reveals that miR-615-5p could interact with circSPECC1 and huntingtin-interacting protein-1 (HIP-1). Then we determine the interactions between miR-615-5p, circSPECC1, and HIP1 using dual luciferase reporter system and pull-down assays. Mechanistic analysis indicates that the downregulation of circSPECC1 results in a decreased HIP1 expression. This study demonstrates that circSPECC1 modulated by YB-1 is increased in glioma cell lines. In addition, circSPECC1 promotes glioma growth through the upregulation of HIP1 by sponging miR-615-5p and targeting the HIP1/AKT pathway. This indicates that YB-1 and circSPECC1 may both be promising targets for glioma treatment.

Huntingtin-interacting protein family members have a conserved pro-viral function from Caenorhabditis elegans to humans.

Huntingtin-interacting protein family members are evolutionarily conserved from yeast to humans, and they are known to be key factors in clathrin-mediated endocytosis. Here we identified the Caenorhabditis elegans protein huntingtin-interacting protein-related 1 (HIPR-1) as a host factor essential for Orsay virus infection of C. elegans Ablation of HIPR-1 resulted in a greater than 10,000-fold reduction in viral RNA, which could be rescued by ectopic expression of HIPR-1. Viral RNA replication from an endogenous transgene replicon system was not affected by lack of HIPR-1, suggesting that HIPR-1 plays a role during an early, prereplication virus life-cycle stage. Ectopic expression of HIPR-1 mutants demonstrated that neither the clathrin light chain-binding domain nor the clathrin heavy chain-binding motif were needed for virus infection, whereas the inositol phospholipid-binding and F-actin-binding domains were essential. In human cell culture, deletion of the human HIP orthologs HIP1 and HIP1R led to decreased infection by Coxsackie B3 virus. Finally, ectopic expression of a chimeric HIPR-1 harboring the human HIP1 ANTH (AP180 N-terminal homology) domain rescued Orsay infection in C. elegans, demonstrating conservation of its function through evolution. Collectively, these findings further our knowledge of cellular factors impacting viral infection in C. elegans and humans.

Clinicopathological features and resistance mechanisms in HIP1-ALK-rearranged lung cancer: A multicenter study.

Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) respond well to ALK tyrosine kinase inhibitors (TKIs), and echinoderm microtubule-associated protein-like 4 (EML4)-ALK-rearranged NSCLC accounts for the majority of those patients. However, few studies have evaluated ALK-TKIs treatment for patients with huntingtin-interacting protein 1 (HIP1)-ALK fusions. This retrospective study evaluated the clinicopathological characteristics, genomic features, response to ALK-TKIs, and resistance mechanisms in 11 cases with HIP1-ALK fusions from five Chinese centers. Patients who received crizotinib at the Chinese centers had an objective response rate of 90% [9/10 cases, 95% confident index (CI): 54.1%-99.5%], median progression-free survival of 17.9 months (95% CI: 5.8-NA months), and median overall survival of 58.8 months (95% CI: 24.7-NA months). One patient who received first-line lorlatinib treatment achieved partial response for > 26.5 months. Despite the small sample size, HIP1-ALK (H21:A20) variant was the most common variant (four of 11 cases, 36.4%) and associated with better outcomes. Among the 11 cases, there were eight patients having available specimens for genetic testing before ALK-TKIs treatment and four patients undergoing biopsy after ALK-TKIs failure. The most common coexisting gene was TP53 among 11 patients and two of four patients after crizotinib failure harbored acquired ALK mutations (e.g., L1152V/Q1146K and L1196M). Brigatinib treatment appeared to be effective for a patient who failed crizotinib treatment because of the L1152V/Q1146K mutations, which might be related to increased binding affinity to these mutants. Although HIP1-ALK-rearranged NSCLC appears to initially respond well to ALK-TKIs, crizotinib resistance may be correlated with the AKAP9-BRAF fusion, ALK compound mutations (L1152V/Q1146K), and the ALK L1196M mutation. Larger studies are needed to evaluate the significance of HIP1-ALK-rearranged NSCLC.

A novel long non-coding RNA PCLN16 facilitates androgen receptor signaling in prostate cancer.

