A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

Succinate Dehydrogenase-Deficient Renal Cancer Featuring Fructose-1,6-Biphosphatase Loss, Pyruvate Kinase M2 Overexpression, and SWI/SNF Chromatin Remodeling Complex Aberrations: A Rare Case Report.

Succinate dehydrogenase (SDH)-deficient renal cancer is a rare renal cancer subtype recently accepted by the World Health Organization as a unique subtype of renal cell carcinoma (RCC). Here we report a case of 17-year-old man. The detailed evaluation indicated occurrence of the SDHB-deficient RCC. The genetic testing revealed no germline mutation in SDH genes. Immunohistochemistry showed SDHB deficiency, overexpression of pyruvate kinase M2 and dramatic downregulation of fructose-1,6-bisphosphatase metabolic enzymes, and unaltered levels of phosphorylated AMP-activated protein kinase and mammalian target of rapamycin. Strong upregulation of INI1 and BRG1 and overexpression of BAF180, subunits of SWI/SNF ATP-dependent chromatin remodeling complex, were also found. The identified tumor pathologically did not resemble clear cell renal cell carcinoma (ccRCC), but some metabolic alterations are common for both cancer types. Thus, we postulate that the phenotypical differences between ccRCC and SDHB-deficient RCC may be related to distinct molecular and metabolic alterations. IMPLICATIONS FOR PRACTICE: Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is a rare renal tumor occurring even in young patients. Until now, in all described and genetically tested cases, mutations and deletions in SDH genes have been found. This article describes SDHB-deficient RCC without any germline mutations in SDH genes. Therefore, genetic analysis for germline mutations in SDH genes in SDH-deficient RCC, especially in young individuals, should be strongly recommended, although as of now it is not obligatory. This knowledge will allow improvement of patient monitoring including both disease recurrence and new cancer appearance.

Cardiotoxicity danger in immunotherapy.

Immunotherapy based on immune checkpoint inhibitors (ICIs) is currently broadly used in the treatment of different types of cancer. The treatment targeting programmed cell death protein 1/programmed death-ligand 1 axis is already approved by Food and Drug Administration for numerous cancers. These kinds of therapy brought spectacular results in the treatment of non-small cell lung cancer where systemic therapy was ineffective. However, a wide range of applied therapies based on ICIs in the clinic have led to unexpected side effects, such as severe cardiotoxicity. It needs to be underlined that the molecular mechanism of myocarditis in response to ICIs is still not fully understood. Lack of sufficient knowledge, especially concerning the kind of risk factors increasing probability of myocarditis, poses currently a large clinical problem. Continuous cardiac monitoring of patients who undergo ICI treatment presents another problem as it is cost-ineffective for the healthcare system. Herein, we highlight the risks of use of anticancer therapy based on ICIs. We also stress that detailed monitoring of any event of cardiotoxicity following ICIs treatment should be carefully investigated and registered to give a global overview of the frequency of myocarditis occurrence. Moreover, we propose that the extension of molecular and systemic knowledge of etiology of myocarditis as a side effect, including the role of protein kinases, will be highly beneficial for the medical field. Last but not least, better understanding of mechanisms of cardiotoxicity induction will improve the safety of cancer patients and will help clinicians in prediction of unexpected side effect occurrence.

Various forms of HIF-1α protein characterize the clear cell renal cell carcinoma cell lines.

Renal cell carcinoma (RCC) represents around 2-3% of all malignancies diagnosed in adult patients. Most frequent (around 70-80% cases) and the most aggressive subtype is clear cell RCC (ccRCC). Mutations in VHL (von Hippel Lindau) gene, characteristic for this cancer type, lead to altered activity of the trimeric VBC (pVHL-elongin B-C) complex and consequently to HIF-1α stabilization. In this study, we present results of exhaustive investigation of HIF-1α alternative transcript variants abundance in A498, CAKI-1, and 786-O ccRCC cell lines. We proved the existence of truncated HIF-1α protein form (HIF1A∆-6) in A498 and HIF1A gene rearrangements in 786-O cell lines. Subsequently, we found that HIF1A∆2-6 was more stable than the full-length HIF-1α. Moreover, the shorter HIF-1α was insensitive for hypoxia and was overaccumulated after proteasome inhibitor treatment indicative of potential diversified roles of full-length and truncated HIF-1α forms in the cell. We also showed that A498, CAKI-1, and 786-O exhibit differential expression of various regulatory genes involved in the control of metabolic processes, that is, glucose and lipid metabolism, and encoding subunits of such machineries like SWI/SNF chromatin remodeling complex. Furthermore, these cell lines exhibited differential responses to axitinib, everolimus, and sunitinib-anticancer drugs-in normoxia and hypoxia as well as various alterations in metabolism-related regulatory processes. Finally, we have shown that overexpression of truncated HIF1A∆2-6 form may affect the protein level of endogenous full-length HIF-1α protein. Thus, our study proves an important role of HIF-1α in the ccRCC development.

