Analysis of paired primary-metastatic renal cancer lesions reveals RCN3-MMP10 axis facilitates lung metastasis.

POLE2 exhibits oncogenic properties. This study aimed to clarify its effects and underlying mechanisms in renal cell carcinoma (RCC). Using bioinformatics analyses, we predicted the relationship between POLE2 and autophagy, epithelial-mesenchymal transformation (EMT), and the AKT/mTOR pathway. The expression pattern of POLE2 was further verified in the clinical cohort comprising 94 tumor samples from patients with RCC. Following, we constructed in vivo and in vitro models to further investigate the potential mechanisms of POLE2 using a series of molecular biology approaches. The results showed that GINS1 was the downstream target of POLE2, and its overexpression reversed the inhibitory effects of POLE2 knockdown on RCC proliferation, metastasis, and EMT, while restoring the autophagy suppression. Furthermore, POLE2/GINS1 inhibited AKT/mTOR-mediated autophagy, thereby promoting EMT and lung metastasis of RCC. These findings provide a more comprehensive perspective on the genetic function of POLE2 in RCC progression.

Renal Cell Carcinoma: Diagnosis Based on Metastatic Manifestations

Renal cell carcinoma (RCC) is a major healthcare burden globally. Tumor-derived extracellular vesicles (EVs) contribute to the formation of a pro-metastatic microenvironment. In the present study, we explored the role and mechanism of RCC cell 786-O-derived EVs (786-O-EVs) in RCC. First, 786-O-EVs were extracted and identified, and EV internalization of RCC cells was observed. RCC cell malignant behaviors and long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression patterns were detected before and after 786-O-EV treatment. MALAT1 was intervened to evaluate RCC cell behaviors. The downstream mechanism involving MALAT1 was predicted. In addition, the relationship among MALAT1, transcription factor CP2 like 1 (TFCP2L1) and ETS proto-oncogene 1, transcription factor (ETS1) was analyzed. TFCP2L1 expression patterns were measured after 786-O-EV exposure. Tumor xenograft formation assay and lung metastasis model were adopted to verify the role of 786-O-EVs in vivo in RCC. It was found that 786-O-EVs could be internalized by RCC cells. 786-O-EVs promoted RCC cell malignant behaviors, accompanied by elevated MALAT1 expression levels. The 786-O-EVs with MALAT1 knockdown attenuated the promotive effect of sole 786-O-EVs on RCC cells. MALAT1 located ETS1 in the TFCP2L1 promoter and negatively regulated TFCP2L1, and ETS1 protein could specifically bind to MALAT1. 786-O-EVs enhanced the binding of ETS1 and the TFCP2L1 promoter and decreased TFCP2L1 expression. In vivo, 786-O-EVs promoted tumor growth and RCC lung metastasis, which was suppressed following inhibition of MALAT1. Our findings indicated that 786-O-EVs promoted RCC invasion and metastasis by transporting MALAT1 to promote the binding of transcription factor ETS1 and TFCP2L1 promoter.

Identification of Genomic Alterations Associated With Metastasis and Cancer Specific Survival in Clear Cell Renal Cell Carcinoma

Renal cell carcinoma (RCC) has the highest mortality rate among urological cancers and tumor angiogenesis that plays a critical role in RCC progress. Epidermal growth factor-like domain multiple 7 (EGFL7) has been recently identified as a regulator in RCC tumor angiogenesis and progression. Long noncoding RNA (LncRNA) HOTAIR has been considered as a pro-oncogene in multiple cancers, but its precise mechanism of tumor angiogenesis has rarely been reported. MicroRNA-126 (miR-126) functions as a tumor suppressor in RCC. However, the underlying tumor angiogenesis mechanism of HOTAIR/miR-126 axis in RCC has not been studied. The proliferation, migration, angiogenesis, and expression of EGFL7 and related proteins in extracellular signal-regulated kinase (ERK)/activators of transcription 3 (STAT3) signal pathway were determined to examine the effect and mechanism of HOTAIR and miR-126 on RCC progress. The regulatory relationship of HOTAIR and miR-126, as well as miR-126 and EGFL7 were tested using dual-luciferase reporter assay. Aenograft RCC mice model was used to examine the effect of HOTAIR on RCC tumor growth and metastasis in vivo. HOTAIR knockdown and miR-126 overexpression suppressed the proliferation, migration, and angiogenesis of RCC cells. HOTAIR regulated EGFL7 expression by competitively binding to miR-126. Knockdown of HOTAIR significantly suppressed the RCC tumor progression and lung metastasis in vivo. These findings suggest that lncRNA HOTAIR regulate RCC angiogenesis through miR-126/EGFL7 axis and provide a new perspective on the molecular pathways of angiogenesis in RCC development, which might be potential therapeutic targets for RCC treatment.

