The role of AIMP3, a putative tumour suppressor gene, as a predictive biomarker of response to chemo/radiotherapy in vitro and in patients with muscle-invasive bladder cancer.

Bladder cancer is the second most common urological cancer after prostate cancer and is one of the leading causes of cancer mortality in most western countries. For organ-confined, muscle-invasive disease, the standard of care, in terms of definitive cure, remains radical surgery (cystectomy) with lymphadenectomy. However, survival rates remain poor following supposedly curative treatment. Radical radiotherapy and more recently, multimodality treatment incorporating chemo-radiotherapy, are alternatives which allow bladder preservation in those choosing not to undergo or are unsuitable for radical surgery. However, survival rates following radiotherapy are generally lower relative to radical cystectomy and multimodality treatments can only be offered to select cases in few institutions. Biomarkers which can accurately predict tumour response to radiotherapy or chemotherapy can aid the selection of patients who are likely to respond well to treatment options incorporating radiotherapy and/or chemotherapy, as alternatives to radical cystectomy, in the management of bladder cancer. Such a strategy would allow personalised cancer care with patients likely to benefit from treatments that they are likely to respond well to and concomitantly avoid complications arising from other treatments less likely to benefit them. This thesis investigated the novel tumour suppressor gene, AIMP3 which is involved in the DNA damage response (DDR) pathway following exposure to genotoxic insults such as irradiation and chemotherapy. The expression and cellular localisation of AIMP3 protein was characterised in a panel of bladder cancer cell lines. Expression of AIMP3 was altered by gene knockdown with siRNA transfection and survival outcomes assessed following irradiation and chemotherapy. The predictive value of AIMP3 expression in determining survival outcome of patients with muscle-invasive bladder cancer who had undergone radical radiotherapy, with or without carbogen supplementation, in the BCON trial, was assessed. Prognostic significance was evaluated by interrogating a control cohort of patients who had undergone radical cystectomy and had not had exposure to radiotherapy or either neoadjuvant or adjuvant chemotherapy. Reportedly important DDR proteins, including Mre11, p53 and ERCC1, were also interrogated in the BCON, Radical Cystectomy, Neodjuvant and LaMB trial TMA datasets. Clonogenic survival outcomes following AIMP3 knockdown were also investigated in cisplatin-sensitive (RT112) and cisplatin-resistant (RT112CP) cell lines following cisplatin exposure. Survival outcome, stratified for AIMP3 as well as ERCC1, Mre11 and p53 status, were interrogated in the Neoadjuvant set, which incorporated a cohort of patients who had undergone cisplatin-based neoadjuvant chemotherapy prior to radical treatment. This was validated in a second cohort of patients who had undergone cisplatin-based chemotherapy as part of the LaMB trial

Increased HSF1 expression predicts shorter disease-specific survival of prostate cancer patients following radical prostatectomy

Bjork, Johanna K Ahonen, Ilmari Mirtti, Tuomas Erickson, Andrew Rannikko, Antti Butzow, Anna Nordling, Stig Lundin, Johan Lundin, Mikael Sistonen, Lea Nees, Matthias Akerfelt, Malin eng Oncotarget. 2018 Jul 27;9(58):31200-31213. doi: 10.18632/oncotarget.25756. eCollection 2018 Jul 27.Prostate cancer is a highly heterogeneous disease and the clinical outcome is varying. While current prognostic tools are regarded insufficient, there is a critical need for markers that would aid prognostication and patient risk-stratification. Heat shock transcription factor 1 (HSF1) is crucial for cellular homeostasis, but also a driver of oncogenesis. The clinical relevance of HSF1 in prostate cancer is, however, unknown. Here, we identified HSF1 as a potential biomarker in mRNA expression datasets on prostate cancer. Clinical validation was performed on tissue microarrays from independent cohorts: one constructed from radical prostatectomies from 478 patients with long term follow-up, and another comprising of regionally advanced to distant metastatic samples. Associations with clinical variables and disease outcomes were investigated. Increased nuclear HSF1 expression correlated with disease advancement and aggressiveness and was, independently from established clinicopathological variables, predictive of both early initiation of secondary therapy and poor disease-specific survival. In a joint model with the clinical Cancer of the Prostate Risk Assessment post-Surgical (CAPRA-S) score, nuclear HSF1 remained a predictive factor of shortened disease-specific survival. The results suggest that nuclear HSF1 expression could serve as a novel prognostic marker for patient risk-stratification on disease progression and survival after radical prostatectomy.Peer reviewe