The prostate cancer (PCa) poses serious threat to men's health. The androgen receptor (AR) is essential for normal prostate development and prostate cancer progression. We identified a novel lncRNA PCLN16 which is significantly correlated with AR signaling during prostate cancer progression. The AR-regulated PCLN16 was abundantly overexpressed in localized or metastatic prostate cancer tissues and AR-dependent cell lines. PCLN16 silence suppressed AR signaling and tumor growth. PCLN16 interacted with Huntingtin interacting protein 1 (HIP1) transcript to reduce HIP1 degradation. Therefore, PCLN16 could augment AR signaling via a novel positive feedback loop. Our experiments support an oncogenic role for PCLN16 and suggest that PCLN16 might serve as a potential target for therapeutic intervention.

The long non-coding RNA PCAL7 promotes prostate cancer by strengthening androgen receptor signaling.

BACKGROUND: The prostate cancer (PCa) has been a global problem to men health. Notably, the androgen receptor (AR) is essential for both normal development of prostate and prostate cancer progression. METHODS: The RNA sequencing was used to identify the novel long non-coding RNA (lncRNA) termed PCAL7. The RT-qPCR was performed to quantify PCAL7 expression. Migration and proliferation assays were used to examine the function of PCAL7. Fluorescence in situ hybridization (FISH) was used to determine subcellular localization. RESULTS: By RNA sequencing, the differentially expressed lncRNAs were identified (top 10 upregulated lncRNAs: PCAL7, AC083843.1, CTC-338M12.3, RP11-443B7.1, RP11-1008C21.2, RN7SL329P, RP4-773N10.4, RP11-264B17.2, KB-1507C5.2, and RP11-20B24.6; top 10 downregulated lncRNAs: RP11-77H9.2, RAB11FIP1P1, AP001625.6, CTA-217C2.1, RP11-603J24.7, RP11-315I20.1, AC092839.1, RP4-758J18.10, RP11-259O2.3, and HMGN2P17). PCAL7 was the lncRNA with the highest fold upregulation and significantly correlated with AR signaling during prostate cancer progression. The AR-regulated PCAL7 was abundantly overexpressed in prostate cancer tissues and AR-dependent cell lines. PCAL7 knockdown inhibited cell migration and proliferation. Consistently, the migration and proliferation were promoted by PCAL7 overexpression. PCAL7 depletion via antisense oligonucleotides (ASOs) markedly suppressed AR signaling and tumor growth. Mechanistically, PCAL7 interacted with Huntingtin-interacting protein 1 (HIP1) to stabilize HIP1. Therefore, PCAL7 could advance AR signaling via a novel positive feedback loop. CONCLUSION: Our experiments support an oncogenic role for PCAL7 which promotes prostate cancer progression suggesting PCAL7 may serve as a potential therapeutic target.

Long non-coding RNA DNM3OS/miR-204-5p/HIP1 axis modulates oral cancer cell viability and migration.

BACKGROUND: Non-coding RNAs play a critical role in the occurrence and development of oral cancer. The present study is aimed to identify long non-coding RNA (lncRNA) that might be novel effective targets for the treatments of oral cancer and the underlying mechanism. METHODS: The microarray profiling and RNA-sequencing analysis were performed to identify lncRNAs related to oral cancer development, and lncRNA DNM3OS was selected. DNM3OS knockdown was generated in cancer cell lines, and the specific effects of DNM3OS knockdown on cell phenotype were examined. DNM3OS targeted miRNA and miRNA targeted downstream mRNA were selected, the predicted bindings were verified, and the specific effects of miRNA on oral cancer cells were examined. Finally, the dynamic effects of DNM3OS and miRNA on target mRNA expression and oral cancer cell phenotype were examined. RESULTS: DNM3OS was upregulated in oral cancer tissues and cells. DNM3OS knockdown in CAL27 and SCC-9 cells inhibited cell viability and migration. DNM3OS targeted miR-204-5p to inhibit miR-204-5p expression. miR-204-5p overexpression suppressed oral cancer cell aggressiveness. miR-204-5p targeted HIP1 to inhibit HIP1 expression. HIP1 knockdown inhibited oral cancer cell viability and migration. The effects of DNM3OS knockdown were significantly reversed by miR-204-5p inhibition. Within oral carcinoma tissue samples, expression of DNM3OS and HIP1 was increased whereas the miR-204-5p expression was downregulated; miR-204-5p had a negative correlation with DNM3OS and HIP1, respectively, while DNM3OS and HIP1 were positively correlated with each other. CONCLUSION: Long non-coding RNA DNM3OS, miR-204-5p, and HIP1 form an axis that modulates oral cancer cell viability and migration.

