Single-cell analysis reveals metastatic cell heterogeneity in clear cell renal cell carcinoma

Renal cell carcinoma (RCC) is one of the leading causes of cancer-related death worldwide. Tumour metastasis and heterogeneity lead to poor survival outcomes and drug resistance in patients with metastatic RCC (mRCC). In this study, we aimed to assess intratumoural heterogeneity (ITH) in mRCC cells by performing a combined analysis of bulk data and single-cell RNA-sequencing data, and develop novel biomarkers for prognosis prediction on the basis of the potential molecular mechanisms underlying tumorigenesis. Eligible single-cell cohorts related to mRCC were acquired using the Gene Expression Omnibus (GEO) dataset to identify potential mRCC subpopulations. We then performed gene set variation analysis to understand the differential function in primary RCC and mRCC samples. Subsequently, we applied weighted correlation network analysis to identify coexpressing gene modules that were related to the external trait of metastasis. Protein-protein interactions were used to screen hub subpopulation-difference (sub-dif) markers (ACTG1, IL6, CASP3, ACTB and RAP1B) that might be involved in the regulation of RCC metastasis and progression. Cox regression analysis revealed that ACTG1 was a protective factor (HR < 1), whereas the other four genes (IL6, CASP3, ACTB and RAP1B) were risk factors (HR > 1). Kaplan-Meier survival analysis suggested the potential prognostic value of these sub-dif markers. The expression of sub-dif markers in mRCC was further evaluated in clinical samples by immunohistochemistry (IHC). Additionally, the genetic features of sub-dif marker expression patterns, such as genetic variation profiles, correlations with tumour-infiltrating lymphocytes (TILs), and targeted signalling pathway activities, were assessed in bulk RNA-seq datasets. In conclusion, we established novel subpopulation markers as key prognostic factors affecting EMT-related signalling pathway activation in mRCC, which could facilitate the implementation of a treatment for mRCC patients.     

Resistance to sunitinib in renal clear cell carcinoma results from sequestration in lysosomes and inhibition of the autophagic flux

Metastatic renal cell carcinomas (mRCC) are highly vascularized tumors that are a paradigm for the treatment with antiangiogenesis drugs targeting the vascular endothelial growth factor (VEGF) pathway. The available drugs increase the time to progression but are not curative and the patients eventually relapse. In this study we have focused our attention on the molecular mechanisms leading to resistance to sunitinib, the first line treatment of mRCC. Because of the anarchic vascularization of tumors the core of mRCC tumors receives only suboptimal concentrations of the drug. To mimic this in vivo situation, which is encountered in a neoadjuvant setting, we exposed sunitinib-sensitive mRCC cells to concentrations of sunitinib below the concentration of the drug that gives 50% inhibition of cell proliferation (IC50). At these concentrations, sunitinib accumulated in lysosomes, which downregulated the activity of the lysosomal protease CTSB (cathepsin B) and led to incomplete autophagic flux. Amino acid deprivation initiates autophagy enhanced sunitinib resistance through the amplification of autolysosome formation. Sunitinib stimulated the expression of ABCB1 (ATP-binding cassette, sub-family B [MDR/TAP], member 1), which participates in the accumulation of the drug in autolysosomes and favor its cellular efflux. Inhibition of this transporter by elacridar or the permeabilization of lysosome membranes with Leu-Leu-O-methyl (LLOM) resensitized mRCC cells that were resistant to concentrations of sunitinib superior to the IC50. Proteasome inhibitors also induced the death of resistant cells suggesting that the ubiquitin-proteasome system compensates inhibition of autophagy to maintain a cellular homeostasis. Based on our results we propose a new therapeutic approach combining sunitinib with molecules that prevent lysosomal accumulation or inhibit the proteasome.     

Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

Vascular endothelial growth factor (VEGF) and mammalian target of rapamycin are well-known therapeutic targets for renal cell carcinoma (RCC). Sunitinib is an agent that targets VEGF receptors and is considered to be a standard treatment for metastatic or unresectable clear cell RCC (ccRCC). However, ccRCC eventually develops resistance to sunitinib in most cases, and the mechanisms underlying this resistance are not fully elucidated. In the present study, we established unique primary xenograft models, KURC1 (Kyoto University Renal Cancer 1) and KURC2, from freshly isolated ccRCC specimens. The KURC1 xenograft initially responded to sunitinib treatment, however finally acquired resistance. KURC2 retained sensitivity to sunitinib for over 6 months. Comparing gene expression profiles between the two xenograft models with different sensitivity to sunitinib, we identified interleukin 13 receptor alpha 2 (IL13RA2) as a candidate molecule associated with the acquired sunitinib-resistance in ccRCC. And patients with high IL13RA2 expression in immunohistochemistry in primary ccRCC tumor tends to have sunitinib-resistant metastatic site. Next, we showed that sunitinib-sensitive 786-O cells acquired resistance in vivo when IL13RA2 was overexpressed. Conversely, shRNA-mediated knockdown of IL13RA2 successfully overcame the sunitinib-resistance in Caki-1 cells. Histopathological analyses revealed that IL13RA2 repressed sunitinib-induced apoptosis without increasing tumor vasculature in vivo. To our knowledge, this is a novel mechanism of developing resistance to sunitinib in a certain population of ccRCC, and these results indicate that IL13RA2 could be one of potential target to overcome sunitinib resistance.     

The Role of Angiopoietins as Potential Therapeutic Targets in Renal Cell Carcinoma

Angiopoietin 2 (Ang2) is a secreted glycoprotein upregulated at sites of angiogenesis and has been implicated in cancer neovascularization. Recent studies have suggested efficacy of combined Ang and vascular endothelial growth factor receptor (VEGFR) inhibition for patients with metastatic renal cell carcinoma (mRCC). We measured Ang2 expression in human tissue and plasma, and tested the effect of dual Ang1/2 (trebananib; AMG386) or Ang2 alone (L1-7) inhibition with VEGFR inhibition on murine RCC growth and blood flow. Ang2 levels were higher in human tumors than normal tissues with RCC ranking highest for Ang2 expression across all tumor types tested. Plasma Ang2 was significantly higher in patients with mRCC compared to controls or patients with stage I disease. Plasma Ang2 decreased with sunitinib treatment and increased at time of disease progression. In the RCC mouse, dual Ang1/2 and Ang2 inhibition improved the activity of sunitinib. Combined Ang1/2 and VEGFR inhibition prevented the resumption of blood flow associated with sunitinib resistance. Thus, Ang2 inhibition, independent of Ang1 inhibition, improves the activity of sunitinib and plasma Ang2 increases in the setting of progression on sunitinib possibly contributing to resistance. Further, arterial spin-labeled perfusion magnetic resonance imaging might be a non-invasive marker of the antiangiogenic activity of Ang inhibitors.     

FKBP51 promotes invasion and migration by increasing the autophagic degradation of TIMP3 in clear cell renal cell carcinoma

The occurrence of metastasis is a serious risk for renal cell carcinoma (RCC) patients. In order to develop novel therapeutic approaches to control the progression of metastatic RCC, it is of urgent need to understand the molecular mechanisms underlying RCC metastasis and identify prognostic markers of metastatic risk. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been known to be closely associated with extracellular matrix (ECM) turnover, which plays a highly active role in tumor metastasis. Recent studies have shown that immunophilin FK-506-binding protein 51 (FKBP51) may be important for the regulation of ECM function, and exert effects on the invasion and migration of tumor cells. However, the mechanisms underlying these activities remain unclear. The present study detected the role of FKBP51 in clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC, and found that FKBP51 significantly promotes ccRCC invasion and migration by binding with the TIMP3, connecting TIMP3 with Beclin1 complex and increasing autophagic degradation of TIMP3. Given the important roles that TIMPs/MMPs play in ECM regulation and remodeling, our findings will provide new perspective for future investigation of the regulation of metastasis of kidney cancer and other types of cancer.Subject terms: Renal cell carcinoma, Extracellular matrix     

Association of Angiopoietin-2 and Ki-67 Expression with Vascular Density and Sunitinib Response in Metastatic Renal Cell Carcinoma

The Angiopoietin-2 (Ang2, Angpt2) growth factor is a context-dependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase and known to promote tumour angiogenesis and metastasis. Angiopoietin antagonists have been tested in clinical cancer trials in combination with VEGF-based anti-angiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. Here, we evaluated, using immunohistochemistry, the expression of Ang2, CD31 and the cell proliferation marker Ki-67 in the primary kidney cancer from 136 mRCC patients, who received first-line sunitinib after nephrectomy. Ang2 protein expression was restrained to RCC tumour vessels, and correlated with tumour vascularization and response to sunitinib. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS, 15.7 vs. 28.5 months, P = 0.015). In summary, in this study to investigate endothelial Ang2 in mRCC patients treated with first-line sunitinib, high cancer Ang2 expression was associated with the CBR, but not PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS.     