Loss of N-WASP drives early progression in an Apc model of intestinal tumourigenesis

N‐WASP (WASL) is a widely expressed cytoskeletal signalling and scaffold protein also implicated in regulation of Wnt signalling and homeostatic maintenance of skin epithelial architecture. N‐WASP mediates invasion of cancer cells in vitro and its depletion reduces invasion and metastatic dissemination of breast cancer. Given this role in cancer invasion and universal expression in the gastrointestinal tract, we explored a role for N‐WASP in the initiation and progression of colorectal cancer. While deletion of N‐wasp is not detectably harmful in the murine intestinal tract, numbers of Paneth cells increased, indicating potential changes in the stem cell niche and migration up the crypt‐villus axis was enhanced. Loss of N‐wasp promoted adenoma formation in an adenomatous polyposis coli (Apc) deletion model of intestinal tumourigenesis. Thus, we establish a tumour suppressive role of N‐WASP in early intestinal carcinogenesis despite its later pro‐invasive role in other cancers. Our study highlights that while the actin cytoskeletal machinery promotes invasion of cancer cells, it also maintains normal epithelial tissue function and thus may have tumour suppressive roles in pre‐neoplastic tissues

Role of Wnt5a in Prostate Cancer

Wnt5a is a non-canonical secreted glycoprotein of the Wnt family that plays important roles in organ development and tissue orientation. Previous studies have reported that Wnt5a was upregulated at both mRNA and protein levels in prostate cancer, but information regarding its role in predicting clinical outcome in patients after radical prostatectomy is limited. The aim of the present thesis is to define the role of Wnt5a protein expression in prostate cancer. We started by evaluating Wnt5a protein expression by immunohistochemistry in a large, well-defined and population-based cohort and found Wnt5a protein expression to be upregulated in prostate cancer cells compared to benign epithelium. Interestingly, it predicted a favorable outcome for patients after radical prostatectomy as patients with preserved overexpression of Wnt5a protein in tumor cells had longer biochemical recurrence free time compared to patients with low Wnt5a protein expression. We demonstrated that this effect may be explained by the ability of Wnt5a to impair invasion in prostate cancer cells as recombinant Wnt5a treatment decreased invasion in 22Rv1 and DU145 cells while Wnt5a knockdown resulted in increase in invasion in LNCaP and 22Rv1 cells. In the light of conflicting reports on the role of Wnt5a in prostate cancer outcome, we validated our findings in an external population-based cohort. Again, we showed that Wnt5a protein expression was predictive of recurrence after radical prostatectomy in patients with low-grade prostate cancer and this was further enhanced whenWnt5a was combined with prostate cancer tissue biomarkers of known predictive value. We also demonstrated a positive correlation between Wnt5a and ERG protein expressions and that high Wnt5a protein and presence of ERG expression predicted a more favorable outcome. Despite this we observed that rWnt5a treatment of VCaP cells significantly decreased their ERG protein expression. Therefore, the relation between Wnt5a and ERG clearly need further exploration to better understand their functional interplay. In conclusion, our study indicates a tumor suppressor function of Wnt5a protein in localized PCa and that it can be used as a predictive tissue biomarker. Further, we suggest a novel therapeutic approach for patients with localized PCa targeting Wnt5a signaling to impair progression of PCa in these patients by using a Wnt5a mimicking peptide (Foxy5)

POU2F1 activity regulates HOXD10 and HOXD11 promoting a proliferative and invasive phenotype in head and neck cancer