Divergent methylation of CRISPR repeats and cas genes in a subtype I-D CRISPR-Cas-system.

BACKGROUND: The presence and activity of CRISPR-Cas defense systems is a hallmark of many prokaryotic microorganisms. Here, the distribution of sequences related to the highly iterated palindrome 1 (HIP1) element and the DNA methylation of CGATCG motifs embedded within HIP1 as a vital part of the CRISPR1 repeat sequence was analyzed in the cyanobacterium Synechocystis sp. PCC 6803. Previously suggested functions of HIP1 include organization of chromosomal structure, DNA recombination or gene regulation, all of which could be relevant in CRISPR-Cas functionality. RESULTS: The CRISPR1 repeat-spacer array contains more than 50 CGATCG elements that are double-methylated (5mCG6mATCG) by the enzymes M.Ssp6803I and M.Ssp6803III. Hence, more than 200 possible methylation events cluster over a stretch of 3600 bp of double-stranded DNA. Bisulfite sequencing showed that these motifs were highly methylated at the m5CGATCG positions whereas specific motifs within the CRISPR1 cas genes were hypomethylated suggesting a lowered accessibility for the DNA methylase to these regions. Assays for conjugation and CRISPR1-mediated DNA interference revealed a 50% drop in conjugation efficiency in the mutant lacking the 5mC methylation of CGATCG motifs, while the highly efficient DNA interference activity was not affected by the lack of m5CGATCG DNA-methylation, nor was the capability to differentiate between self and non-self targets based on the protospacer adjacent motifs (PAMs) GTA and GTC versus the non-PAM AGC. A third DNA methylation mediated by M.Ssp6803II modifies the first cytosine in the motif GGCC yielding GGm4CC. We found a remarkable absence of GGCC motifs and hence the corresponding methylation over an 11 kb stretch encompassing all the cas genes involved in interference and crRNA maturation but not adaptation of the CRISPR1 system. CONCLUSIONS: The lack of GGCC tetranucleotides along the CRISPR1 interference and maturation genes supports the reported hybrid character of subtype I-D CRISPR-Cas systems. We report tight and very high 5mC methylation of the CRISPR1 repeat sequences. Nevertheless, cells lacking the 5mC methylation activity were unaffected in their CRISPR1-mediated interference response but the efficiency of conjugation was reduced by 50%. These results point to an unknown role of m5CGATCG DNA-methylation marks in conjugation and DNA transformation.

Distinct c-Met activation mechanisms induce cell rounding or invasion through pathways involving integrins, RhoA and HIP1.

Many carcinomas have acquired oncogenic mechanisms for activating c-Met, including c-Met overexpression and excessive autocrine or paracrine stimulation with hepatocyte growth factor (HGF). However, the biological outcome of c-Met activation through these distinct modes remains ambiguous. Here, we report that HGF-mediated c-Met stimulation triggers a mesenchymal-type collective cell invasion. By contrast, the overexpression of c-Met promotes cell rounding. Moreover, in a high-throughput siRNA screen that was performed using a library of siRNAs against putative regulators of integrin activity, we identified RhoA and the clathrin-adapter protein HIP1 as crucial c-Met effectors in these morphological changes. Transient RhoA activation was necessary for the HGF-induced invasion, whereas sustained RhoA activity regulated c-Met-induced cell rounding. In addition, c-Met-induced cell rounding correlated with the phosphorylation of filamin A and the downregulation of active cell-surface integrins. By contrast, a HIP1-mediated increase in ß1-integrin turnover was required for the invasion triggered by HGF. Taken together, our results indicate that c-Met induces distinct cell morphology alterations depending on the stimulus that activates c-Met.

Epilepsy is a possible feature in Williams-Beuren syndrome patients harboring typical deletions of the 7q11.23 critical region.