SWI/SNF chromatin remodeling complex and glucose metabolism are deregulated in advanced bladder cancer.

Bladder cancer (BC) is a frequently diagnosed malignancy affecting predominantly adult and elderly populations. It is expected that due to the longer life time, BC will become even more frequent in the future; thus in consequence, it will represent serious health problem of older society part. The treatment of advanced BC is mostly ineffective due to its very aggressive behavior. So far, no effective targeted therapy is used for BC treatment. Here, we found that BC is characterized by lower protein levels of BRM, INI1, and BAF155 main subunits of SWI/SNF chromatin remodeling complex (CRC) which is involved in global control of gene expression and influences various important cellular processes like: cell cycle control, apoptosis, DNA repair, etc. Moreover, the expression of SMARCA2, a BRM encoding gene, strongly correlated with BC metastasis and expression of such metabolic genes as PKM2 and PRKAA1. Furthermore, the analysis of T24 and 5637 commonly used BC cell lines revealed different expression levels of metabolic genes including FBP1 gene encoding Frutose-1,6-Bisphosphatase, an enzyme controlling glycolysis flux and gluconeogenesis. The tested BC cell lines exhibited various molecular and metabolic alterations as well as differential glucose uptake, growth rate, and migration potential. We have shown that BRM subunit is involved in the transcriptional control of genes encoding metabolic enzymes. Moreover, we found that the FBP1 expression level and the SWI/SNF CRCs may serve as markers of molecular subtypes of BC. Collectively, this study may provide a new knowledge about the molecular and metabolic BC subtypes which likely will be of high importance for the clinic in the future.

Role of Vitamin D and Its Receptors in the Pathophysiology of Chronic Rhinosinusitis.

OBJECTIVES: Chronic rhinosinusitis (CRS) is a disease that represents a challenging therapeutic problem. Vitamin D and its receptors (VDR) are involved in the regulation of the immune system and may play role in CRS. Objectives of this study were to assess the relationships between the total concentration of vitamin D (25VD3) in sera, vitamin D receptor (VDR) expression, 1α-hydroxylase expression, and clinical data, including age, gender, Sino-Nasal Outcome Test (SNOT-22), computerized tomography (CT) scan, allergy status, and vitamin D supplementation in CRS patients with (CRSwNP) and without nasal polyps (CRSsNP), and in a control group. METHODS: The studied group comprised 52 patients with CRS without nasal polyps (sNP), 55 with CRS with nasal polyps (wNP), and 59 in the control group. The endpoints were determined by appropriate methods. We conducted immunohistochemical staining of gathered tissue from the ostiomeatal complex for determination of VDR and 1α-hydroxylase. Analytical results were compared with clinical data as already noted. RESULTS: A decrease in VDR nuclear staining occurred in CRS patients as compared to controls. Insignificant differences were observed in 1α-hydroxylase, expression in all studied groups, while VDR and cytochrome CYP27B1 protein expression (1α-hydroxylase) correlated with clinical data. CONCLUSIONS: The data provide evidence that indicates that vitamin D and its receptor and enzymes may play a role in CRS.

Molecular characterization and patient outcome of melanoma nodal metastases and an unknown primary site.