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.     

CircCYP24A1 hampered malignant phenotype of renal cancer carcinoma through modulating CMTM-4 expression via sponging miR-421

Renal cell carcinoma (RCC) is a lethal urinary malignancy. Circular RNAs (circRNAs) contribute to the malignant phenotype and progression of several types of human cancers, including RCC. In this study, we identified relatively low hsa_circ_0060927 (circCYP24A1) expression in RCC tissue through high-throughput sequencing and RT–qPCR. Fluorescence in situ hybridization (FISH) was used to validate the expression and subcellular localization of circCYP24A1 in RCC tissues. CCK-8, Transwell, EdU, and wound-healing assays indicated that circCYP24A1 overexpression inhibited the proliferation, invasion, and migration of RCC cells. Dual-luciferase reporter, RNA immunoprecipitation (RIP), FISH, and RNA-pulldown assays verified that circCYP24A1 inhibited RCC progression by sponging miR-421, thus inducing CMTM-4 expression. Xenograft assays and metastasis models further indicated that circCYP24A1 significantly inhibited the metastasis and proliferation of RCC cells in vivo. Taken together, circCYP24A1 is a prognosis-related circRNA in RCC that functions through the circCYP24A1/miR-421/CMTM-4 axis to modulate RCC progression.Subject terms: Renal cell carcinoma, Cancer metabolism     

p21-activated kinase 1 determines stem-like phenotype and sunitinib resistance via NF-κB/IL-6 activation in renal cell carcinoma

