Targeting Epithelial-to-Mesenchymal Transition to Treat Advanced Renal Cell Carcinoma: From Mice to Men!

Prosjektnummer: 912167
Ansvarlig person: Hans-Peter Marti
Institusjon: Helse Bergen HF
Prosjektkategori: Åpen prosjektstøtte
Helsekategori: Cancer, Renal and Urogenital
Forskningsaktivitet: 4. Detection and Diagnosis, 5. Treatment Developement

2020 - sluttrapport

A.) Aim: Analyses and targeting of EMT in experimental and human kidney carcinoma to develop new therapy and prognostic markers. B.) Resultater Disputas: Lea Landolt, PHD: "Epithelial-to-mesenchymal transition in clear cell renal cell cancer and renal fibrosis", 16. januar 2019. All manuscripts with short summary: 1. AXL INHIBITORS ATTENUATE EXPERIMENTAL RENAL CELL CARCINOMA DEVELOPMENT In an ongoing study, we have shown that the two Axl inhibitors from Bergenbio AS, Bergen, such as small the molecular Bemcentinib and the anti-AXL monoclonal antibody BGB149, inhibited growth of experimental carcinoma by >75% in the orthotopic murine model of renal cell carcinoma (786-O human tumor cells) in athymic nude (bcg Balb/c) mice. Publication: Manuscript in preparation and conference abstract submitted. 2. AGAP2-AS1 AS PROGNOSTIC BIOMARKER IN LOW-RISK CLEAR CELL RENAL CELL CARCINOMA PATIENTS WITH PROGRESSING DISEASE We performed RNA sequencing from formalin-fixed samples obtained from 8 low-risk patients with progressing tumours and 16 matched stable patients. A total of 1167 genes, were differentially expressed. Overexpression of AGAP2-AS1 correctly classifies 23 of 24 samples without requiring larger gene panels. Selected immunohistochemistry supported, at the protein level, observed gene expression patterns. Therefore, AGAP2-AS1 may represent a biomarker identifying high risk patients currently classified as low risk at time of surgery. Manuscript submitted and under review at Scientific Reports. 3. TRANSCRIPTOME-PROTEOME INTEGRATION OF ARCHIVAL HUMAN RENAL CELL CARCINOMA (RCC) BIOPSIES ENABLES IDENTIFICATION OF MOLECULAR MECHANISMS Paired core biopsies of RCC-affected and nontumor tissue were analysed Fromm 11 patients. Proteomics confirmed metabolic and oxidative stress-related pathways in RCC. Unlike proteomics or mRNA sequencing alone, a combinatorial approach captured a spectrum, such as mitochondrial damage, repression of apoptosis, and immune system pathways. Sirtuins, immunoproteasome genes, and CD74 are potential therapy targets. Publication: Am J Physiol Renal Physiol. 2019 May 1;316(5):F1053-F1067. 4. 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 (ccRCC) Formalin-fixed paraffin-embedded biopsy tissues are a source of miRNA-seq. Our results support a role for miR-155 as a promising cancer classifier and potentially as a therapeutic target in ccRC. Publication: Int J Mol Sci. 2018 Mar 10;19(3):803. doi: 10.3390/ijms19030803. 5. RENAL CARCINOMA/KIDNEY PROGENITOR CELL CHIMERA ORGANOID AS A NOVEL TUMORIGENESIS GENE DISCOVERY MODEL Comparing renal ontogenesis control genes to the genes involved in kidney cancer may provide new growth-associated gene screens and that 3D RCC chimera organoids can serve as a novel model with which to investigate the behavioral roles of cancer cells within the context of emergent complex tissue structures. Publication: Dis Model Mech. 2017 Dec 19;10(12):1503-1515. doi: 10.1242/dmm.028332. 6. DEVELOPMENT AND CONFIRMATION OF POTENTIAL GENE CLASSIFIERS OF HUMAN CLEAR CELL RENAL CELL CARCINOMA USING NEXT-GENERATION RNA SEQUENCING This study provides confirmatory data on the potential use of CA9 and TNFAIP6 as biomarkers of ccRCC. Thus, next-generation sequencing expands the application of tissue analyses. Publication: Scand J Urol. 2016 Dec;50(6):452-462. doi: 10.1080/21681805.2016.1238007. 7. 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 RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens. Publication: PLoS One. 2016 Feb 22;11(2)e0149743. doi: 10.1371/journal.pone.0149743. We now have a possible rationale to plan and perform an early the clinical phase 1b/2 trial in patients with advanced renal cell carcinoma using Axl inhibitors from Bergenbio.