BACKGROUND: Melanoma of unknown primary site (MUP) is not a completely understood entity with nodal metastases as the most common first clinical manifestation. The aim of this multicentric study was to assess frequency and type of oncogenic BRAF/NRAS/KIT mutations in MUP with clinically detected nodal metastases in relation to clinicopathologic features and outcome. MATERIALS AND METHODS: We analyzed series of 103 MUP patients (period: 1992-2010) after therapeutic lymphadenectomy (LND): 40 axillary, 47 groin, 16 cervical, none treated with BRAF inhibitors. We performed molecular characterization of BRAF/NRAS/KIT mutational status in nodal metastases using direct sequencing of respective coding sequences. Median follow-up time was 53 months. RESULTS: BRAF mutations were detected in 55 cases (53 %) (51 V600E, 93 %; 4 others, 7 %), and mutually exclusive NRAS mutations were found in 14 cases (14 %) (7 p.Q61R, 4 p.Q61K, 2 p.Q61H, 1 p.Q13R). We have not detected any mutations in KIT. The 5-year overall survival (OS) was 34 %; median was 24 months. We have not found significant correlation between mutational status (BRAF/NRAS) and OS; however, for BRAF or NRAS mutated melanomas we observed significantly shorter disease-free survival (DFS) when compared with wild-type melanoma patients (p = .04; 5-year DFS, 18 vs 19 vs 31 %, respectively). The most important factor influencing OS was number of metastatic lymph nodes >1 (p = .03). CONCLUSIONS: Our large study on molecular characterization of MUP with nodal metastases showed that MUPs had molecular features similar to sporadic non-chronic-sun-damaged melanomas. BRAF/NRAS mutational status had negative impact on DFS in this group of patients. These observations might have potential implication for molecular-targeted therapy in MUPs.

[AMP-activated protein kinase (AMPK) as therapeutic target]. / Kinaza bialkowa aktywowana przez AMP (AMPK) jako cel terapeutyczny.

AMP-activated protein kinase (AMPK) is one of the major energy sensor at both: cellular and whole body level. It exists as heterotrimer containing three subunits: the catalytic α subunit, ß and regulatory γ. AMPK is localized both in the cytoplasm and in the nucleus. It is activated by increasing concentrations of AMP during the energy shortage, causing activation of catabolic pathways and inhibition of energy consuming processes. AMPK activity can be regulated allosterically: by binding AMP to a regulatory γ subunit, as well as by phosphorylation on Thr172 of the catalytic α subunit by other kinases. Activated AMPK can effectively inhibit the mTOR pathway which is hyperactive in many types of cancer. On the other hand AMPK inactivation associates with the type II diabetes, diet-induced obesity, insulin resistance and the development of other metabolic disorders. The AMPK dysfunction is also observed in inflammation. It was discovered during last years that abnormalities in the AMPK function can induce the metabolic reprogramming in cancer cells known as the Warburg effect. Additionally, AMPK is activated during irradiation. Its activation leads to inhibition of growth. On the other hand, active AMPK enables cells to survive in difficult conditions such as hypoxia, or glucose deprivation. Because of its crucial role in maintaining of the energy homeostasis AMPK is an excellent therapeutic target. However, it still remains unknown what is better: to activate or inhibit the AMPK function.

Recurrent and novel SS18-SSX fusion transcripts in synovial sarcoma: description of three new cases.

Synovial sarcoma (SS) is an aggressive type of tumor, comprising approximately 10 % of soft tissue sarcomas. Over 90 % of SS cases are characterized by the t(X;18)(p11.2;q11.2) translocation, which results mainly in the formation of oncogenic SS18-SSX1 or SS18-SSX2 fusions. In a typical SS18-SSX fusion transcript, exon 10 of SS18 is fused to exon 6 of SSX1/2. However, several variant fusion transcripts have been already described. In the present study, we examined the fusion transcript type in a series of 40 primary untreated SS tumor specimens using reverse transcription polymerase chain reaction and fluorescence in situ hybridization assay. We detected SS18-SSX1 transcript in 22 (55 %) patients and SS18-SSX2 transcript in 17 (42.5 %) patients, while in one patient, none of SS18-SSX1/2 fusion transcripts were identified. Among the cases under study, two tumors carried novel SS18-SSX1 and SS18-SSX2 variant translocations that were allegedly created by an alternative splicing, and in additional case, an unusual translocation variant previously described by other group was found. Our data suggest that alternative splicing may play an important role in novel fusion transcript formation, and additionally we show that it may be a recurrent event in SS. Furthermore, we describe the first case of a complex rearrangement possibly linking SS to REPS2 gene.

The outcome and predictive factors of sunitinib therapy in advanced gastrointestinal stromal tumors (GIST) after imatinib failure - one institution study.

