Caracterización de NF-КB en cáncer de mama: posible factor pronóstico y predictivo

La activación del factor de transcripción NF-kB juega un papel importante en el proceso de la oncogénesis. El propósito del presente estudio fue determinar si NF-kB se encuentra activado en cáncer de mama e identificar la correlación de su actividad con las características clínico-patológicas del tumor. Además, se analizaron aquellos genes cuya expresión está regulada por NF-kB en tejido tumoral, tejido adyacente al tumor y tejido normal procedente de reducciones mamarias. Finalmente se estudio si NF-kB o alguno de sus genes diana podría tener un papel en el tratamiento del cáncer de mama. Para el estudio de la activación de NF-kB se utilizaron muestras de tejido congelado procedentes de cirugía que fueron incluidas en OCT (compuesto para la temperatura óptima de corte) y a las que se les realizó un análisis histopatológico para comprobar la calidad. De las 450 muestras que analizamos, seleccionamos 148 muestras de carcinoma mamario y 16 de tejido adyacente no tumoral. Se obtuvieron los extractos proteicos de todas ellas y la determinación de la activación de las cinco subunidades de NF-kB se realizó mediante ELISA y se validó mediante western blot. Para la determinación de la expresión génica utilizamos tejido parafinado. Se extrajo el ARN y se realizó la transcripción inversa. La calidad de la muestra se determinó mediante el kit comercial “RT2 RNA QC PCR array”. El perfil génico se realizó con “RT2 Profiler PCR Array Human NF-kB Signaling Targets”. La sensibilidad al tratamiento con el fármaco quimioterápico doxorubicina se realizó con las líneas celulares derivadas de carcinoma mamario MCF7, MDAMB231, MDAMB468, SKBR3 y BT474. La activación de NF-kB inducida por la doxorubicina se evaluó dichas líneas celulares, realizándose también la comparación de la expresión génica antes y después del tratamiento. Se observó que las subunidades de NF-kB p65, p50, p52 y relB se encuentran activadas de forma significativa en tejido tumoral frente a tejido no tumoral, siendo el valor-p ≤ 0,005 en todos los casos. Además, en carcinoma ductal invasivo la activación de relB estaba asociada al tamaño tumoral (p=0,004), la invasión de tejidos adyacentes (p=0,01) y afectación ganglionar (p= 0,003) mientras que el aumento de la activación de la subunidad p65 estaba asociado a invasión linfática (p=0,033). Asimismo los niveles de activación de p52 y c-rel fueron más altos en carcinoma ductal invasivo que en carcinoma ductal in situ con valores-p de 0,008 y p=0,019 respectivamente. Por otra parte, valores de activación altos para p52 y relB en tumores positivos para receptores de estrógenos estuvieron significativamente asociados a recidiva tras el tratamiento. Respecto al análisis de expresión de genes diana de NF-kB, encontramos diferencias significativas entre la expresión de tejido normal/adyacente y tumoral en los genes CXCL10, CXCL9, MMP9 y LTA cuya expresión fue mayor en tejido tumoral y MYC, EGFR y MAP2k6 cuya expresión disminuía. También observamos que la expresión de MMP9, CXCL9 y MYC en tejido adyacente al tumor estaba aumentada respecto a tejido normal. En líneas celulares observamos que el tratamiento con doxorubicina producía la activación de NF-kB y de la expresión de genes asociados a actividades quimocina y citocina, señalización intercelular y respuesta inflamatoria mientras que la expresión de genes relacionados con actividades anti-apoptóticas y transducción de señales estaba disminuída. Estos datos indican que NF-kB está constitutivamente activado en cáncer de mama y sugiere que la actividad de NF-kB está asociada con la progresión tumoral y la supervivencia libre de enfermedad. Por otro lado, algunos genes asociados