Enhanced IMP3 Expression Activates NF-кB Pathway and Promotes Renal Cell Carcinoma Progression

Background

Insulin-like growth factor 2 mRNA binding protein 3 (IMP3) is expressed in metastatic and a subset of primary renal cell carcinoma (RCC). However, the role of IMP3 in RCC progression was poorly understood. We aim to uncover the mechanism of IMP3 in regulating clear cell RCC (CCRCC) progression and validate the prognostic significance of IMP3 in localized CCRCC.

Methods

Caki-1 cells stably overexpressing IMP3 and Achn cells with knockdown of IMP3 were analyzed for cell migration and invasion by Transwell assay. RNA-seq was used to profile gene expression in IMP3-expressing Caki-1 cells. A cohort of 469 localized CCRCC patients were examined for IMP3 expression by immunohistochemistry using tumor tissue array.

Results

IMP3 promoted Caki-1 cell migration and invasion, whereas knockdown of IMP3 by RNAi inhibited Achn cell migration and invasion. Enhanced IMP3 expression activated NF-кB pathway and through which, it functioned in promoting the RCC cell migration. IMP3 expression in localized CCRCC was found to be associated with higher nuclear grade, higher T stage, necrosis and sarcomatoid differentiation (p< 0.001). Enhanced IMP3 expression was correlated with shorter recurrence-free and overall survivals. Multivariable analysis validated IMP3 as an independent prognostic factor for localized CCRCC patients.

Conclusion

IMP3 promotes RCC cell migration and invasion by activation of NF-кB pathway. IMP3 is validated to be an independent prognostic marker for localized CCRCC.     

TGF-β1 Expression in Chromophobe Renal Cell Carcinoma and Renal Oncocytoma

Distinguishing renal oncocytoma (RO) from the eosinophilic variant of chromophobe renal cell carcinoma (ChRCC) under the light microscope is a common diagnostic problem. Our recent research has shown significant difference between the presence of tumor fibrous capsule in ChRCCs and ROs. Transforming growth factor beta 1 (TGF-β1) is a potent cytokine involved in regulating a number of cellular processes. Two main purposes of this research were to investigate whether the TGF-β1 staining could be related to the presence of tumor fibrous capsule and if it could be used in the differential diagnosis between ChRCC and RO. We investigated 34 cases: 16 ChRCCs (8 eosinophilic and 8 classic) and 18 ROs. All available slides of each tumor, routinely stained with hematoxylin and eosin (H&E) were first analyzed to note the presence of tumor fibrous capsule. One paraffin embedded tissue block matching the representative H&E slide was selected for the immunohistochemical analysis. TGF-β1 expression was analyzed semiquantitatively in the tumor tissue, the tumor fibrous capsule, if present and the peritumoral renal parenchyma. Intensity of TGF-β1 expression was weaker in ChRCCs than the one observed in ROs (P<0.05). The type of reaction in ChRCCs was predominantly membranous unlike in ROs, which exhibited a predominantly cytoplasmic reaction (P<0.05). Moreover, none of the ROs showed membranous type of reaction for TGF-β1. In the group of ChRCCs, tumors with capsule had statistically significant higher quantity of TGF-β1 expression in tumor tissue and in peritumoral renal parenchyma compared to the tumors without capsule (P<0.05). Our results showed different types of TGF-β1 expression in ChRCCs and ROs: ChRCCs had predominantly membranous type of reaction, and ROs predominantly cytoplasmic. Furthermore, ChRCCs with capsule had statistically significant higher quantity of TGF-β1 expression in tumor tissue and in peritumoral renal parenchyma compared to the tumors without capsule. Based on these findings we can speculate that it could be possible that TGF-β1 plays a role in the formation of fibrous capsule in ChRCCs.Key words: capsule, chromophobe renal cell carcinoma, renal oncocytoma, TGF-β1     

TGFβ Signaling in Myeloid Cells Regulates Mammary Carcinoma Cell Invasion through Fibroblast Interactions

