Fibrosis and cancer: shared features and mechanisms suggest common targeted therapeutic approaches.

Epidemiological studies support a strong link between organ fibrosis and epithelial cancers. Moreover, clinical and experimental investigations consistently indicate that these diseases intertwine and share strikingly overlapping features. As a deregulated response to injury occurring in all body tissues, fibrosis is characterized by activation of fibroblasts and immune cells, contributing to progressive deposition of extracellular matrix (ECM) and inflammation. Cancers are driven by genetic alterations resulting in dysregulated cell survival, proliferation and dissemination. However, non-cancerous components of tumour tissues including fibroblasts, inflammatory cells and ECM play key roles in oncogenesis and cancer progression by providing a pro-mutagenic environment where cancer cells can develop, favouring their survival, expansion and invasiveness. Additional commonalities of fibrosis and cancer are also represented by overproduction of growth factors, like transforming growth factor ß, epithelial-to-mesenchymal transition, high oxidative stress, Hippo pathway dysfunctions and enhanced cellular senescence. Here, we review advances in the analysis of cellular and molecular mechanisms involved in the pathogenesis of both organ fibrosis and cancer, with particular reference to chronic kidney diseases and renal cell cancers. Most importantly, improved understanding of common features is contributing to the development of innovative treatment strategies targeting shared mechanisms.

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.

Transcriptome-proteome integration of archival human renal cell carcinoma biopsies enables identification of molecular mechanisms.

Renal cell cancer is among the most common forms of cancer in humans, with around 35,000 deaths attributed to kidney carcinoma in the European Union in 2012 alone. Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and the most lethal of all genitourinary cancers. Here, we apply omics technologies to archival core biopsies to investigate the biology underlying ccRCC. Knowledge of these underlying processes should be useful for the discovery and/or confirmation of novel therapeutic approaches and ccRCC biomarker development. From partial or full nephrectomies of 11 patients, paired core biopsies of ccRCC-affected tissue and adjacent ("peritumorous") nontumor tissue were both sampled and subjected to proteomics analyses. We combined proteomics results with our published mRNA sequencing data from the same patients and with published miRNA sequencing data from an overlapping patient cohort from our institution. Statistical analysis and pathway analysis were performed with JMP Genomics and Ingenuity Pathway Analysis (IPA), respectively. Proteomics analysis confirmed the involvement of metabolism and oxidative stress-related pathways in ccRCC, whereas the most affected pathways in the mRNA sequencing data were related to the immune system. Unlike proteomics or mRNA sequencing alone, a combinatorial cross-omics pathway analysis approach captured a broad spectrum of biological processes underlying ccRCC, such as mitochondrial damage, repression of apoptosis, and immune system pathways. Sirtuins, immunoproteasome genes, and CD74 are proposed as potential targets for the treatment of ccRCC.

Expanding the Utilization of Formalin-Fixed, Paraffin-Embedded Archives: Feasibility of miR-Seq for Disease Exploration and Biomarker Development from Biopsies with Clear Cell Renal Cell Carcinoma.

Novel predictive tools for clear cell renal cell carcinoma (ccRCC) are urgently needed. MicroRNAs (miRNAs) have been increasingly investigated for their predictive value, and formalin-fixed paraffin-embedded biopsy archives may potentially be a valuable source of miRNA sequencing material, as they remain an underused resource. Core biopsies of both cancerous and adjacent normal tissues were obtained from patients (n = 12) undergoing nephrectomy. After small RNA-seq, several analyses were performed, including classifier evaluation, obesity-related inquiries, survival analysis using publicly available datasets, comparisons to the current literature and ingenuity pathway analyses. In a comparison of tumour vs. normal, 182 miRNAs were found with significant differential expression; miR-155 was of particular interest as it classified all ccRCC samples correctly and correlated well with tumour size (R² = 0.83); miR-155 also predicted poor survival with hazard ratios of 2.58 and 1.81 in two different TCGA (The Cancer Genome Atlas) datasets in a univariate model. However, in a multivariate Cox regression analysis including age, sex, cancer stage and histological grade, miR-155 was not a statistically significant survival predictor. In conclusion, formalin-fixed paraffin-embedded biopsy tissues are a viable source of miRNA-sequencing material. Our results further support a role for miR-155 as a promising cancer classifier and potentially as a therapeutic target in ccRCC that merits further investigation.

RNA extraction for RNA sequencing of archival renal tissues.