BACKGROUND: Gastrointestinal stromal tumors (GIST) mutational status is recognized factor related to the results of tyrosine kinase inhibitors therapy such as imatinib (IM) or sunitinib (SU). Arterial hypertension (AH) is common adverse event related to SU, reported as predictive factor in renal cell carcinoma. The aim of the study was to analyze the outcomes and factors predicting results of SU therapy in inoperable/metastatic CD117(+) GIST patients after IM failure. METHODS: We identified 137 consecutive patients with advanced inoperable/metastatic GIST treated in one center with SU (2nd line treatment). Median follow-up time was 23 months. Additionally, in 39 patients there were analyzed selected constitutive single nucleotide polymorphisms (SNPs) of VEGFA and VEGFR2 genes. RESULTS: One year progression-free survival (PFS; calculated from the start of SU) rate was 42% and median PFS was 43 weeks. The estimated overall survival (OS, calculated both from start of SU or IM) was 74 weeks and 51 months, respectively. One-year PFS was 65% (median 74 weeks) in 55 patients with AH vs. 22% (median 17 weeks) in patients without AH. Patients with primary tumors carrying mutations in KIT exon 9 or wild-type had substantially better 1-year PFS (68% and 57%; median 65.5 and 50.5 weeks, respectively) than patients having tumors with KIT exon 11 or PDGFRA mutations (34% and 15%; median 36.8 and 9 weeks, respectively). We identified two independent factors with significant impact on PFS and OS in univariate and multivariate analysis: primary tumor genotype and presence of AH. The most common adverse events during therapy were: fatigue, AH, hypothyroidism, hand and foot syndrome, mucositis, skin reactions, dyspepsia, and diarrhea. Two deaths were assessed as related to tumor rupture caused by reaction to SU therapy. The presence of C-allele in rs833061 and the T-allele in rs3025039 polymorphism of VEGFA were associated with significantly higher risk of hypothyroidism (OR: 10.0 p = 0.041 and OR: 10.5; p = 0.015, respectively). CONCLUSIONS: We confirmed that many advanced GIST patients benefit from SU therapy with OS > 1.5 year. Primary tumor KIT/PDGFRA genotype and SU-induced AH, as surrogate of its antiangiogenic activity are two independent factors influencing both PFS and OS.

Lack of microsatellite instability in squamous cell vulvar carcinoma.

Mutator phenotypes with microsatellite instability (MSI) are observed in a subset of solid tumors. The objective of our study was to investigate the occurrence of MSI in vulvar squamous cell carcinoma (VSCC) and a possible relation between MSI and the presence of human papilloma virus (HPV). DNA samples from 44 tissue specimens of the primary VSCC as well as from six metastatic lymph node samples were analysed and compared with matched reference DNA from blood samples. The MSI status was examined by polymerase chain reaction (PCR) using the Bethesda panel of five microsatellite markers. PCR products were analysed by fluorescent capillary electrophoresis. No microsatellite instability was detected in tumor samples or in metastatic lymph nodes from any of the VSCC patients examined. Microsatellite instability seems not to play a major role in the carcinogenesis of VSCC and is probably not associated with the HPV-related genetic background of this neoplasm.

Downstream and intermediate interactions of synovial sarcoma-associated fusion oncoproteins and their implication for targeted therapy.

Synovial sarcoma (SS), an aggressive type of soft tissue tumor, occurs mostly in adolescents and young adults. The origin and molecular mechanism of the development of SS remain only partially known. Over 90% of SS cases are characterized by the t(X;18)(p11.2;q11.2) translocation, which results mainly in the formation of SS18-SSX1 or SS18-SSX2 fusion genes. In recent years, several reports describing direct and indirect interactions of SS18-SSX1/SSX2 oncoproteins have been published. These reports suggest that the fusion proteins particularly affect the cell growth, cell proliferation, TP53 pathway, and chromatin remodeling mechanisms, contributing to SS oncogenesis. Additional research efforts are required to fully explore the protein-protein interactions of SS18-SSX oncoproteins and the pathways that are regulated by these partnerships for the development of effective targeted therapy.

Clinical utility of the new American Joint Committee on Cancer staging system for gastrointestinal stromal tumors: current overall survival after primary tumor resection.