Background:Disabled homolog 2-interacting protein is a new member of the Ras GTPase superfamily involved in the regulation of cell proliferation, apoptosis, and metastasis. However, the expression of disabled homolog 2-interacting protein in renal cell carcinoma, its correlation with cancer prognosis, and tumor infiltrating lymphocytes remains unclear.Methods:The expression of disabled homolog 2-interacting protein was analyzed by UALCAN database, GEPIA database and the evaluation of disabled homolog 2-interacting protein effects on clinical prognosis. Prognostic factor analysis was used to identify the correlations between disabled homolog 2-interacting protein and cancer immune infiltration via the TIMER database. In addition, COXPRESdb database was used to analyze the enrichment of disabled homolog 2-interacting protein co-expression genes.Results:Compared to the normal tissues, the messenger RNA expression levels of DAB2IP are higher in 8 while lower in 15 types of tumor tissues. Furthermore, disabled homolog 2-interacting protein has high expression in kidney chromophobe and low expression in both kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. The messenger RNA expression levels of disabled homolog 2-interacting protein decrease gradually due to the increasing tumor staging which positively correlates with disease-free survival and overall survival in both kidney renal clear cell carcinoma and kidney renal papillary cell carcinoma. The expression levels of disabled homolog 2-interacting protein also positively correlate with the tumor purity of kidney chromophobe, kidney renal clear cell carcinoma, and kidney renal papillary cell carcinoma samples. Besides, the expression of disabled homolog 2-interacting protein in renal cell carcinoma has negative correlation with the immune infiltration, and the immune infiltration of B cells and CD8+ T cells affects the prognosis of kidney renal papillary cell carcinoma. Enrichment analysis of disabled homolog 2-interacting protein co-expressed genes suggested that its biological role was mainly in regulating GTPase activity.Conclusions:These findings suggest that disabled homolog 2-interacting protein functions as a tumor suppressor in the progression of renal cell carcinoma, and the expression of disabled homolog 2-interacting protein is related to the immune infiltrating cells and affects the survival of renal cell carcinoma. Disabled homolog 2-interacting protein can be a novel clinical biomarker for patients with renal cell carcinoma, which also provides new insights for the future treatments of renal cell carcinoma.

4662 Background: Pulmonary metastases of renal cell carcinoma (RCC) are associated with poor prognosis especially when the disease is rapidly progressing. The lung is a common site for metastases and respiratory failure is a common cause of death in patients with RCC. Inhalation therapy with IL-2 is thus an appealing method for palliation. This multi-center study summarizes the national experience with IL-2 inhalation in patients with lung metastases of RCC. Methods: All the patients had to have radiologically documented lung metastases, had to be able to comply with inhalation technique, and were not candidates for other treatment options. Treatment included 3 daily inhalations of 18 MU IL-2 by using a Salvia Lifetec Jetair inhalator. Treatment had to be continued until progression or complete response or life threatening toxicity or patient's refusal. Response was evaluated according to RECIST. Results: Forty patients (median 66.5 years), with histological or cytological diagnosis of RCC were enrolled. Nephrectomy was performed in a total of 32 patients. Previous treatments for metastatic disease included various combinations of systemic chemo-immunotherapy in 12 patients. Twenty-eight patients were systemic-treatment-naïve.The observed true response rate was 2.5% and disease stabilization rate was 55%. The disease-control rate (partial response plus disease stabilization rate) reached 57.5%. Time to progression ranged from 0.3 (in case of rapidly progressing disease) to more than 43 months, with a median of 8.7 months. The side effects included cough in 8 patients, weakness in 9, dyspnea in 3, fever in 2, sleepiness in 1, asthenia in 1, decreased appetite in 1, and abdominal pain in 1. Conclusions: Inhalation of IL-2 for treating pulmonary metastases of RCC is feasible, tolerable, and efficacious in controlling a progressive disease for considerable periods of time. Definition of response of biological therapy by RECIST system should be re-assessed and modified: stable disease should be regarded as a favorable and an important response. No significant financial relationships to disclose.