HOX genes are master regulators of organ morphogenesis and cell differentiation during embryonic development, and continue to be expressed throughout post-natal life. To test the hypothesis that HOX genes are dysregulated in head and neck squamous cell carcinoma (HNSCC) we defined their expression profile, and investigated the function, transcriptional regulation and clinical relevance of a subset of highly expressed HOXD genes. Two HOXD genes, D10 and D11, showed strikingly high levels in HNSCC cell lines, patient tumor samples and publicly available datasets. Knockdown of HOXD10 in HNSCC cells caused decreased proliferation and invasion, whereas knockdown of HOXD11 reduced only invasion. POU2F1 consensus sequences were identified in the 5’ DNA of HOXD10 and D11. Knockdown of POU2F1 significantly reduced expression of HOXD10 and D11 and inhibited HNSCC proliferation. Luciferase reporter constructs of the HOXD10 and D11 promoters confirmed that POU2F1 consensus binding sites are required for optimal promoter activity. Utilizing patient tumor samples a significant association was found between immunohistochemical staining of HOXD10 and both the overall and the disease-specific survival, adding further support that HOXD10 is dysregulated in head and neck cancer. Additional studies are now warranted to fully evaluate HOXD10 as a prognostic tool in head and neck cancers

Opposing prognostic relevance of junction plakoglobin in distinct prostate cancer patient subsets

Both oncogenic and tumor suppressor functions have been described for junction plakoglobin (JUP), also known as γ-catenin. To clarify the role of JUP in prostate cancer, JUP protein expression was immunohistochemically detected in a tissue microarray containing 11 267 individual prostatectomy specimens. Considering all patients, high JUP expression was associated with adverse tumor stage (P = 0.0002), high Gleason grade (P < 0.0001), and lymph node metastases (P = 0.011). These associations were driven mainly by the subset without TMPRSS2:ERG fusion, in which high JUP expression was an independent predictor of poor prognosis (multivariate analyses, P = 0.0054) and early biochemical recurrence (P = 0.0003). High JUP expression was further linked to strong androgen receptor expression (P < 0.0001), high cell proliferation, and PTEN and FOXP1 deletion (P < 0.0001). In the ERG-negative subset, high JUP expression was additionally linked to MAP3K7 (P = 0.0007) and CHD1 deletion (P = 0.0021). Contrasting the overall prognostic effect of JUP, low JUP expression indicated poor prognosis in the fraction of CHD1-deleted patients (P = 0.039). In this subset, the association of high JUP and high cell proliferation was specifically absent. In conclusion, the controversial biological roles of JUP are reflected by antagonistic prognostic effects in distinct prostate cancer patient subsets

DNA content based flow sorting combined with genomic high-resolution profiling in the context of the development of castration resistance in prostate cancer

Prostate cancer (PCa) is a common malignant disease in men. Due to increased screening efforts, PCa is often diagnosed in early stages and patients are therefore eligible for curative surgical approaches. Yet many patients, mostly due to age or extended disease, are treated primarily using androgen deprivation therapy (ADT). While this approach usually leads to disease stability or even reduction, unfortunately, this effect is usually not sustainable and tumors acquire resistance toward ADT commonly referred to as castration resistance. The resulting tumor progression usually leads to long term disease progression with increased morbidity and eventually the death of the patient. Among other factors, changes in the tumor genome may confer resistance towards ADT, similar to what has previously been demonstrated for therapy resistance in other malignancies. This thesis focuses on different translational approaches aimed at tracking down the phenotypic and genomic changes occurring in PCa upon ADT. In the first paper included in the thesis (“ERG rearrangement and protein expression in the progression to castration-resistant prostate cancer”), we explored one of the prototypic genomic alterations, namely the TMPRSS2-ERG gene fusion, and its role in the development of castration-resistant PCa. We used a tissue microarray (TMA) that comprised clinical samples of PCa prior to and after ADT using classical molecular analysis methods such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Interestingly we were able to identify a PCa subgroup that appeared to be associated with a partial shutdown of the androgen receptor (AR)-driven signaling axis. Further, the notion of tumor evolution and intra-tumor heterogeneity has spawned an increased interest in recent years. The advent of high resolution, high throughput genomic analysis methods have provided the necessary tools to decipher the clonal complexity of tumors. This led to an improved understanding of the clonal evolution of malignant diseases. While most computational approaches focus on the bulk of the tumor, we developed and refined a combined approach relying on prior flow sorting of cytogenetically distinct tumor subpopulations and subsequent molecular and computational analyses to obtain a different perspective on intra-tumor heterogeneity. The resulting findings are summarized in the second paper entitled “Delineation of human prostate cancer evolution identifies chromothripsis as a polyclonal event selecting for FKBP4 driven castration resistance”. To the best of our knowledge, this was the first paper describing tumor populations exhibiting features of chromothripsis, the catastrophic shattering, and the reconnection of chromosomes, that are related but distinct in the different subpopulations. This gains further clinical significance as in the process the tumor acquired a new amplification of a chaperone protein, namely FKBP4, which is involved in AR signaling and highly correlates with patients’ survival. Taken together, these findings, therefore, might open new doors in the development of prognostic and therapeutic tools