BACKGROUND: The objectives of the current study were to assess the reliability of the new revision of the American Joint Committee on Cancer (AJCC) staging system for gastrointestinal stromal tumors (GISTs) based on the National Comprehensive Cancer Network-Armed Forces Institute of Pathology risk classification and to analyze the factors that influence after resection for primary GISTs in 2 AJCC groups: patients with GISTs originating from the stomach and omentum (G-GISTs) and patients with other primary GISTs located mainly in the small bowel (nongastric GISTs [NG-GISTs]). METHODS: The authors prospectively analyzed a group of 640 patients with primary, CD117-positive GISTs who underwent surgery with curative intention (R0/R1 resection), including 340 G-GISTs (55.5%) and 300 NG-GISTs (44.5%). Factors were explored that had an effect on disease-free survival time (DFS), which was calculated from the date of radical operation to the date of recurrence or last follow-up. The median follow-up was 39 months. RESULTS: Compared with NG-GISTs, G-GISTs were characterized by a significantly lower median size (5.3 cm and 8.5 cm, respectively; P < .0001) and lower mitotic activity (median, 3 in 50 high-power fields [HPF] vs 5 in 50 HPF; P < .0001), and they were diagnosed in older patients (median age, 62 years vs 57 years; P = .002). The most commonly detected mutations in G-GIST were those located in KIT exon 11 (60.5%) and platelet-derived growth factor receptor alpha (PDGFRA) exon 18 (19%) versus KIT exons 11 and 9 in NG-GISTs (72% and 17.4%, respectively). The prognosis of patients who had G-GISTs was significantly better compared that of patients who had NG-GISTs, with 5-year DFS rates of 69% (median, 83 months) versus 43% (median, 33 months), respectively (P < .00001). The most significant prognostic factors that correlated with shorter DFS in both G-GISTs and NG-GISTs were primary tumor size >5 cm and >10 cm (P < .0001) and mitotic index >5 in 50 HPF and >10 in 50 HPF (P < .0001). The 5-year DFS rates in G-GISTs according to AJCC stage categories were as follows: 96% for stage IA tumors, 92% for stage IB tumors, 51% for II tumors, 22% for stage IIIA tumors, and 22% for stage IIIB tumors (P < .0001). The 5-year DFS rates in NG-GISTs according to AJCC categories were as follows: 92% for stage I tumors, 66% for stage II tumors, 28% for IIIA tumors, and 16% for IIIB tumors (P < .0001). The high prognostic significance of the AJCC classification also was confirmed for overall survival data, including the impact of therapy with tyrosine kinase inhibitors. CONCLUSIONS: The reliability of AJCC risk classification after resection of primary GIST was confirmed for DFS and overall survival. Patients with primary G-GISTs had a better prognosis than patients with NG-GISTs. In both groups, primary tumor size and mitotic activity were the most important prognostic factors in terms of DFS.

Methyl-CpG binding column-based identification of nine genes hypermethylated in colorectal cancer.

DNA methylation is an epigenetic event that plays a role in gene expression regulation. Alterations in DNA methylation contribute to cancer development and progression. The aim of this study was to identify gene promoters aberrantly methylated in colorectal tumor tissue in comparison to normal colonic mucosa. Analyses were performed on two pooled DNA samples: from normal and cancerous tissue obtained from CRC patients. DNA was fractionated according to methylation degree with the use of affinity column containing methyl-CpG binding domain. To identify novel hypermethylated gene promoters, methylated DNA from normal and from cancerous tissues were analyzed with the use of promoter microarrays. We identified nine novel genes hypermethylated in colorectal cancer. The frequency of their promoter methylation was assessed in the larger group of patients (n = 77): KCNK12 (methylated in 41% of CRC patients), GPR101 (40%), CDH2 (45%), BARX1 (56%), CNTFR (22%), SYT6 (64%), SMO (21%), EPHA5 (43%), and GSPT2 (21%). The results of gene expression level analysis suggest the role of promoter methylation in downregulation of six out of nine genes examined. We did not find correlation between gene methylation and age, gender, tumor grade or stage. Importantly, in stage IV CRC methylation of GPR101 correlated with longer time to progression (P = 0.0042; HR = 2.5468; 95% CI 1.5391-10.0708).

Detection of Melanoma Cells in Lymphatic Drainage (LD) After Lymph Nodes Dissection Via Nested RT-PCR Analysis of Molecular Melanocytic Markers.

We present the assay based on multimarker analysis of mRNA transcripts associated with melanocytic cells detected in lymphatic fluid collected after lymph node dissection. Positive results of reverse transcriptase polymerase chain reaction (RT-PCR) test have a strong relationship with melanoma recurrence and disease-specific survival time in stage III melanoma.