a NF-kB con expresión diferente entre tejido tumoral y normal podrían actuar como dianas terapeuticas potenciales en cáncer de mama ya que su expresión se modifica tras el tratamiento con doxorubicina. Finalmente se sugiere que el microambiente tumoral podría tener un papel destacado en la progresión del cáncer y la resistencia a fármacos.Activation of transcription factor NF-kB has been shown to play a role in oncogenesis. The purpose of the study was to determine whether NF-kB is activated in breast tumour carcinoma tissues and to identify any correlation between its activity and the clinical-pathological features of tumours. Besides we analyse genes whose expression is regulated by NF-kB in tumoral tissue, adjacent tumoral tissue and normal breast tissue. Finally we studied if NF-KB or its target genes could have a role in the treatment of breast cancer. For the study of activation of NF-kB we used frozen breast tissues from surgery. For quality control the frozen tissues were included in optimal cutting temperature compound (OCT) and a histopathological analysis was performed. Of them, we select 148 human breast carcinoma tissues and 16 adjacent non-tumour tissues. Nuclear protein extracts were obtained and NF-kB activation was determined by ELISA analysis and data were validated by western blot. To determine gene expression we used paraffin-embedded breast cancer tissues. The RNA was extracted and reverse transcription performed. The quality of samples was determined with RT2 RNA QC PCR array. The RNA profile was identified with RT2 Profiler PCR Array Human NF-kB Signaling Targets. Sensibility to doxorubicin was tested in MCF7, MDAMB231, MDAMB468, SKBR3 and BT474 breast cancer-derived cell lines. Doxorubicin-induced NF-kB activation was assessed in cells lines and a comparison between gene expression before and after the treatment with doxorubicin was performed. NF-kB study showed that p65, p50, p52 and relB subunits activation was significantly higher in breast tumours than in normal adjacent tissues (p≤0.005 in all cases). In invasive ductal carcinoma (IDC) relB activation was related to tumor size (p=0.004), invasion of adjacent tissues (p=0.01) and lymph nodes invasion (p= 0.003) and p65 increased levels were associated with lymph nodes invasion (p=0.033). p52 and c-rel levels were higher in invasive ductal carcinoma (IDC) than in ductal carcinoma in situ (DCIS) with p=0.008 and p=0.019 respectively. Higher p52 and relB activation levels in oestrogen receptor-positive tumour tissues were significantly related to patients relapse. On the other hand we detect significant differences in the expression between normal/adjacent and tumoral tissues in genes CXCL10, CXCL9, MMP9 and LTA whose levels were higher in tumoral tissue and MYC, EGFR and MAP2k6 whose levels were lower. We also found that MYC, CXCL9 and MMP9 expression was higher in adjacent tissue than normal tissue, pointing the importance of microenvironment in cancer. In cell lines we found that doxorubicin treatment enhanced NF-kB activation and expression of genes related to chemokine and cytokine activities, cell-cell signalling and inflammatory response whereas gene expression associated with anti-apoptosis activity and signal transduction was decreased. These data indicate NF-kB is constitutively activated in human breast carcinoma tissues and suggest NF-kB activity is related to tumour progression and disease-free patient survival. On the other hand, some genes related to NF-KB with differential expression between tumor and normal tissue could be a potential target in breast cancer as its expression is modified by doxorubicin. Finally our data suggest microenvironment could have a role in cancer progression and drugs’ resistance