BACKGROUND: Next generation sequencing (NGS) and especially ribonucleic acid (RNA) sequencing is a powerful tool to acquire insights into molecular disease mechanisms. Therefore, it is of interest to optimize methods for RNA extraction from archival, formalin fixed and paraffin embedded (FFPE) tissues. This is challenging due to RNA degradation and chemical modifications. The aim of this study was to find the most appropriate method to extract RNA from FFPE renal tissue to enable NGS. METHOD: We evaluated seven commercially available RNA extraction kits: High Pure FFPE RNA Isolation (Roche), ExpressArt Clear FFPE RNAready (Amsbio), miRNeasy FFPE, RNeasy FFPE (Qiagen), PureLink FFPE Total RNA (Invitrogen), RecoverAll Total Nucleic Acid Isolation (Ambion) and Absolutely RNA FFPE Kit (Agilent). RNA was obtained from tissue blocks of two healthy, male Wistar rats and from normal renal tissue of patients undergoing nephrectomy. Yield and quality of RNA extracted from rat whole kidney sections, human kidney core biopsies and laser capture microdissected (LCM) glomerular cross-sections were assessed: Analyses of RNA quantity were performed using NanoDrop and Qubit. RNA quality is reflected by DV200 values (% of RNA fragments >200 nucleotides) utilizing the Agilent 2100 BioAnalyzer. RNA of human LCM samples was subsequently sequenced using the Illumina TruSeq(®) RNA Access Library Preparation Kit. CONCLUSION: Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.

Transcriptomic analysis reveals partial epithelial-mesenchymal transition and inflammation as common pathogenic mechanisms in hypertensive nephrosclerosis and Type 2 diabetic nephropathy.

Hypertensive nephrosclerosis (HN) and Type 2 diabetic nephropathy (T2DN) are the leading causes of chronic kidney disease (CKD). To explore shared pathogenetic mechanisms, we analyzed transcriptomes of kidney biopsies from patients with HN or T2DN. Total RNA was extracted from 10 µm whole kidney sections from patients with HN, T2DN, and normal controls (Ctrl) (n = 6 for each group) and processed for RNA sequencing. Differentially expressed (log2 fold change >1, adjusted p < 0.05) genes (DEG) and molecular pathways were analyzed, and selected results were validated by immunohistochemistry (IHC). ELISA on serum samples was performed on a related cohort consisting of patients with biopsy-proven HN (n = 13) and DN (n = 9), and a normal control group (n = 14). Cluster analysis on RNA sequencing data separated diseased and normal tissues. RNA sequencing revealed that 88% (341 out of 384) of DEG in HN were also altered in T2DN, while gene set enrichment analysis (GSEA) showed that over 90% of affected molecular pathways, including those related to inflammation, immune response, and cell-cycle regulation, were similarly impacted in both HN and T2DN samples. The increased expression of genes tied to interleukin signaling and lymphocyte activation was more pronounced in HN, while genes associated with extracellular matrix organization were more evident in T2DN. Both HN and T2DN tissues exhibited significant upregulation of genes connected with inflammatory responses, T-cell activity, and partial epithelial to mesenchymal transition (p-EMT). Immunohistochemistry (IHC) further confirmed T-cell (CD4+ and CD8+ ) infiltration in the diseased tissues. Additionally, IHC revealed heightened AXL protein expression, a key regulator of inflammation and p-EMT, in both HN and T2DN, while serum analysis indicated elevated soluble AXL levels in patients with both conditions. These findings underline the shared molecular mechanisms between HN and T2DN, hinting at the potential for common therapeutic strategies targeting both diseases.

Lipoproteins in Staphylococcus aureus mediate inflammation by TLR2 and iron-dependent growth in vivo.

Lipoproteins (Lpp) are ligands of TLR2 and signal by the adaptor MyD88. As part of the bacterial cell envelope, Lpp are mainly involved in nutrient acquisition for Staphylococcus aureus. The impact of Lpp on TLR2-MyD88 activation for S. aureus in systemic infection is unknown. S. aureus strain SA113 deficient in the enzyme encoded by the prolipoprotein diacylglyceryl transferase gene (Deltalgt), which attaches the lipid anchor to pro-Lpp, was used to study benefits and costs of Lpp maturation. Lpp in S. aureus induced early and strong cytokines by TLR2-MyD88 signaling in murine peritoneal macrophages. Lpp contributed via TLR2 to pathogenesis of sepsis in C57BL/6 mice with IL-1beta, chemokine-mediated inflammation, and high bacterial numbers. In the absence of MyD88-mediated inflammation, Lpp allowed bacterial clearing from liver devoid of infiltrating cells, but still conferred a strong growth advantage in mice, which was shown to rely on iron uptake and storage in vitro and in vivo. With iron-restricted bacteria, the Lpp-related growth advantage was evident in infection of MyD88(-/-), but not of C57BL/6, mice. On the other hand, iron overload of the host restored the growth deficit of Deltalgt in MyD88(-/-), but not in immunocompetent C57BL/6 mice. These results indicate that iron acquisition is improved by Lpp of S. aureus but is counteracted by inflammation. Thus, lipid anchoring is an evolutionary advantage for S. aureus to retain essential proteins for better survival in infection.

Ultrasound and Microbubbles Enhance Uptake of Doxorubicin in Murine Kidneys.