Seizures are rarely reported in Williams-Beuren syndrome (WBS)--a contiguous-gene-deletion disorder caused by a 7q11.23 heterozygous deletion of 1.5-1.8 Mb--and no previous study evaluated electro-clinical features of epilepsy in this syndrome. Furthermore, it has been hypothesized that atypical deletion (e.g., larger than 1.8 Mb) may be responsible for a more pronounced neurological phenotypes, especially including seizures. Our objectives are to describe the electro-clinical features in WBS and to correlate the epileptic phenotype with deletion of the 7q11.23 critical region. We evaluate the electro-clinical features in one case of distal 7q11.23 deletion syndrome and in eight epileptic WBS (eWBS) patients. Additionally, we compare the deletion size-and deleted genes-of four epileptic WBS (eWBS) with that of four non-epileptic WBS (neWBS) patients. Infantile spasms, focal (e.g., motor and dyscognitive with autonomic features) and generalized (e.g., tonic-clonic, tonic, clonic, myoclonic) seizures were encountered. Drug-resistance was observed in one patient. Neuroimaging discovered one case of focal cortical dysplasia, one case of fronto-temporal cortical atrophy and one case of periventricular nodular heterotopia. Comparison of deletion size between eWBS and neWBS patients did not reveal candidate genes potentially underlying epilepsy. This is the largest series describing electro-clinical features of epilepsy in WBS. In WBS, epilepsy should be considered both in case of typical and atypical deletions, which do not involve HIP1, YWHAG or MAGI2.

Low HIP1R mRNA and protein expression are associated with worse survival in diffuse large B-cell lymphoma patients treated with R-CHOP.

Huntingtin-interacting protein 1-related (HIP1R) is an endocytic protein involved in receptor trafficking, including regulating cell surface expression of receptor tyrosine kinases. We have previously shown that low HIP1R protein expression was associated with poorer survival in diffuse large B-cell lymphoma (DLBCL) patients from Denmark treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). In this multicenter study, we extend these findings and validate the prognostic and subtyping utility of HIP1R expression at both transcript and protein level. Using data mining on three independent transcriptomic datasets of DLBCL, HIP1R transcript was preferentially expressed in germinal center B-cell (GCB)-like DLBCL subtype (P<0.01 in all three datasets), and lower expression was correlated with worse overall survival (OS; P<0.01) and progression-free survival (PFS; P<0.05) in a microarray-profiled DLBCL dataset. At the protein level examined by immunohistochemistry, HIP1R expression at 30% cut-off was associated with GCB-DLBCL molecular subtype (P=0.0004; n=42), and predictive of OS (P=0.0006) and PFS (P=0.0230) in de novo DLBCL patients treated with R-CHOP (n=73). Cases with high FOXP1 and low HIP1R expression frequency (FOXP1(hi)/HIP1R(lo) phenotype) exhibited poorer OS (P=0.0038) and PFS (P=0.0134). Multivariate analysis showed that HIP1R<30% or FOXP1(hi)/HIP1R(lo) subgroup of patients exhibited inferior OS and PFS (P<0.05) independently of the International Prognostic Index. We conclude that HIP1R expression is strongly indicative of survival when utilized on its own or in combination with FOXP1, and the molecule is potentially applicable for subtyping of DLBCL cases.

Regulation of the scp Genes in the Cyanobacterium Synechocystis sp. PCC 6803--What is New?

In the cyanobacterium Synechocystis sp. PCC 6803 there are five genes encoding small CAB-like (SCP) proteins, which have been shown to be up-regulated under stress. Analyses of the promoter sequences of the scp genes revealed the existence of an NtcA binding motif in two scp genes, scpB and scpE. Binding of NtcA, the key transcriptional regulator during nitrogen stress, to the promoter regions was shown by electrophoretic mobility shift assay. The metabolite 2-oxoglutarate did not increase the affinity of NtcA for binding to the promoters of scpB and scpE. A second motif, the HIP1 palindrome 5' GGCGATCGCC 3', was detected in the upstream regions of scpB and scpC. The transcription factor encoded by sll1130 has been suggested to recognize this motif to regulate heat-responsive genes. Our data suggest that HIP1 is not a regulatory element within the scp genes. Further, the presence of the high light regulatory (HLR1) motif was confirmed in scpB-E, in accordance to their induced transcriptions in cells exposed to high light. The HLR1 motif was newly discovered in eight additional genes.