Hippo pathway effectors YAP1/TAZ induce an EWS–FLI1‐opposing gene signature and associate with disease progression in Ewing sarcoma

YAP1 and TAZ (WWTR1) oncoproteins are the final transducers of the Hippo tumor suppressor pathway. Deregulation of the pathway leads to YAP1/TAZ activation fostering tumorigenesis in multiple malignant tumor types, including sarcoma. However, oncogenic mutations within the core components of the Hippo pathway are uncommon. Ewing sarcoma (EwS), a pediatric cancer with low mutation rate, is characterized by a canonical fusion involving the gene EWSR1 and FLI1 as the most common partner. The fusion protein is a potent driver of oncogenesis, but secondary alterations are scarce, and little is known about other biological factors that determine the risk of relapse or progression. We have observed YAP1/TAZ expression and transcriptional activity in EwS cell lines. Analyses of 55 primary human EwS samples revealed that high YAP1/TAZ expression was associated with progression of the disease and predicted poorer outcome. We did not observe recurrent SNV or copy number gains/losses in Hippo pathway‐related loci. However, differential CpG methylation of the RASSF1 locus (a regulator of the Hippo pathway) was observed in EwS cell lines compared with mesenchymal stem cells, the putative cell of origin of EwS. Hypermethylation of RASSF1 correlated with the transcriptional silencing of the tumor suppressor isoform RASFF1A, and transcriptional activation of the pro‐tumorigenic isoform RASSF1C, which promotes YAP1/TAZ activation. Knockdown of YAP1/TAZ decreased proliferation and invasion abilities of EwS cells and revealed that YAP1/TAZ transcription activity is inversely correlated with the EWS–FLI1 transcriptional signature. This transcriptional antagonism could be explained partly by EWS–FLI1‐mediated transcriptional repression of TAZ. Thus, YAP1/TAZ may override the transcriptional program induced by the fusion protein, contributing to the phenotypic plasticity determined by dynamic fluctuation of the fusion protein, a recently proposed model for disease dissemination in EwS

Development of a prognostic model for Macrophage Activation Syndrome in Systemic Juvenile Idiopathic Arthritis