Association of redox and inflammation-related biomarkers with prognosis in IgA nephropathy: A prospective observational study

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AGAP2-AS1 as a prognostic biomarker in low-risk clear cell renal cell carcinoma patients with progressing disease

Background Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer and one of the most common cancers. While survival for localized ccRCC is good, the survival of metastatic disease is not, and thirty percent of patients with ccRCC develop metastases during follow-up. Although current scoring methods accurately identify patients at low progression risk, a small subgroup of those patients still experience metastasis. We therefore aimed to identify ccRCC progression biomarkers in "low-risk" patients who were potentially eligible for adjuvant treatments or more intensive follow-up. Methods We assembled a cohort of ccRCC patients (n = 443) and identified all "low-risk" patients who later developed progressing tumors (n = 8). Subsequently, we performed genome-wide expression profiling from formalin-fixed samples obtained at initial surgery from these "low-risk" patients and 16 matched patients not progressing to recurrence with metastasis. The patients were matched for Leibovich sore, creatinine, age, sex, tumor size and tumor stage. Key results were confirmed with qPCR and external data from The Cancer Genome Atlas. Results Principal component analysis indicated that systematic transcriptomic differences were already detectable at the time of initial surgery. One thousand one hundred sixty-seven genes, mainly associated with cancer and immune-related pathways, were differentially expressed between progressors and nonprogressors. A search for a classifier revealed that overexpression of AGAP2-AS1, an antisense long noncoding RNA, correctly classified 23 of 24 samples, years (4.5 years average) in advance of the discovery of metastasis and without requiring larger gene panels. Subsequently, we confirmed AGAP2-AS1 gene overexpression by qPCR in the same samples (p < 0.05). Additionally, in external data from The Cancer Genome Atlas, overexpression of AGAP2-AS1 is correlated with overall unfavorable survival outcome in ccRCC, irrespective of other prognostic predictors (p = 2.44E-7). Conclusion AGAP2-AS1 may represent a novel biomarker identifying high-risk ccRCC patients currently classified as "low risk" at the time of surgery.Peer reviewe

A multiomics disease progression signature of low‑risk ccRCC

Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer. Identification of ccRCC likely to progress, despite an apparent low risk at the time of surgery, represents a key clinical issue. From a cohort of adult ccRCC patients (n = 443), we selected low-risk tumors progressing within a 5-years average follow-up (progressors: P, n = 8) and non-progressing (NP) tumors (n = 16). Transcriptome sequencing, miRNA sequencing and proteomics were performed on tissues obtained at surgery. We identified 151 proteins, 1167 mRNAs and 63 miRNAs differentially expressed in P compared to NP low-risk tumors. Pathway analysis demonstrated overrepresentation of proteins related to “LXR/ RXR and FXR/RXR Activation”, “Acute Phase Response Signaling” in NP compared to P samples. Integrating mRNA, miRNA and proteomic data, we developed a 10-component classifier including two proteins, three genes and five miRNAs, effectively differentiating P and NP ccRCC and capturing underlying biological differences, potentially useful to identify “low-risk” patients requiring closer surveillance and treatment adjustments. Key results were validated by immunohistochemistry, qPCR and data from publicly available databases. Our work suggests that LXR, FXR and macrophage activation pathways could be critically involved in the inhibition of the progression of low-risk ccRCC. Furthermore, a 10-component classifier could support an early identification of apparently low-risk ccRCC patients.Peer reviewe

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-tomesenchymal transition (EMT) and inflammation – both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (aSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfb), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.publishedVersio

Axl-inhibitor bemcentinib alleviates mitochondrial dysfunction in the unilateral ureter obstruction murine model

Renal fibrosis is a progressive histological manifestation leading to chronic kidney disease (CKD) and associated with mitochondrial dysfunction. In previous work, we showed that Bemcentinib, an Axl receptor tyrosine kinase inhibitor, reduced fibrosis development. In this study, to investigate its effects on mitochondrial dysfunction in renal fibrosis, we analysed genome-wide transcriptomics data from a unilateral ureter obstruction (UUO) murine model in the presence or absence of bemcentinib (n = 6 per group) and SHAM-operated (n = 4) mice. Kidney ligation resulted in dysregulation of mitochondria-related pathways, with a significant reduction in the expression of oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), citric acid cycle (TCA), response to reactive oxygen species and amino acid metabolism-related genes. Bemcentinib treatment increased the expression of these genes. In contrast, AKT/PI3K signalling pathway genes were up-regulated upon UUO, but bemcentinib largely inhibited their expression. At the functional level, ligation reduced mitochondrial biomass, which was increased upon bemcentinib treatment. Serum metabolomics analysis also showed a normalizing amino acid profile in UUO, compared with SHAM-operated mice following bemcentinib treatment. Our data suggest that mitochondria and mitochondria-related pathways are dramatically affected by UUO surgery and treatment with Axl-inhibitor bemcentinib partially reverses these effects.publishedVersio

Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-кB target genes in human breast cancer

NF-кB has been linked to doxorubicin resistance in breast cancer patients. NF- кB nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined; however its functional relevance at transcriptional level on NF-кB -dependent genes and the biological consequences are unclear. We studied NF-кB -dependent gene expression induced by doxorubicin in breast cancer cells and fresh human cancer specimens with different genetic backgrounds focusing on their p53 status. NF-кB –dependent signature of doxorubicin was identified by gene expression microarrays in breast cancer cells treated with doxorubicin and the IKKβ-inhibitor MLN120B, and confirmed ex vivo in human cancer samples. The association with p53 was functionally validated. Finally, NF-кB activation and p53 status was determined in a cohort of breast cancer patients treated with adjuvant doxorubicin-based chemotherapy. Doxorubicin treatment in the p53-mutated MDA-MB-231 cells resulted in NF NF- кB driven-gene transcription signature. Modulation of genes related with invasion, metastasis and chemoresistance (ICAM-1, CXCL1, TNFAIP3, IL8) were confirmed in additional doxorubicin-treated cell lines and fresh primary human breast tumors. In both systems, p53-deficient background correlated with the activation of the NF-кB –dependent signature. Furthermore, restoration of p53WT in the mutant p53 MDAMB- 231 cells impaired NF-кB driven transcription induced by doxorubicin. Moreover, a p53 deficient background and nuclear NF-кB /p65 in breast cancer patients correlated with reduced disease free-survival. This study supports that p53 deficiency is necessary for a doxorubicin driven NF-кB -response that limits doxorubicin cytotoxicity in breast cancer and is linked to an aggressive clinical behavior.Financial support: This work was supported by RD12/0036/0051 (J.A.), RD09/0076/0101, RD09/0076/0036, RD12/0036/0054 (A.B), RD12/0036/0070 (A. Ll), PI12/00680 (J.A.), PI12/01552 (F.R.), PI12/01421 (A.Ll.), 2009 SGR 321 (J.A.), FMM 9757/002 (F.R.), and the “Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC). J.A. and F.R. are recipients of intensification program ISCIII/FEDER. We thank Fundació Cellex (Barcelona) for a generous donation to the Hospital del Mar Medical Oncology Service. We thank Millenium for generously providing MLN120B

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (alpha SMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgf beta), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.Peer reviewe

Variable expression of eighteen common housekeeping genes in human non-cancerous kidney biopsies

Housekeeping, or reference genes (RGs) are, by definition, loci with stable expression profiles that are widely used as internal controls to normalize mRNA levels. However, due to specific events, such as pathological changes, or technical procedures, their expression might be altered, failing to fulfil critical normalization pre-requisites. To identify RG genes suitable as internal controls in human non-cancerous kidney tissue, we selected 18 RG candidates based on previous data and screen them in 30 expression datasets (>800 patients), including our own, publicly available or provided by independent groups. Datasets included specimens from patients with hypertensive and diabetic nephropathy, Fabry disease, focal segmental glomerulosclerosis, IgA nephropathy, membranous nephropathy, and minimal change disease. We examined both microdissected and whole section-based datasets. Expression variability of 4 candidate genes (YWHAZ, SLC4A1AP, RPS13 and ACTB) was further examined by qPCR in biopsies from patients with hypertensive nephropathy (n = 11) and healthy controls (n = 5). Only YWHAZ gene expression remained stable in all datasets whereas SLC4A1AP was stable in all but one Fabry dataset. All other RGs were differentially expressed in at least 2 datasets, and in 4.5 datasets on average. No differences in YWHAZ, SLC4A1AP, RPS13 and ACTB gene expression between hypertensive and control biopsies were detected by qPCR. Although RGs suitable to all techniques and tissues are unlikely to exist, our data suggest that in non-cancerous kidney biopsies expression of YWHAZ and SLC4AIAP genes is stable and suitable for normalization purposes