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter.

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (-386/-374), non-perfect DR1 (-118/-106) and HNF4α-specific binding motif (H4-SBM) (-26/-14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.

Successful treatment of a non-small-cell lung cancer patient harboring HIP1-ALK (H28:A20) and CTNNB1 p.S45del with alectinib.

This is the first case report of a non-small-cell lung cancer (NSCLC) patient harboring HIP1-ALK (H28:A20) and CTNNB1 p.S45del treated with first-line alectinib. Approximately 5% of NSCLC patients are reported to have anaplastic lymphoma kinase (ALK) rearrangements, and among these EML4-ALK is the most frequent fusion variant. However, in recent years the use of next-generation sequencing (NGS) in clinical laboratories has become increasingly widespread, identifying a lot of new ALK fusion partners as well as a large quantity of co-occurring genomic alterations. Unfortunately, the growing number of genomic alterations detected by NGS does not always correspond to adequate knowledge of their clinical significance, often resulting in an empiric treatment of patients harboring uncommon mutations.

HIP1-ALK-Rearranged Lung Cancer in a Young Adult With BRAF V600E Mutation Detected After ALK Tyrosine Kinase Inhibitor Therapy: A Case Report.

HIP1-ALK is a relatively rare fusion pattern in ALK-rearranged NSCLC. Existing studies on the efficacy of ALK tyrosine kinase inhibitor (TKI) resistance mechanisms and treatment strategies in HIP1-ALK-rearranged lung cancer are limited. Here, we report the case of an 18-year-old man with HIP1-ALK-rearranged adenocarcinoma who developed BRAF V600E and V1180L mutations after ALK TKI therapy, in whom the administration of BRAF and MEK inhibitors was ineffective. Brigatinib was effective after chemotherapy with cytotoxic drugs. Development of effective treatments is desirable for rare variants of ALK-rearranged lung cancer after acquiring resistance to ALK TKIs.

Detection of polyreactive immunoglobulin G facilitates diagnosis in children with autoimmune hepatitis.

OBJECTIVE: The detection of autoantibodies is essential to diagnose autoimmune hepatitis (AIH). Particularly in children, specificity of autoantibodies decreases due to lower titers being diagnostic and being present not only in AIH but also in other liver diseases. Recently, quantification of polyreactive IgG (pIgG) for detection of adult AIH showed the highest overall accuracy compared to antinuclear antibodies (ANA), anti-smooth muscle antibodies (anti-SMA), anti-liver kidney microsomal antibodies (anti-LKM) and anti-soluble liver antigen/liver pancreas antibodies (anti-SLA/LP). We aimed to evaluate the diagnostic value of pIgG for pediatric AIH. DESIGN: pIgG, quantified using HIP1R/BSA coated ELISA, and immunofluorescence on rodent tissue sections were performed centrally. The diagnostic fidelity to diagnose AIH was compared to conventional autoantibodies of AIH in training and validation cohorts from a retrospective, European multi-center cohort from nine centers from eight European countries composed of existing biorepositories from expert centers (n = 285). RESULTS: IgG from pediatric AIH patients exhibited increased polyreactivity to multiple protein and non-protein substrates compared to non-AIH liver diseases and healthy children. pIgG had an AUC of 0.900 to distinguish AIH from non-AIH liver diseases. pIgG had a 31-73% higher specificity than ANA and anti-SMA and comparable sensitivity that was 6-20 times higher than of anti-SLA/LP, anti-LC1 and anti-LKM. pIgG had a 21-34% higher accuracy than conventional autoantibodies, was positive in 43-75% of children with AIH and normal IgG and independent from treatment response. CONCLUSION: Detecting pIgG improves the diagnostic evaluation of pediatric AIH compared to conventional autoantibodies, primarily owing to higher accuracy and specificity.

Flurbiprofen inhibits cell proliferation in thyroid cancer through interrupting HIP1R-induced endocytosis of PTEN.