2019

Targeting Epithelial-to-Mesenchymal-Transition to Treat Advanced Renal Cell Carcinoma: From Mice to MenPlease find below the report focusing on investigations related to human and experimental renal cell carcinoma. Thus, the current report contains two parts: 1. Therapy of orthotopic clear cell renal cell carcinoma (ccRCC) with Axl inhibion, and 2. RNA sequencing of human renal tissue samples with ccRCC. In addition, we completed a publication.1. Axl inhibition reduces experimental, orthotopic renal cell carcinoma Introduction: Our overall goal is to find a new way to treat patients with advanced ccRCC. For that, we wanted to evaluate first the small molecule BGB324 in a preclinical situation. Method: Induction of renal tumor was achieved using the 786-0-Luc renal carcinoma cells. Animals were injected with 1x106 cells into the renal capsule. After 4-7 days growth tumor size was estimated and animals were splitted into 4 groups. Desired tumor size at the beginning of the treatment was 6mm or around. GROUP 1 - Treatment with BGB324 (Bemcentinib, oral Axl receptor tyrosine kinase inhibitor, BerGenBio, AS) - 50mg/kg oral gavage 2 x per day from Monday till Friday. Inhibitor should be diluted to the final concentration of 5mg/ml. BGB324 will be dissolved in 0.5% (w/w) hydroxylpropyl-methylcellulose (HPMC) in 0.1% (w/w) tween 80 in water, according to the manufacturer. For every gavage fresh stock will be prepared. GROUP 2 - Control. Oral gavage without active compound. GROUP 3 - Combined treatment with BGB324 and PD-1 Ab treatment. Animals will receive 10mg/kg IP injection at the first day of the treatment followed by 5mg/kg every 5th day of the pembrolizumab (Keytruda). Treatment was provided for 8 weeks with once a week tumor visualisation and measurements. Results: First, we confirmed the expression of AXL in 786-O-Luc renal carcinoma cells using PCR and Western blot. Thereafter, animal experiments were performed, as described above. Growth of tumor was visualized using Bioluminescence In Vivo Imaging Analysis (IVIS analysis), every 7-10 days and measurements were done using ultrasound analysis (VEVO system). Mass of the tumor was significantly decreased >50% during BGB324 treatment and there was no difference between BGB324 treatment and the combination of Axl (BGB324) and PD-1 (Keytruda). Conclusion: Axl-inhibition represents a novel avenue to treat renal cell carcinoma. 2. Transcriptomic classifier of Low Risk Renal Cell Carcinoma Patients with subsequent Progressive Disease Introduction: We aimed to characterize the gene expression profile of low-risk patients both with and without progressive disease to define predictive markers. Methods: Formalin-fixed tissue blocks from ccRCC patients (n=24, eight progressors and 16 non-progressors) with a low Leibovich score were collected. Patients had a mean age of 65 years (5 females and 19 males). Total RNA was extracted (miRNeasy FFPE Kit, Qiagen) and sequenced (TruSeq RNA Access Library Kit, Illumina). Results: 1167 differentially expressed genes were detected. Progressors overexpressed genes related to cancer, B-cell infiltration and other immune-system related pathways. Principal component analyses and hierarchical clustering depicted a systematic transcriptomic difference between progressors and non-progressors. The AGAP2-AS1 mRNA classified 23 out of 24 samples correctly. The correlation between sample status as either progressor or non-progressor and AGAP2-AS1 level was R2 =0.69, p<0.01. Patients were split into groups based on AGAP2-AS1 expression (cut-off log2cpm > 1), where higher expression correlated with shorter survival. Immunohistochemistry of AGAP2, USP10 and KI-67 confirmed mRNA results. Conclusion: RNA-seq results show a transcriptomic difference between low-risk ccRCC progressors and low-risk non-progressors. AGAP2-AS1 may serve as a potential classifier fro identify low-risk progressors.