Differential microRNA Expression in USP8-Mutated and Wild-Type Corticotroph Pituitary Tumors Reflect the Difference in Protein Ubiquitination Processes.

BACKGROUND: USP8 mutations are the most common driver changes in corticotroph pituitary tumors. They have direct effect on cells' proteome through disturbance of ubiquitination process and also influence gene expression. The aim of this study was to compare microRNA profiles in USP8-mutated and wild-type tumors and determine the probable role of differential microRNA expression by integrative microRNA and mRNA analysis. METHODS: Patients with Cushing's disease (n = 28) and silent corticotroph tumors (n = 20) were included. USP8 mutations were identified with Sanger sequencing. MicroRNA and gene expression was determined with next-generation sequencing. RESULTS: USP8-mutated patients with Cushing's disease showed higher rate of clinical remission and trend towards lower tumor volume than wild-type patients. Comparison of microRNA profiles of USP8-mutated and wild-type tumors revealed 68 differentially expressed microRNAs. Their target genes were determined by in silico prediction and microRNA/mRNA correlation analysis. GeneSet Enrichment analysis of putative targets showed that the most significantly overrepresented genes are involved in protein ubiquitination-related processes. Only few microRNAs influence the expression of genes differentially expressed between USP8-mutated and wild-type tumors. CONCLUSIONS: Differences in microRNA expression in corticotropinomas stratified according to USP8 status reflect disturbed ubiquitination processes, but do not correspond to differences in gene expression between these tumors.

RRM2 gene expression depends on BAF180 subunit of SWISNF chromatin remodeling complex and correlates with abundance of tumor infiltrating lymphocytes in ccRCC.

About 40% of clear cell renal cell carcinoma (ccRCC) cases carry the pbrm1 mutation inactivating BAF180 subunit of the SWI/SNF chromatin remodeling complex (CRC). Here we show that the majority of transcriptomic changes appear at the stage I of ccRCC development. By contrast, the stage II ccRCC exhibits hyperactivation of DNA replication demonstrated by the overexpression of several genes, e.g., RRM1 and RRM2 genes encoding subunits of ribonucleotide reductase (RNR) complex. We found that the degree of RRM1 and RRM2 upregulation in ccRCC patients depends on pbrm1 mutation. We show that the BAF180 protein product of the PBRM1 gene directly binds to RRM1 and RRM2 loci. The BAF180 binding regions are targeted by regulatory proteins previously reported as SWI/SNF CRC interacting partners. BAF180 binding to RRMs loci correlates with enrichment of H3K27me3 in case of RRM1 and H3K14Ac on RRM2, indicating the existence of differential regulatory mechanism controlling expression of these genes. We found that the strong overexpression of RRM2 in ccRCC patient samples correlates with T cell infiltration. Surprisingly, the majority of tumor infiltrating lymphocytes (TILs) consisted of CD4+ T cells. Furthermore, we show that exhausted CD4+ T cells induced the expression of the RRM2 gene in the primary ccRCC cell line. Collectively, our results provide the link between PBRM1 loss, RRM2 expression and T cell infiltration, which may lead to the establishment of new treatment of this disease.

PD-L1 Overexpression, SWI/SNF Complex Deregulation, and Profound Transcriptomic Changes Characterize Cancer-Dependent Exhaustion of Persistently Activated CD4+ T Cells.

Growing tumors avoid recognition and destruction by the immune system. During continuous stimulation of tumor-infiltrating lymphocytes (TILs) by tumors, TILs become functionally exhausted; thus, they become unable to kill tumor cells and to produce certain cytokines and lose their ability to proliferate. This collectively results in the immune escape of cancer cells. Here, we show that breast cancer cells expressing PD-L1 can accelerate exhaustion of persistently activated human effector CD4+ T cells, manifesting in high PD-1 and PD-L1 expression level son T cell surfaces, decreased glucose metabolism genes, strong downregulation of SWI/SNF chromatin remodeling complex subunits, and p21 cell cycle inhibitor upregulation. This results in inhibition of T cell proliferation and reduction of T cell numbers. The RNAseq analysis on exhausted CD4+ T cells indicated strong overexpression of IDO1 and genes encoding pro-inflammatory cytokines and chemokines. Some interleukins were also detected in media from CD4+ T cells co-cultured with cancer cells. The PD-L1 overexpression was also observed in CD4+ T cells after co-cultivation with other cell lines overexpressing PD-L1, which suggested the existence of a general mechanism of CD4+ T cell exhaustion induced by cancer cells. The ChIP analysis on the PD-L1 promoter region indicated that the BRM recruitment in control CD4+ T cells was replaced by BRG1 and EZH2 in CD4+ T cells strongly exhausted by cancer cells. These findings suggest that epi-drugs such as EZH2 inhibitors may be used as immunomodulators in cancer treatment.