BACKGROUND: The incidence of thyroid cancer, a most common tumor in the endocrine system, has increased in recent years. A growing number of studies have focused on the molecular mechanisms of thyroid cancer subtypes, aiming to identify effective therapeutic targets. Endocytosis is of vital significance in the malignant development of tumors, although its involvement in thyroid cancer has been rarely reported. METHODS: HIP1R expressions in thyroid cancer from the TCGA database were analyzed by UALCAN software. Thyroid epithelial and cancer cell lines were cultured in vitro. Western blotting and quantitative PCR were used to analyze protein and mRNA levels, respectively. Cell viability was measured by CCK-8 assay. Immunofluorescence staining indicated protein distribution in cell. Co-immunoprecipitation was used to study protein-protein interaction. Immunohistochemical staining was used to analyze protein expression in clinical tissues. Differences between groups were compared using the two-tailed Student's t test, and those among three or more groups were compared by one-way or two-way ANOVA. RESULTS: In the present study, HIP1R (Huntingtin Interacting Protein 1 Related) was found upregulated in thyroid cancer tissues and cell lines compared with that in the controls, while knockdown of HIP1R significantly inhibited the proliferation of thyroid cancer cells. Since HIP1R is essential for the clathrin-dependent endocytic process, we thereafter explored the effect of HIP1R on the endocytosis of thyroid cancer cells. Interestingly, knockdown of HIP1R significantly reduced the number of clathrin-coated pits (CCPs) in thyroid cancer cells. In addition, the interaction between HIP1R and PTEN (phosphatase and tensin homolog) was identified in thyroid cancer cells. Knockdown of HIP1R downregulated intracellular PTEN in thyroid cancer cells, but upregulated membrane-binding PTEN. Notably, flurbiprofen, a commonly used analgesic, significantly inhibited the proliferation of thyroid cancer cells and interfered with the interaction between HIP1R and PTEN, thereby enhancing the binding of PTEN to cell membrane. However, the proliferation inhibitory effect of flurbiprofen was attenuated when knocking down HIP1R or PTEN. CONCLUSIONS: Upregulated HIP1R in thyroid cancer cells promotes cell proliferation and mediates the endocytosis of PTEN. Flurbiprofen may exert an anti-tumor effect on thyroid cancer by blocking the interaction between HIP1R and PTEN.

HIP1R and vimentin immunohistochemistry predict 1p/19q status in IDH-mutant glioma.

BACKGROUND: IDH-mutant gliomas are separate based on the codeletion of the chromosomal arms 1p and 19q into oligodendrogliomas IDH-mutant 1p/19q-codeleted and astrocytomas IDH-mutant. While nuclear loss of ATRX expression excludes 1p/19q codeletion, its limited sensitivity prohibits to conclude on 1p/19q status in tumors with retained nuclear ATRX expression. METHODS: Employing mass spectrometry based proteomic analysis in a discovery series containing 35 fresh frozen and 72 formalin fixed and paraffin embedded tumors with established IDH and 1p/19q status, potential biomarkers were discovered. Subsequent validation immunohistochemistry was conducted on two independent series (together 77 oligodendrogliomas IDH-mutant 1p/19q-codeleted and 92 astrocytomas IDH-mutant). RESULTS: We detected highly specific protein patterns distinguishing oligodendroglioma and astrocytoma. In these patterns, high HIP1R and low vimentin levels were observed in oligodendroglioma while low HIP1R and high vimentin levels occurred in astrocytoma. Immunohistochemistry for HIP1R and vimentin expression in 35 cases from the FFPE discovery series confirmed these findings. Blinded evaluation of the validation cohorts predicted the 1p/19q status with a positive and negative predictive value as well as an accuracy of 100% in the first cohort and with a positive predictive value of 83%; negative predictive value of 100% and an accuracy of 92% in the second cohort. Nuclear ATRX loss as marker for astrocytoma increased the sensitivity to 96% and the specificity to 100%. CONCLUSIONS: We demonstrate that immunohistochemistry for HIP1R, vimentin, and ATRX predict 1p/19q status with 100% specificity and 95% sensitivity and therefore, constitutes a simple and inexpensive approach to the classification of IDH-mutant glioma.