2018

Targeting Epithelial-to-Mesenchymal Transition to Treat Advanced Renal Cell Carcinoma: From Mice to Men!We have focused i) on the role of miRNA in clear cell renal cell carcinoma with respect to diagnosis, prognosis and therapy, and ii) on the use of fine needle aspirates to perform next generation sequencing of mRNA and to compare results with core biopsies. We are also establishing an orthotopic renal cancer model in mice for drug interventions.1. miRNA IN 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. 2. NEXT GENERATION SEQUENCING OF mRNA FROM FINE NEEDLE ASPIRATES Transcriptome analysis is emerging as emerging as a promising tool to enhance precision of diagnosis and monitoring in cancer and in solid organ transplantation. This proof-of-principle study examined whether fine needle aspirates, being less invasive, permit the ascertainment of the identical molecular information as core biopsies. We collected fine needles aspirates from various needle sizes (G19, 21, 23, 25) and the corresponding core biopsies (G16 needle) of non-tumor tissue of full nephrectomy specimens from patients suffering from clear cell renal cell carcinoma (ccRCC; n = 11). RNA expression patterns of two gene sets (156 genes) were executed using targeted RNA sequencing in samples from fine needle vs. core needle samples. A subgroup of kidneys (n = 6) also underwent whole transcriptome RNA sequencing from core biopsies of tumor (ccRCC) and peri-tumoral normal tissue (Tru Seq RNA Access, Illumina). Samples from all needle sizes except two G25 aspirates yielded RNA potentially suitable for sequencing of both gene sets. The mRNA expression patterns of the two gene sets were highly correlated between fine needle aspirates (G23) and corresponding (G16) core biopsies (r = 0.985 and 0.982, respectively). This close correlation was further documented by heat map, Principal Component Analyses (PCA) and whole transcription RNA sequencing. The similarity between fine needle aspirates and core needle biopsies was additionally confirmed in the subgroup with complete RNA sequencing. In conclusion, fine needle biopsies yield similar genomic information to core needle biopsies. The less invasive nature of fine needle biopsies may therefore permit more frequent molecular monitoring and a more targeted use of core needle biopsies in native kidneys (e.g. of multimorbid patients with suspicious lesions) and in transplanted kidneys.

2017

Advances in diagnosis, pathophysiology and therapy of kidney cancerWe have made progress regarding the detection of diagnostic markers and of pathophysiological processes in human kidney cancer focussing on clear cell renal cell carcinoma. This is reflected by two manuscripts submitted to publication. Furthermore we have started to build up an experimental mouse model of kidney cancer using human cancer cells.We are working both on human and experimental clear cell renal cell carcinoma. Thus, we present the results separately. Note, Jessica Furriol, PhD, has been elected as the technician on the project. She started to work in our group on 1st July 2017. 1. Human Kidney Carcinoma Late in the year 2017 we have submitted two articles for publication describing the use of miRNA in the diagnosis and prognosis of kidney cancer and the use of fine needle aspirates for RNA sequencing. The first article (in revision): Core biopsies (n=24) of both cancerous and adjacent normal tissues were obtained from patients undergoing nephrectomy. After full sequencing, 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 predicted poor survival (hazard ratios of 2.58 and 1.81), classified all ccRCC samples correctly, and correlated well with tumour size (R2=0.83). Therefore, formalin-fixed paraffin-embedded biopsy tissues are a viable source of miRNA sequencing material. miRNA-155-5p represents a promising cancer classifier, prognostic marker and therapeutic target in ccRCC that merits further investigation. The second article (submitted): Molecular monitoring in kidney diseases predominantly relies on core needle biopsies. This proof of principle study tests whether the less invasive fine needle aspirates provide the same molecular information. Fine needles aspirates (using G19, 21, 23, 25 needles) and corresponding regular core biopsies (using a G16 needle) were obtained from non-tumor tissues of radical nephrectomy specimens of patients with clear cell renal cell carcinoma (n=11). Targeted RNA sequencing was performed for two gene sets (Illumina), such as cell cycle panel, n=63 genes, and Wnt panel, n=93 genes. Expression patterns in fine needle (G23) vs. core needle (G16) samples were compared. A subset of six subjects also underwent whole transcriptome RNA sequencing. The mRNA abundances of the two predefined gene sets correlated very well between fine needle aspirates and corresponding core biopsies. Pearson correlation coefficients (r) were 0.985 for the cell cycle panel and 0.982 for the Wnt panel. This close correlation was supported by overlap in the heat map and Principal Component Analyses (PCA). The internal comparator group with complete RNA sequencing confirmed similarity between fine and core needle biopsies. In conclusion, minimally invasive fine needle biopsies provide similar genomic information to core needle biopsies and hence might allow a more targeted use of core needle biopsies and potentially provide sufficient information for molecular diagnostics in kidney diseases, such as cancer but also renal transplantation. 2. Experimental Kidney Carcinoma In the second half of 2017, Jessica Furriol, PhD, focussed on cultivating two different cell lines of human renal cell carcinoma. After successful cultivation, aliquots of these cells were injected subcutaneously into mice. Thereby, we have used two different strains of mice for this first evaluation (n=6, per group). At the time time, we evaluate ultrasound in addition to MRI imaging to measure subcutaneous tumor growth. These studies are still in progress.