The p21-activated kinase 1 (PAK1), a serine/threonine kinase that orchestrates cytoskeletal remodeling and cell motility, has been shown to function as downstream node for various oncogenic signaling pathways to promote cell proliferation, regulate apoptosis and accelerate mitotic abnormalities, resulting in tumor formation and invasiveness. Although alterations in PAK1 expression and activity have been detected in various human malignancies, its potential biological and clinical significance in renal cell carcinoma (RCC) remains obscure. In this study, we found increased PAK1 and phosphorylated PAK1 levels in tumor tissues according to TNM stage progression. Elevated phosphorylated PAK1 levels associated with progressive features and indicated unfavorable overall survival (OS) as an independent adverse prognosticator for patients with RCC. Moreover, PAK1 kinase activation with constitutive active PAK1 mutant T423E promoted growth, colony formation, migration, invasion and stem-like phenotype of RCC cells, and vice versa, in PAK1 inhibition by PAK1 kinase inactivation with specific PAK1 shRNA, dead kinase PAK1 mutant K299R or allosteric inhibitor IPA3. Stem-like phenotype due to sunitinib administration via increased PAK1 kinase activation could be ameliorated by PAK1 shRNA, PAK1 mutant K299R and IPA3. Furthermore, nuclear factor-κB (NF-κB)/interleukin-6 (IL-6) activation was found to be responsible for PAK1-mediated stem-like phenotype following sunitinib treatment. Both IL-6 neutralizing antibody and IPA3 administration enhanced tumor growth inhibition effect of sunitinib treatment on RCC cells in vitro and in vivo. Our results unraveled that oncogenic activation of PAK1 defines an important mechanism for maintaining stem-like phenotype and sunitinib resistance through NF-κB/IL-6 activation in RCC, lending PAK1-mediated NF-κB/IL-6 activation considerable appeal as novel pharmacological therapeutic targets against sunitinib resistance.Arising from the renal tubular epithelial cells, renal cell carcinoma (RCC) accounts for ∼4% of all malignant diseases and 90% of renal malignancies in adults.^{1, 2} Although most RCCs are detected incidentally by the widespread use of abdominal imaging examinations for unrelated symptoms, ∼25–30% of patients are still diagnosed with metastatic disease.³ In addition, 20% of patients with localized RCC undergoing radical surgery experience relapse and develop metastatic RCC (mRCC) during follow-up.^{4, 5} Unfortunately, mRCC is refractory chemotherapy and radiotherapy with a 5-year survival rate of ^<10%.⁶ Immunotherapy with interleukin 2 (IL-2) and/or interferon-α (IFN-α) is the standard treatment for mRCC and has limited efficacy by substantial number of adverse effects.⁷ Despite the significant improvement in mRCC treatment with antiangiogenesis drugs such as sunitinib and sorafinib, its duration of therapeutic effect is often short.⁸ Clearly, this dire situation mandates better understanding of the molecular mechanism of RCC carcinogenesis so that novel targets could be identified for effective therapies.The p21-activated kinases (PAKs) are a family of conversed nonreceptor serine/threonine kinases that function as key regulators of pleiotropic physiological processes including cytoskeleton dynamics and cell polarity, motility, invasion and survival.⁹ Currently, 6 PAKs have been classified into group I PAKs (PAK1–3) and group II PAKs (PAK4–6) on the basis of structural and functional similarities.¹⁰ As the best-characterized member of the PAK family, PAK1 was identified as a protein that interacts with cell division cycle 42 (CDC42) and RAC1.¹¹ In addition to CDC42 and RAC1, other signaling including PI3K/Akt can also lead to the activation of PAK1.¹² PAK1 phosphorylation at threonine-423 (T423) by upstream signaling has been linked to its activation, as substitution of the acidic residue glutamic acid (E) at this site yields a constitutively active PAK1 T423E enzyme.¹³ Activation and localization of PAK1 lead to mediated physiological effects of downstream signaling via activating additional kinases and other effectors by phosphorylating them at specific serine and threonine residues or through protein–protein interaction.⁹PAK1 expression and activity are upregulated in different human tumors, such as breast, lung, colorectal, liver and kidney cancers,^{14, 15, 16} and are associated with tumor invasiveness, metastasis and poor prognosis. Besides, PAK1 is also a component of various signaling pathways, including mitogen-activated protein kinase (MAPK), JUN N-terminal Kinase (JNK) and nuclear factor-κB (NF-κB) pathways, all of which are believed to be important in carcinogenesis.⁹ Moreover, PAK1 has been found to play critical roles in anoikis resistance that facilitates metastasis by allowing tumor cells to survive following detachment from the matrix in original tissue and travelling to distant sites. Resistance to anoikis program represents a molecular basis for cancer progression and drug resistance.^{15, 17} The regulation of phosphorylation and function of Snail by PAK1 signaling kinase may contribute to the process of epithelial–mesenchymal transition (EMT) that plays a pivotal role in the conversion of early-stage tumors into invasive malignancies.¹⁸ EMT induction in cancer cells results in the acquisition of stem-like phenotype and drug resistance trait.^{19, 20}Based on these previous findings, we hypothesized that PAK1-mediated stem-like phenotype might induce sunitinib resistance and involve in RCC tumor progression. Our present study revealed that upregulation of PAK1 kinase activity conferred stem-like phenotype via NF-κB/IL-6 activation in vitro and in vivo that defines a novel potential mechanism underlying tumor metastasis and sunitinib resistance in RCC patients.     

Crosstalk between Epidermal Growth Factor Receptors (EGFR) and integrins in resistance to EGFR tyrosine kinase inhibitors (TKIs) in solid tumors

Cell adhesion to the extracellular matrix (ECM) is important in a variety of physiological and pathologic processes, including development, tumor invasion, and metastasis. Integrin-mediated attachment to ECM proteins has emerged to cue events primitively important for the transformed phenotype of human cancer cells. Cross-talk between integrins and growth factor receptors takes an increasingly prominent role in defining adhesion, motility, and cell growth. This functional interaction has expanded beyond to link integrins with resistance to Tyrosine kinase inhibitors (TKIs) of Epidermal Growth Factor Receptors (EGFRs). In this regard, integrin-mediated adhesion has two separate functions one as a clear collaborator with growth factor receptor signaling and the second as a basic mechanism contributing in Epithelial to Mesenchymal Transition (EMT) which affects response to chemotherapy. This review provides an overview of these mechanisms and describes treatment options for selectively targeting and disrupting integrin interaction to EGFR for cancer therapy.     