4662 Background: Pulmonary metastases of renal cell carcinoma (RCC) are associated with poor prognosis especially when the disease is rapidly progressing. The lung is a common site for metastases and respiratory failure is a common cause of death in patients with RCC. Inhalation therapy with IL-2 is thus an appealing method for palliation. This multi-center study summarizes the national experience with IL-2 inhalation in patients with lung metastases of RCC. Methods: All the patients had to have radiologically documented lung metastases, had to be able to comply with inhalation technique, and were not candidates for other treatment options. Treatment included 3 daily inhalations of 18 MU IL-2 by using a Salvia Lifetec Jetair inhalator. Treatment had to be continued until progression or complete response or life threatening toxicity or patient's refusal. Response was evaluated according to RECIST. Results: Forty patients (median 66.5 years), with histological or cytological diagnosis of RCC were enrolled. Nephrectomy was performed in a total of 32 patients. Previous treatments for metastatic disease included various combinations of systemic chemo-immunotherapy in 12 patients. Twenty-eight patients were systemic-treatment-naïve.The observed true response rate was 2.5% and disease stabilization rate was 55%. The disease-control rate (partial response plus disease stabilization rate) reached 57.5%. Time to progression ranged from 0.3 (in case of rapidly progressing disease) to more than 43 months, with a median of 8.7 months. The side effects included cough in 8 patients, weakness in 9, dyspnea in 3, fever in 2, sleepiness in 1, asthenia in 1, decreased appetite in 1, and abdominal pain in 1. Conclusions: Inhalation of IL-2 for treating pulmonary metastases of RCC is feasible, tolerable, and efficacious in controlling a progressive disease for considerable periods of time. Definition of response of biological therapy by RECIST system should be re-assessed and modified: stable disease should be regarded as a favorable and an important response. No significant financial relationships to disclose.

BackgroundTo determine safety and efficacy of radiofrequency ablation (RFA) for local treatment of lung metastases of renal cell carcinoma (RCC), sequenced or combined with systemic treatments.MethodsRetrospectively, we studied 53 patients treated by RFA for a maximum of six lung metastases of RCC. The endpoints were local efficacy, overall (OS), disease-free (DFS), pulmonary progression-free (PPFS) and systemic treatment-free (STFS) survivals, complications graded by the CTCAE classification and factors associated with survivals. Potential factors analysed were: clinical and pathological data, tumoral staging of TNM classification, primary tumor histology, Fuhrman’s grade, age, number and size of lung metastases and extra-pulmonary metastases pre-RFA.ResultsOne hundred metastases were treated by RFA. Median follow-up time was 61 months (interquartile range 90–34). Five-year OS was 62% (95% confidence interval (CI): 44–75). Median DFS was 9.9 months (95% CI: 6–16). PPFS at 1 and 3 years was 58.9% (95%CI: 44.1–70.9) and 35.2% (95%CI: 21.6–49.1), respectively. We observed 3% major complications (grade 3 and 4 of CTCAE classification). Local efficacy was 91%. Median STFS was 28.3 months. Thirteen patients (25%) with lung recurrence could be treated by another RFA. T3/T4 tumors had significantly worse OS, PPFS and STFS. Having two or more lung metastases increased the risk of pulmonary progression more than threefold.ConclusionIntegrated to systemic treatment strategy, RFA is safe and effective for the treatment strategy of lung metastasis from RCC with good OS and long systemic treatment-free survival. RFA offers the possibility of repeat procedures, with low morbidity.