Metastasis is the most devastating aspect of cancer, however we know very little about the mechanisms of local invasion, the earliest step of metastasis. During tumor growth CD11b⁺Gr1⁺ cells, known also as MDSCs, have been shown to promote tumor progression by a wide spectrum of effects that suppress the anti-tumor immune response. In addition to immunosuppression, CD11b⁺Gr1⁺ cells promote metastasis by mechanisms that are currently unknown. CD11b⁺Gr1⁺ cells localize near fibroblasts, which remodel the ECM and leave tracks for collective cell migration of carcinoma cells. In this study we discovered that CD11b⁺Gr1⁺ cells promote invasion of mammary carcinoma cells by increasing fibroblast migration. This effect was directed by secreted factors derived from CD11b⁺Gr1⁺ cells. We have identified several CD11b⁺Gr1⁺ cell secreted proteins that activate fibroblast migration, including CXCL11, CXCL15, FGF2, IGF-I, IL1Ra, Resistin, and Shh. The combination of CXCL11 and FGF2 had the strongest effect on fibroblast migration that is associated with Akt1 and ERK1/2 phosphorylation. Analysis of subsets of CD11b⁺Gr1⁺ cells identified that CD11b⁺Ly6C^highLy6G^low cells increase fibroblast migration more than other myeloid cell populations. Additionally, tumor-derived CD11b⁺Gr1⁺ cells promote fibroblast migration more than splenic CD11b⁺Gr1⁺ cells of tumor-bearing mice. While TGFβ signaling in fibroblasts does not regulate their migration toward CD11b⁺Gr1⁺ cells, however deletion of TGFβ receptor II on CD11b⁺Gr1⁺ cells downregulates CXCL11, Shh, IGF1 and FGF2 resulting in reduced fibroblast migration. These studies show that TGFβ signaling in CD11b⁺Gr1⁺ cells promotes fibroblast directed carcinoma invasion and suggests that perivascular CD11b⁺Ly6C^highLy6G^low cells may be the stimulus for localized invasion leading to metastasis.     

Does Bone-targeted Therapy Benefit Patients with Metastatic Renal Cell Carcinoma?

INTRODUCTION: In metastatic renal cell carcinoma (mRCC), the bone is the second most common site of metastasis and is associated with increased morbidity and poorer quality of life. Bone-targeted therapies (BTTs) such as denosumab and zoledronic acid may prevent skeletal-related events (SREs). However, the benefit of BTTs in combination with tyrosine kinase inhibitors (TKIs) remains unclear. METHODS: We performed a retrospective chart review at the Urologic Cancer Centre for Research and Innovation. Patients with mRCC were included if they had bone metastases treated with TKIs between 2010 and 2017. Our primary outcome was overall survival (OS), defined as the time elapsed from clinical diagnosis of mRCC to death, and modelled using the Kaplan–Meier method. Secondary outcomes included the median time to SRE and the analysis of prognostic factors of OS using Cox proportional hazards regression. RESULTS: In total, 230 patients with mRCC were identified; of which, 46 had bone metastases treated with TKIs and were included in the study (TKI-only, n = 37; TKI + BTT, n = 9). In the TKI + BTT cohort, patients received either denosumab (n = 5) or zoledronic acid (n = 4). At the time of analysis, 63% of patients were deceased. We observed an OS trend favouring the TKI + BTT cohort (13.8 months [95% confidence interval {CI}: 12.3–15.2] vs. 29.6 months [95% CI: 7.2–51.9], hazard ratio [HR]: 1.66 (95% CI: 0.62–4.45), P = 0.31). When patients in the TKI + BTT cohort were stratified by type of therapy (denosumab or zoledronic acid), the median time to SRE was similar between the groups (4.2 months [95% CI: 2.28–6.14] vs. 2.2 months [95% CI: not available], P = 0.71]. On univariate or multivariate analysis, it was found that age, gender, comorbidities, International metastatic RCC database consortium (IMDC) prognostic group and pathologic tumour grade were not significant predictors of worse OS. Pathologic stage 3 or 4 was an independent predictor of worse OS (HR: 5.8, 95% CI: 1.41–24.03, P = 0.015). CONCLUSION: BTTs may have a continued role in the era of targeted therapy and immunotherapy. Further prospective data are required to validate our findings.     

Estrogen Receptor-α Correlates with Higher Fungal Cell Number in Oral Paracoccidioidomycosis in Women

Background

Paracoccidioidomycosis is a neglected tropical fungal infection with great predilection for adult men, indicating the participation of female hormone estrogen in preventing paracoccidioidomycosis development in women. Estrogen has an immunologic effect leading to polarization toward the Th2 immune response, which favors the disease evolution.

Objectives

To evaluate estrogen and progesterone receptors in oral paracoccidioidomycosis lesions and to verify any association with tissue fungi counting in women and men.

Methods

Thirty-two cases of chronic oral paracoccidioidomycosis were included. Immunohistochemical analyses for anti-estrogen receptor-α, anti-progesterone receptor and anti-Paracoccidioides brasiliensis antibodies were performed. The differences between women and men and the relations among the immunomarkers for each gender were also evaluated.

Results

A significant positive correlation was observed between estrogen receptor-α and the amount of fungi in women. In addition, estrogen receptor-α was mildly expressed in the inflammatory cells of female patients, while progesterone receptor was expressed in both genders, with similar expression between women and men. Moreover, fungi counting revealed no differences between genders.

Conclusions

Estrogen receptor-α was expressed only in women and showed a positive correlation with the amount of fungi in oral paracoccidioidomycosis, while progesterone receptor was observed in both genders and exhibited no correlation with estrogen receptor-α or fungi counting.