Recent advancements in the treatment of renal cell carcinoma--focus on international guidelines

Recent advances in understanding the fundamental biology underlying clear-cell RCC have opened the door to a series of targeted agents, such as tyrosine kinase inhibitors (TKIs) or mTOR inhibitors. These new agents have become the standard of care in managing advanced clear-cell RCC. Choice of initial medical management in patients with metastatic clear-cell RCC should be guided by randomised studies. On the evidence available, the first-line therapy in patients with good- or intermediate-risk mRCC should be either sunitinib or pazopanib, or bevacizumab plus interferon. In selected patients sorafenib is an option, as is high-dose interleukin-2 if performance status is good. In patients with poor prognosis, temsirolimus is recommended. In cytokine refractory patients, sorafenib, when patients have progressed on a tyrosine kinase inhibitor everolimus is the agent of choice. Biró K, Küronya Z. Recent advancements in the treatment of renal cell carcinoma - focus on international guidelines.     

Starvation and rapamycin differentially regulate host cell lysosome exocytosis and invasion by Trypanosoma cruzi metacyclic forms

The molecular mechanisms of host cell invasion by T. cruzi metacyclic trypomastigotes (MT), the developmental forms that initiate infection in the mammalian host, are only partially understood. Here we aimed at further identifying the target cell components involved in signalling cascades leading to MT internalization, and demonstrate for the first time the participation of mammalian target of rapamycin (mTOR). Treatment of human epithelial HeLa cells with mTOR inhibitor rapamycin reduced lysosomal exocytosis and MT invasion. Downregulation of phosphatidylinositol 3-kinase and protein kinase C also impaired exocytosis and MT internalization. The recombinant protein based on gp82, the MT surface molecule that mediates cell adhesion/invasion, induced exocytosis in HeLa cells. Such an effect has not previously been attributed to any T. cruzi surface molecule. Rapamycin treatment diminished gp82 binding as well. Cell invasion assays under conditions that promoted lysosome exocytosis, such as 1 h incubation in starvation medium PBS(++) , increased MT invasion, whereas pre-starvation of cells for 1-2 h had an opposite effect. In contrast to MT, invasion of tissue culture trypomastigotes (TCT) increased upon host cell pre-starvation or treatment with rapamycin, a novel finding that discloses quite distinctive features of the two infective forms in a key process for infection.     

Activation of G protein coupled estrogen receptor (GPER) promotes the migration of renal cell carcinoma via the PI3K/AKT/MMP-9 signals

Renal cell carcinoma (RCC) is the third most frequent malignancy within urological oncology. However, the mechanisms responsible for RCC metastasis are still needed further illustration. Our present study revealed that a seven-transmembrane receptor G-protein coupled estrogen receptor (GPER) was highly detected in various RCC cell lines such as ACHN, OS-RC-2 and SW839. The activation of GPER by its specific agonist G-1 significantly promoted the in vitro migration and invasion of ACHN and OS-RC-2 cells. G-1 also up regulated the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. The inhibitor of MMP-9 (Cat-444278), but not MMP-2 (Sc-204092), abolished G-1 induced cell migration, which suggested that MMP-9 is the key molecule mediating G-1 induced RCC progression. Further, G-1 treatment resulted in phosphorylation of AKT and ERK in RCC cells. PI3K/AKT inhibitor (LY294002), while not ERK inhibitor (PD98059), significantly abolished G-1 induced up regulation of MMP-9 in both AHCN and OS-RC-2 cells. Generally, our data revealed that activation of GPER by its specific agonist G-1 promoted the metastasis of RCC cells through PI3K/AKT/MMP-9 signals, which might be a promising new target for drug discovery of RCC patients.     

N-Cadherin Expression Is Associated with Acquisition of EMT Phenotype and with Enhanced Invasion in Erlotinib-Resistant Lung Cancer Cell Lines

Background

The epidermal growth-factor receptor tyrosine kinase inhibitors have been effective in non-small cell lung cancer patients. However, acquired resistance eventually develops in most patients despite an initial positive response. Emerging evidence suggests that there is a molecular connection between acquired resistance and the epithelial–mesenchymal transition (EMT). N-cadherin is involved in the EMT and in the metastasis of cancer cells. Here, we analyzed N-cadherin expression and function in erlotinib-resistant lung cancer cell lines.

Methods

H1650 cell lines were used to establish the subline resistant to erlotinib(H1650ER). Then, induction of the EMT was analyzed using immunostaining and western blots in H1650ER cells. N-cadherin expression in the resistant cells was examined using FACS and western blot. In addition, an invasion assay was performed to characterize the resistant cells. The effects of N-cadherin on cell proliferation and invasion were analyzed. The association of N-cadherin expression with the EMT phenotype was investigated using immunohistochemical analysis of 13 archived, lung adenocarcinoma tissues, before and after treatment with erlotinib.