The use of ultrasound and microbubble-enhanced drug delivery, commonly referred to as sonoporation, has reached numerous clinical trials and has shown favourable results. Nevertheless, the microbubbles and acoustic path also pass through healthy tissues. To date, the majority of studies have focused on the impact to diseased tissues and rarely evaluated the impact on healthy and collateral tissue. The aim of this study was to test the effect and feasibility of low-intensity sonoporation on healthy kidneys in a mouse model. In our work here, we used a clinical diagnostic ultrasound system (GE Vivid E9) with a C1-5 ultrasound transducer combined with a software modification for 20-µs-long pulses to induce the ultrasound-guided drug delivery of doxorubicin (DOX) in mice kidneys in combination with SonoVue® and Sonazoid™ microbubbles. The acoustic output settings were within the commonly used diagnostic ranges. Sonoporation with SonoVue® resulted in a significant decrease in weight vs. DOX alone (p = 0.0004) in the first nine days, whilst all other comparisons were not significant. Ultrasound alone resulted in a 381% increase in DOX uptake vs. DOX alone (p = 0.0004), whilst SonoVue® (p = 0.0001) and Sonazoid™ (p < 0.0001) further increased the uptake nine days after treatment (419% and 493%, respectively). No long-standing damage was observed in the kidneys via histology. In future sonoporation and drug uptake studies, we therefore suggest including an "ultrasound alone" group to verify the actual contribution of the individual components of the procedure on the drug uptake and to perform collateral damage studies to ensure there is no negative impact of low-intensity sonoporation on healthy tissues.

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 (αSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfß), 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.

Clear Cell Renal Cell Carcinoma is linked to Epithelial-to-Mesenchymal Transition and to Fibrosis.

Clear cell renal cell carcinoma (ccRCC) represents the most common type of kidney cancer with high mortality in its advanced stages. Our study aim was to explore the correlation between tumor epithelial-to-mesenchymal transition (EMT) and patient survival. Renal biopsies of tumorous and adjacent nontumorous tissue were taken with a 16 g needle from our patients (n = 26) undergoing partial or radical nephrectomy due to ccRCC RNA sequencing libraries were generated using Illumina TruSeq® Access library preparation protocol and TruSeq Small RNA library preparation kit. Next generation sequencing (NGS) was performed on Illumina HiSeq2500. Comparative analysis of matched sample pairs was done using the Bioconductor Limma/voom R-package. Liquid chromatography-tandem mass spectrometry and immunohistochemistry were applied to measure and visualize protein abundance. We detected an increased generic EMT transcript score in ccRCC Gene expression analysis showed augmented abundance of AXL and MMP14, as well as down-regulated expression of KL (klotho). Moreover, microRNA analyses demonstrated a positive expression correlation of miR-34a and its targets MMP14 and AXL Survival analysis based on a subset of genes from our list EMT-related genes in a publicly available dataset showed that the EMT genes correlated with ccRCC patient survival. Several of these genes also play a known role in fibrosis. Accordingly, recently published classifiers of solid organ fibrosis correctly identified EMT-affected tumor samples and were correlated with patient survival. EMT in ccRCC linked to fibrosis is associated with worse survival and may represent a target for novel therapeutic interventions.

Transcriptome Sequencing (RNAseq) Enables Utilization of Formalin-Fixed, Paraffin-Embedded Biopsies with Clear Cell Renal Cell Carcinoma for Exploration of Disease Biology and Biomarker Development.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens.

Development and confirmation of potential gene classifiers of human clear cell renal cell carcinoma using next-generation RNA sequencing.

OBJECTIVE: A previous study by this group demonstrated the feasibility of RNA sequencing (RNAseq) technology for capturing disease biology of clear cell renal cell carcinoma (ccRCC), and presented initial results for carbonic anhydrase-9 (CA9) and tumor necrosis factor-α-induced protein-6 (TNFAIP6) as possible biomarkers of ccRCC (discovery set) [Eikrem et al. PLoS One 2016;11:e0149743]. To confirm these results, the previous study is expanded, and RNAseq data from additional matched ccRCC and normal renal biopsies are analyzed (confirmation set). MATERIALS AND METHODS: Two core biopsies from patients (n = 12) undergoing partial or full nephrectomy were obtained with a 16 g needle. RNA sequencing libraries were generated with the Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using linear modeling (voom/Limma; R Bioconductor). RESULTS: The formalin-fixed and paraffin-embedded discovery and confirmation data yielded 8957 and 11,047 detected transcripts, respectively. The two data sets shared 1193 of differentially expressed genes with each other. The average expression and the log2-fold changes of differentially expressed transcripts in both data sets correlated, with R² = .95 and R² = .94, respectively. Among transcripts with the highest fold changes were CA9, neuronal pentraxin-2 and uromodulin. Epithelial-mesenchymal transition was highlighted by differential expression of, for example, transforming growth factor-ß1 and delta-like ligand-4. The diagnostic accuracy of CA9 was 100% and 93.9% when using the discovery set as the training set and the confirmation data as the test set, and vice versa, respectively. These data further support TNFAIP6 as a novel biomarker of ccRCC. TNFAIP6 had combined accuracy of 98.5% in the two data sets. CONCLUSIONS: This study provides confirmatory data on the potential use of CA9 and TNFAIP6 as biomarkers of ccRCC. Thus, next-generation sequencing expands the clinical application of tissue analyses.