     

Single Nucleotide Polymorphisms in the Wilms’ Tumour Gene 1 in Clear Cell Renal Cell Carcinoma

The Wilms’ tumour gene 1 (WT1) single nucleotide polymorphism (SNP) rs16754 has recently been described as an independent prognostic factor in acute myeloid leukaemia (AML) patients. It is of great interest to test whether WT1 SNPs can be used as a molecular marker in other cancer types in order to improve risk and treatment stratification. We performed sequencing analysis on all 10 exons of the WT1 gene in a total of 182 patients with clear cell renal cell carcinoma (ccRCC). Six different SNPs were identified, in descending order for minor allele frequency: rs2234582, rs16754, rs1799925, rs5030315, rs2234583, and rs2234581. At least one minor allele for WT1 SNP was identified in 61% of ccRCC patients. In the entire study population, only 6% carried two copies of the minor allele. The genotypes of WT1 SNPs in 78 tumour-free kidney tissue specimens were found to be in 95% concordance with corresponding tumour samples. No correlation was observed between WT1 SNP genotypes and RNA expression level. WT1 SNP genotypes did not associate with clinical and pathological characteristics. We found favourable outcomes associated with the homozygous minor allele for WT1 SNP. However, SNP genotypes did not show to be of prognostic significance when comparing wild-type versus homozygous or heterozygous for the minor allele in the entire cohort. None of the previously reported WT1 mutations in AML was found in the present study. A novel WT1 missense mutation was identified in only one patient. Our data suggest that common WT1 mutations are not involved in ccRCC. Due to too few cases harbouring the homozygous minor allele, the prognostic impact needs to be verified in larger study populations.     

Minor Type IV Collagen α5 Chain Promotes Cancer Progression through Discoidin Domain Receptor-1

Type IV collagens (Col IV), components of basement membrane, are essential in the maintenance of tissue integrity and proper function. Alteration of Col IV is related to developmental defects and diseases, including cancer. Col IV α chains form α1α1α2, α3α4α5 and α5α5α6 protomers that further form collagen networks. Despite knowledge on the functions of major Col IV (α1α1α2), little is known whether minor Col IV (α3α4α5 and α5α5α6) plays a role in cancer. It also remains to be elucidated whether major and minor Col IV are functionally redundant. We show that minor Col IV α5 chain is indispensable in cancer development by using α5(IV)-deficient mouse model. Ablation of α5(IV) significantly impeded the development of Kras^G12D-driven lung cancer without affecting major Col IV expression. Epithelial α5(IV) supports cancer cell proliferation, while endothelial α5(IV) is essential for efficient tumor angiogenesis. α5(IV), but not α1(IV), ablation impaired expression of non-integrin collagen receptor discoidin domain receptor-1 (DDR1) and downstream ERK activation in lung cancer cells and endothelial cells. Knockdown of DDR1 in lung cancer cells and endothelial cells phenocopied the cells deficient of α5(IV). Constitutively active DDR1 or MEK1 rescued the defects of α5(IV)-ablated cells. Thus, minor Col IV α5(IV) chain supports lung cancer progression via DDR1-mediated cancer cell autonomous and non-autonomous mechanisms. Minor Col IV can not be functionally compensated by abundant major Col IV.     

The Phototoxic Effect of Water-Soluble Porphyrins on Human Clear Cell Renal Cell Carcinoma Line Сaki-1

Biophysics - Two tetrapyridine porphyrins, cationic porphyrin P4 (TMPyP4) and its amphiphilic derivative porphyrin P1 with carboxyl groups, and their zinc-containing analogs ZnP4 and ZnP1 were...     

TGFβ Receptor I Conditional Knockout Mice Develop Spontaneous Squamous Cell Carcinoma

We generated a mouse model with a conditional deletion of TGF-β signaling in the neurons by crossing TGF-β ?receptor I (TβRI) floxed mice with neurofilament-H (NF-H) Cre mice. 35% of F1 conditional knockout (COKO) mice developed spontaneous squamous cell carcinomas (SCCs) in periorbital and/or perianal regions. Transplantation of these tumors into athymic nude mice resulted in 62% tumorigenicity. To determine whether evasion of the immune response plays any role in this tumorigenesis, we analyzed the expression levels of receptors for interleukin-13 (mIL-13R), a key negative regulator of tumor immunosurveillance, and found that 33% of COKO tumors expressed the IL-13Rα2 chain. Primary cultures of the SCCs expressing IL-13Rα2 were sensitive to the cytotoxic effect of IL-13R-directed cytotoxin treatment. This is the first demonstration that loss of TβRI can lead to spontaneous tumor formation. These mice can serve as a unique mouse model of SCC to evaluate the tumorigenicity and effect of anti-cancer therapeutics.     