Multimarker reverse transcriptase-polymerase chain reaction assay in lymphatic drainage and sentinel node tumor burden.

PURPOSE: We assessed molecular (presence of melanoma cells markers in lymph fluid [LY]) and pathological features (sentinel lymph node [SN] tumor burden according to Rotterdam criteria, metastases microanatomic location) and correlated them with survival and melanoma prognostic factors in a group of patients with positive SN biopsy. METHODS: We analyzed 368 consecutive SN-positive patients after completion lymph node dissection (CLND). In 321 patients we obtained data on SLN microanatomic location/tumor burden (only 7 cases had metastases <0.1 mm); in 137 we additionally analyzed 24-hour collected LY after CLND (multimarker reverse transcriptase-polymerase chain reaction [MM-RT-PCR] with primers for tyrosinase, MART1 (MelanA), and uMAGE mRNA (27.7% positive samples)]. Median follow-up time was 41 months. RESULTS: According to univariate analysis, the following factors had a negative impact on overall survival (OS): higher Breslow thickness (P = .0001), ulceration (P < .0001), higher Clark level (P = .008), male gender (P = .0001), metastatic lymph nodes >1 (P < .0001), nodal metastases extracapsular extension (P < .0001), metastases to additional non-SNs (P = .0004), micrometastases size ≥ 0.1 mm (P = .0006), and positive LY MM-RT-PCR (P = .0007). SN tumor burden showed linear correlation with increasing Breslow thickness (P = .01). The 5-year OS rates for SLN tumor burden <0.1 mm, 1-1.0 mm, and >1.0 mm were 84%/66%/44%, respectively, and for positive and negative LY MM-RT-PCR 47%/0%, respectively. The independent factors for shorter OS (multivariate analysis): male gender, primary tumor ulceration, number of involved nodes ≥ 4, micrometastases size >1.0 mm, and, in additional model including molecular analysis-positive MM-RT-PCR results (hazard ratio [HR] 3.2), micrometastases size >1.0 mm (HR 1.13), and primary tumor ulceration (HR 2.17). Similar results were demonstrated for disease-free survival (DFS) data. CONCLUSIONS: SN tumor burden categories according to Rotterdam criteria and the positive result of LY MM-RT-PCR assay demonstrated additional, independent prognostic value in SN-positive melanoma patients, showing significant correlation with shorter DFS and OS.

HAX-1 overexpression, splicing and cellular localization in tumors.

BACKGROUND: HAX-1 has been described as a protein potentially involved in carcinogenesis and especially metastasis. Its involvement in regulation of apoptosis and cell migration along with some data indicating its overexpression in cancer cell lines and tumors suggests that HAX-1 may play a role in neoplastic transformation. Here we present the first systematic analysis of HAX-1 expression in several solid tumors. METHODS: Using quantitative RT-PCR, we have determined the mRNA levels of HAX1 splice variant I in several solid tumors. We have also analyzed by semiquantitative and quantitative RT-PCR the expression of five HAX-1 splice variants in breast cancer samples and in normal tissue from the same individuals. Quantitative PCR was also employed to analyze the effect of estrogen on HAX1 expression in breast cancer cell line. Immunohistochemical analysis of HAX-1 was performed on normal and breast cancer samples. RESULTS: The results reveal statistically important HAX1 up-regulation in breast cancer, lung cancer and melanoma, along with some minor variations in the splicing pattern. HAX-1 up-regulation in breast cancer samples was confirmed by immunohistochemical analysis, which also revealed an intriguing HAX-1 localization in the nuclei of the tumor cells, associated with strong ER status. CONCLUSION: HAX-1 elevated levels in cancer tissues point to its involvement in neoplastic transformation, especially in breast cancer. The connection between HAX-1 nuclear location and ER status in breast cancer samples remains to be clarified.