Introduction: Macrophage activation syndrome (MAS) is a potentially life-threatening complication of systemic juvenile idiopathic arthritis (SJIA) characterized by heterogeneous organ involvement and severity. Early identification of patients at high risk of complicated clinical course may improve outcome by helping initiate prompt, appropriate immunosuppressive and supportive treatments. Yet, despite recent progress in clarifying the underlying immunological mechanisms, factors driving organ damage and severe outcome are not entirely understood, nor has the prognostic value of routinely gathered clinical and laboratory factors been fully explored. Objectives: To develop a prognostic model for SJIA-MAS based on routinely available parameters at disease onset, accounting for patient heterogeneity, possible latent factors, non-linear relationships and confounders. Methods: We examined a retrospective multinational cohort of 362 patients diagnosed with SJIA-MAS. The relationships between demographic, laboratory features at MAS onset (such as hemoglobin, whole blood cells, platelets, ERS, CRP, AST, ALT, bilirubin, fibrinogen, d-dimer, ferritin and creatinine), therapeutic interventions and outcomes were analyzed. Outcomes of interest included a \u201csevere course\u201d (defined as ICU admission or death), occurring of organs failure and CSN dysfunction. To identify potential phenotypes related to clinical features and outcome, we explored laboratory parameter patterns at MAS onset through Latent class modeling, which detects multiple unobserved clusters in heterogeneous populations. A structural causal approach was then used for investigating causal pathways leading to severe outcomes. Directed acyclic graphs (DAGs) were employed to depict possible causal relationships between the candidate biomarkers, potential confounding variables, and the outcomes, and inform the choice of adjustment sets in multivariate regression models. We assessed the possible relationships between variables and outcomes by penalized likelihood logistic regression and identified optimal cut off points for prognostic factors using Multiple Adaptive Regression Splines (MARS) and Classification and Regression Trees (CART). To account for possible treatment confounders, the effect of cyclosporine and etoposide use on outcomes was estimated using augmented inverse probability weighting (IPW) with double robust methods. Finally, results from previous analyses were incorporated in a probabilistic framework through a Bayesian network (BN) model, which provides risk estimates for specific clinical scenarios and quantifies the amount of information contributed from the identified prognostic variables. Results: The latent class model revealed six clusters based on biomarkers at MAS onset, characterized by the following features: mild alterations of white blood cells, platelets, fibrinogen, d-dimer and ferritin values, considered the baseline type (cluster 1, n =115); hyperferritinemia with low organs involvement (cluster 2, n = 101); elevation of inflammatory markers (cluster 3, n =51); hepatobiliary involvement (cluster 4, n = 41); severe pancytopenia, liver and kidney failure with higher elevation of LDH, d-dimer, ferritin (cluster 5, n = 30); biliary and renal dysfunction (cluster 6, n = 24). Cluster 2 and 3 presented lower age and SJIA duration at MAS onset compared to other subgroups. Cluster membership was predictive of severe course (p&lt;0.001), CSN involvement (p&lt;0.001), Hemorrhagic complications (p &lt;0.001) and Heart failure (p&lt;0.001), with patients in cluster 5 showing the highest risk of severe course and heart failure, and increased occurrence of CNS and Hemorrhagic manifestations in both cluster 5 and 6. In multivariate regression models, parameters at onset associated with risk of severe course were creatinine (OR 1,6 [95% CI 1.13\u20132.3]; p = 0.008) and albumin levels (OR 0,65 [95% CI 0.44\u20130.98]; p = 0.044) Higher risk of CNS involvement was found for patients younger at MAS onset (OR 0,62 [95% CI 0.42\u20130.92]; p = 0.018). Na (OR 0.0,89 [95% CI 0.82\u20130.96]; p = 0.006) and creatinine values (OR 1.69 [95% CI 1.14\u20132.5]; p = 0.009) were identified as independent predictors of mortality. There was no evidence for an effect of etoposide (OR 1.03 [95% CI 0.91\u20131.12]) and cyclosporine (OR 1.04 [95% CI 0.92\u20131.19]) on severe course. BNs defined distinct groups with different probability of severe outcomes, achieving a c-index of 0.76 for mortality, 0.81 for severe course and 0.81 for CNS involvement. Adding the obtained latent clusters to the BN model increased the prediction accuracy for severe course up to a c-index of 0.83. Based on information theory metrics (mutual information) from the BN model, decision algorithms for each outcome and a web-based decision support tool for external users were implemented. Conclusions: We developed a probabilistic prognostic model of SJIA-MAS based on routinely available data. This stratification tool may facilitate informed decision-making about the clinical management of these patients. The probabilistic and information-theoretic approach offers a framework for further validation, expansion and integration of the model with emerging molecular biomarkers