At least one-third of renal cell carcinoma (RCC) patients are diagnosed with metastases and an additional 20 to 40% of patients develop metastases after radical nephrectomy with curative intent. Although the treatment of advanced RCC has recently evolved to the use of VEGF-targeted antiangiogenic therapies, there have been no significant improvements in the mortality rate of RCC, most likely because currently available therapies are relatively ineffective. A full understanding of the molecular pathogenesis involved in the progression of RCC is important for the development of innovative treatment options. Tumor Progression Locus 2 (Tpl2/MAP3K8), is a serine-threonine kinase with an important physiological role in tumor necrosis factor, interleukin-1, CD40, Toll-like receptor and G protein-coupled receptor-mediated extracellular signal-regulated kinases (ERK1/ERK2), c-Jun NH2-terminal kinases (JNK), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p38MAPK pathways. Previously identified as an oncogene, its overexpression is reported in many types of cancer and associated with metastatic potential. Inspection of transcriptome profiles registered in the oncomine database indicates that Tpl2 expression is differentially expressed in RCC compared to normal tissue. However, the relevance of Tpl2 in tumor growth and metastasis of RCC and the underlying mechanisms remain to be explored. To investigate a functional role of Tpl2 in the progression of RCC, the molecular effects of Tpl2 inhibition were examined in human RCC preclinical model using Caki-1 cells. In this study, we report a functional role for Tpl2 in RCC tumorigenesis and metastasis. Gene silencing with Tpl2 short hairpin RNA and treatment with a Tpl2 kinase inhibitor remarkably suppressed proliferation, focus formation potential, anoikis-resistance and cell-adhesion as well as in vitro cell migration/invasion capabilities in Caki-1 cells. Cell cycle assay showed that the anti-proliferation effect of Tpl2 shRNA was mediated by arresting cells in the G0/G1 phase. We found that interleukin 1β-induced phosphorylation of ERK or JNK was markedly suppressed by Tpl2 shRNA or Tpl2 kinase inhibitor. Furthermore, the orthotopically xenografted tumor growth of Caki-1 cells was significantly suppressed by Tpl2 knockdown with Tpl2 shRNA. An experimental metastasis assay also shows that Tpl2 inhibition suppressed the ability of Caki-1 cells to form lung metastases in a murine tail vein injection model. In clinical specimens of RCC, immunohistochemistry showed that Tpl2 expression levels in RCC specimens were significantly higher than those in normal renal tissues. These data suggest that Tpl2 may function as an oncogene linked to RCC and potential target for novel therapeutic modalities for advanced RCC. Citation Format: Hye Won Lee, Se Jeong Lee, Mi Young Song, Kyeung Min Joo, Byong Chang Jeong, Do-Hyun Nam. Tumor progression locus 2 (Tpl2) protein kinase contributes tumor growth and metastasis of renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C53.

14572 Background: RCC is often regarded as a radio-resistant tumor. However, brain metastases from RCC have been successful treated with SRS. Therefore, metastases to extra-cranial sites may be treated with similar success using stereotactic body radiation therapy (SBRT), where image-guidance allows for the delivery of precise high dose radiation in a few fractions. We report our experience with SRS/SBRT in the management of primary and metastatic RCC. Methods: The image-guided Novalis radiation therapy system was used. Thirty patients with brain metastases were treated with SRS (16–22 Gy in a single fraction). Five of these patients underwent resection of their metastatic lesions after SRS and their pathology were reviewed. Twenty patients with extra-cranial metastatic lesions (orbits, head and neck, lung, mediatinum, sternum, clavicle, scapula, humerus, rib, spine, abdomen) and 2 patients with biopsy proven primary RCC (not surgical candidates), were treated with SBRT (24–32 Gy in 3–4 fractions over 1–2 weeks). All patients were immobilized in body cast and image-guidance was used for all fractions. 4D-CT was utilized in the treatment planning to assess tumor motion. Results: Of the 30 patients who received SRS to brain metastases, 25 showed decreasing or stable lesion size. Five patients showed an increase in size and underwent resection. Their pathology revealed necrosis in >99% of the specimen, with no viable RCC. Nineteen patients who received SBRT to extra-cranial metastases achieved symptom relief. One patient had local progression, yielding a local control rate of 95%. In the 2 patients with primary RCC, tumor size remained unchanged but their pain improved, and their renal function was unchanged post SBRT. There was no significant treatment related side-effect. Conclusions: Precise high dose radiation can cause significant tumor cell death in “radio-resistant” metastases from RCC. It also offers excellent local control and symptom palliation, without significant toxicity. Therefore, SBRT may represent a novel non-invasive, nephron-sparing option for the treatment of primary RCC as well as extra-cranial metastatic RCC. A prospective clinical trial using SBRT for primary and metastatic RCC is on-going. No significant financial relationships to disclose.