Beskriv brukermedvirkningen i prosjektet

The patient organizations, Nyrekreftforeningen* from the Norwegian Cancer Society and NOFUS** (Norsk forening for pasienter med urologiske sykdommer) will be present in the project steering committee when the subsequent clinical inteventional study can be planned towards the final stages of the project: i) scientific representative, Dr. Andreas Stensvold* (UiO), and ii) a patient selected by Yngvild Ytrehus**. They will help to prepare the clinical phase 1b/2 trial (Work Package-3 of the proposal), e.g. by supporting patient recruitment, and to disseminate results among users. Treating physicians and general practitioners will be involved in the context of the clinical phase 1b/2 trial (Work Package-3 of the proposal).

Vitenskapelige artikler

Landolt L, Spagnoli GC, Hertig A, Brocheriou I, Marti HP

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

Nephrol Dial Transplant 2020 Dec 05. Epub 2020 des 5

PMID: 33280031

Koch E, Finne K, Eikrem Ø, Landolt L, Beisland C, Leh S, Delaleu N, Granly M, Vikse BE, Osman T, Scherer A, Marti HP

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

Am J Physiol Renal Physiol 2019 May 01;316(5):F1053-F1067. Epub 2019 mar 6

PMID: 30838877

Eikrem Ø, Walther TC, Flatberg A, Beisvag V, Strauss P, Farstad M, Beisland C, Koch E, Mueller TF, Marti HP

Fine needle aspirates of kidneys: a promising tool for RNA sequencing in native and transplanted kidneys.

BMC Nephrol 2018 Sep 05;19(1):221. Epub 2018 sep 5

PMID: 30185151

Strauss P, Marti HP, Beisland C, Scherer A, Lysne V, Leh S, Flatberg A, Koch E, Beisvag V, Landolt L, Skogstrand T, Eikrem Ø

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.

Int J Mol Sci 2018 Mar 10;19(3). Epub 2018 mar 10

PMID: 29534467

Eikrem Ø, Walther T.C., Flatberg A., BeisvagV., Strauss Ph., Farstad M., Beisland Ch., Koch E., Mueller T.F., and Marti H.P.

Fine Needle Aspirates of Kidneys are usable for RNAsequencing like Regular Core Biopsies

Submitted for publication

Strauss Ph., Marti HP., Beisland C., Scherer A., Leh S., Flatberg A., Koch E., Beisvag V., Landolt L., Skogstrand T., Eikrem O.

RNA sequencing of formalin-fixed renal carcinoma tissues identifies miR-155 as a predictor of poor survival

in revision

Deltagere

Leif Bostad Prosjektdeltaker
Sigrid Nakken Prosjektdeltaker
Tony Jialiang Chen Prosjektdeltaker
Tarig Al-Hadi Osman Prosjektdeltaker
Jessica Furriol Prosjektdeltaker
Janka Babickova Prosjektdeltaker
Lea Zoe Landolt Prosjektdeltaker
Piotr Mateusz Mydel Prosjektdeltaker
Philipp Strauss Ph.d.-kandidat
Christian Beisland Prosjektdeltaker
Hans-Peter Marti Prosjektleder
Jancka Babickova Prosjektdeltaker
Øystein Solberg Eikrem Ph.d.-kandidat

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