Hepatitis B Virus X Protein Upregulates mTOR Signaling through IKKβ to Increase Cell Proliferation and VEGF Production in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), a major cause of cancer-related death in Southeast Asia, is frequently associated with hepatitis B virus (HBV) infection. HBV X protein (HBx), encoded by a viral non-structural gene, is a multifunctional regulator in HBV-associated tumor development. We investigated novel signaling pathways underlying HBx-induced liver tumorigenesis and found that the signaling pathway involving IκB kinase β (IKKβ), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1), was upregulated when HBx was overexpressed in hepatoma cells. HBx-induced S6K1 activation was reversed by IKKβ inhibitor Bay 11-7082 or silencing IKKβ expression using siRNA. HBx upregulated cell proliferation and vascular endothelial growth factor (VEGF) production, and these HBx-upregulated phenotypes were abolished by treatment with IKKβ inhibitor Bay 11-7082 or mTOR inhibitor rapamycin. The association of HBx-modulated IKKβ/mTOR/S6K1 signaling with liver tumorigenesis was verified in a HBx transgenic mouse model in which pIKKβ, pS6K1, and VEGF expression was found to be higher in cancerous than non-cancerous liver tissues. Furthermore, we also found that pIKKβ levels were strongly correlated with pTSC1 and pS6K1 levels in HBV-associated hepatoma tissue specimens taken from 95 patients, and that higher pIKKβ, pTSC1, and pS6K1 levels were correlated with a poor prognosis in these patients. Taken together, our findings demonstrate that HBx deregulates TSC1/mTOR signaling through IKKβ, which is crucially linked to HBV-associated HCC development.     

Molecular Characterization and Putative Pathogenic Pathways of Tuberous Sclerosis Complex–Associated Renal Cell Carcinoma

Tuberous sclerosis complex–associated renal cell carcinoma (TSC-RCC) has distinct clinical and histopathologic features and is considered a specific subtype of RCC. The genetic alterations of TSC1 or TSC2 are responsible for the development of TSC. In this study, we assessed the mTOR pathway activation and aimed to evaluate molecular characteristics and pathogenic pathways of TSC-RCC. Two cases of TSC-RCC, one from a 31-year-old female and the other from an 8-year-old male, were assessed. The mTOR pathway activation was determined by immunohistochemistry. The mutational spectrum of both TSC-RCCs was evaluated by whole exome sequencing (WES), and pathogenic pathways were analyzed. Differentially expressed genes were analyzed by NanoString Technologies nCounter platform. The mTOR pathway activation and the germline mutations of TSC2 were identified in both TSC-RCC cases. The WES revealed several cancer gene alterations. In Case 1, genetic alterations of CHD8, CRISPLD1, EPB41L4A, GNA11, NOTCH3, PBRM1, PTPRU, RGS12, SETBP1, SMARCA4, STMN1, and ZNRF3 were identified. In Case 2, genetic alterations of IWS1 and TSC2 were identified. Further, putative pathogenic pathways included chromatin remodeling, G protein–coupled receptor, Notch signaling, Wnt/β-catenin, PP2A and the microtubule dynamics pathway in Case 1, and mRNA processing and the PI3K/AKT/mTOR pathway in Case 2. Additionally, the ALK and CRLF2 mRNA expression was upregulated and CDH1, MAP3K1, RUNX1, SETBP1, and TSC1 mRNA expression was downregulated in both TSC-RCCs. We present mTOR pathway activation and molecular characteristics with pathogenic pathways in TSC-RCCs, which will advance our understanding of the pathogenesis of TSC-RCC.     

Association of Dyslipidemia with Renal Cell Carcinoma: A 1∶2 Matched Case-Control Study

Abnormal serum lipid profiles are associated with the risk of some cancers, but the direction and magnitude of the association with renal cell carcinoma is unclear. We explore the relationship between serum lipids and renal cell carcinoma via a matched case-control study. A 1∶2-matched case-control study design was applied, where one renal cell carcinoma patient was matched to two non-renal-cell-carcinoma residents with respect to age (±0 year) and gender. Cases (n = 248) were inpatients with a primary diagnosis of renal cell carcinoma, confirmed by pathology after operations. Controls were sampled from a community survey database matched on age and gender with cases, 2 controls for each case. Stratified Cox proportional hazard regression analysis was used to obtain hazard ratios and corresponding 95% confidence intervals of lipids level and dyslipidemia for the risk of renal cell carcinoma. Elevated serum cholesterol (p<0.001), LDL cholesterol (p<0.001), and HDL cholesterol (p = 0.003) are associated with decreased hazard of renal cell carcinoma, adjusting for obesity, smoke, hypertension and diabetes. However, risk caused by hTG showed no statistical significance (p = 0.263). This study indicates that abnormal lipid profile influences the risk of renal cell carcinoma.