BackgroundThe accumulating evidence confirms that long non-coding RNAs (lncRNAs) play a critical regulatory role in the progression of renal cell carcinoma (RCC). But, the application of lncRNAs in gene therapy remains scarce. Here, we investigated the efficacy of a delivery system by introducing the plasmid-encoding tumor suppressor lncRNA-SLERCC (SLERCC) in RCC cells.MethodsWe performed lncRNAs expression profiling in paired cancer and normal tissues through microarray and validated in our clinical data and TCGA dataset. The Plasmid-SLERCC@PDA@MUC12 nanoparticles (PSPM-NPs) were tested in vivo and in vitro, including cellular uptake, entry, CCK-8 assay, tumor growth inhibition, histological assessment, and safety evaluations. Furthermore, experiments with nude mice xenografts model were performed to evaluate the therapeutic effect of PSPM-NPs nanotherapeutic system specific to the SLERCC.ResultsWe found that the expression of SLERCC was downregulated in RCC tissues, and exogenous upregulation of SLERCC could suppress metastasis of RCC cells. Furthermore, high expression DNMT3A was recruited at the SLERCC promoter, which induced aberrant hypermethylation, eventually leading to downregulation of SLERCC expression in RCC. Mechanistically, SLERCC could directly bind to UPF1 and exert tumor-suppressive effects through the Wnt/β-catenin signaling pathway, thereby inhibiting progression and metastasis in RCC. Subsequently, the PSPM-NPs nanotherapeutic system can effectively inhibit the growth of RCC metastases in vivo.ConclusionsOur findings suggested that SLERCC is a promising therapeutic target and that plasmid-encapsulated nanomaterials targeting transmembrane metastasis markers may open a new avenue for the treatment in RCC.

Laser enucleation of a renal cell carcinoma (RCC) lung metastasis. This video demonstrates a metastasis enucleation using a laser fiber with a power of 60 W. The 64-year-old former heavy smoker male with a history of RCC 18 months prior, was admitted with undefined pulmonary nodules. The procedure started with a uniportal video-assisted thoracic surgery (VATS) wedge resection from segment

Targeting pulmonary metastases of renal cell carcinoma by inhalation of interleukin-2

ABSTRACTTissue transglutaminase (TG2) is the ubiquitously expressed member of transglutaminase family and shown to play a critical role in the development and progression of drug resistance malignancies. We have previously showed the association of TG2 upregulation with progression and metastasis of renal cell carcinoma (RCC) and low disease-free survival. In the present study we further investigate the role of TG2 in cell adhesion, migration and invasion of RCC by silencing TG2 expression in Caki-2 and A-498 primary site and Caki-1 and ACHN metastatic site RCC cell lines. Downregulation of TG2 expression led up to a 60% decrease in actin stress fiber formation and adhesion to β 1 integrin (ITGB1) substrates fibronectin, collagen type I and laminin in both primary and metastatic site RCC cell lines. In addition, treatment with siRNAs against TG2 impaired the migration capacity and cellular invasiveness of ITGB1 substrates in all 4 RCC cell lines. Lastly, the knockdown of TG2 in metastatic Caki-1 cells diminished the expression of CD44, CD73-and CD105 cancer stem cell-like markers. We conclude, for the first time, that TG2 expression is critical for cancer cell adhesion, migration, invasiveness and cancer cell-stemness during RCC progression and dissemination. Therefore, combined targeting of TG2 with drugs widely used in the treatment of RCC may be a promising therapeutic strategy for RCC.