Results

In H1650ER cells, N-cadherin expression was upregulated, paralleled by the reduced expression of E-cadherin. The marked histological change and the development of a spindle-like morphology suggest that H1650ER cells underwent an EMT, accompanied by a decrease in E-cadherin and an increase in vimentin. A change in the EMT status between pre-and post-treatment was observed in 11 out of 13 cases (79%). In biopsies of resistant cancers, N-cadherin expression was increased in 10 out of 13 cases. Induction of the EMT was consistent with aggressive characteristics. Inhibition of N-cadherin expression by siRNA was tested to reduce proliferation and invasion of H1650ER cells in vitro.

Conclusions

Our data provide evidence that induction of the EMT contributes to the acquired resistance to EGFR-TKIs in lung cancer. It suggests that N-cadherin is a potential molecular target in the treatment of NSCLC.     

UBE3C Promotes Growth and Metastasis of Renal Cell Carcinoma via Activating Wnt/β-Catenin Pathway

Renal cell carcinoma (RCC) is the most common primary malignancy of the kidney and one of the most lethal genitourinary malignancies. Clear-cell renal cell carcinoma (ccRCC) has an extremely poor prognosis because of a high potential for tumor growth, vascular invasion, metastasis and recurrence. Unfortunately, the mechanism of RCC growth and metastasis is not well understood. In this report, we for the first time demonstrated ubiquitin protein ligase E3C (UBE3C) as a driving factor for RCC growth and metastasis. UBE3C expression was increased in ccRCC tissues compared with adjacent normal tissues. ccRCC patients with high UBE3C protein expression in tumors were associated with significantly worse postoperative survival. Knockdown of UBE3C expression in ACHN cells inhibited cell proliferation, migrations and invasiveness in vitro while overexpression of UBE3C in 786-O cells exerted the opposite effects. UBE3C up-regulated β-catenin protein levels and promoted β-catenin nuclear accumulation, leading to the activation of the Wnt/β-catenin signal pathway in RCC cells. Collectively, these observations suggest that UBE3C plays an important role in RCC development and progression, and UBE3C may be a novel target for prevention and treatment of ccRCC.     

Specific Inhibition of the Nuclear Exporter Exportin-1 Attenuates Kidney Cancer Growth

Purpose

Despite the advent of FDA-approved therapeutics to a limited number of available targets (kinases and mTOR), PFS of kidney cancer (RCC) has been extended only one to two years due to the development of drug resistance. Here, we evaluate a novel therapeutic for RCC which targets the exportin-1 (XPO1) inhibitor.

Materials and Methods

RCC cells were treated with the orally available XPO1 inhibitor, KPT-330, and cell viability and Annexin V (apoptosis) assays, and cell cycle analyses were performed to evaluate the efficacy of KPT-330 in two RCC cell lines. Immunoblotting and immunofluorescence analysis were performed to validate mechanisms of XPO1 inhibition. The efficacy and on-target effects of KPT-330 were further analyzed in vivo in RCC xenograft mice, and KPT-330-resistant cells were established to evaluate potential mechanisms of KPT-330 resistance.

Results

KPT-330 attenuated RCC viability through growth inhibition and apoptosis induction both in vitro and in vivo, a process in which increased nuclear localization of p21 by XPO1 inhibition played a major role. In addition, KPT-330 resistant cells remained sensitive to the currently approved for RCC multi-kinase inhibitors (sunitinib, sorafenib) and mTOR inhibitors (everolimus, temsirolimus), suggesting that these targeted therapeutics would remain useful as second line therapeutics following KPT-330 treatment.

Conclusion

The orally-available XPO1 inhibitor, KPT-330, represents a novel target for RCC whose in vivo efficacy approaches that of sunitinib. In addition, cells resistant to KPT-330 retain their ability to respond to available RCC therapeutics suggesting a novel approach for treatment in KPT-330-naïve as well as -resistant RCC patients.     

The E-Syt3 cleavage and traffic uncovers the primordial cisterna,a new organelle that mothers the lipid droplets in the adipocyte

Extended synaptotagmins are endoplasmic reticulum proteins consisting of an SMP domain and multiple C2 domains that bind phospholipids and Ca²⁺. E-Syts create contact junctions between the ER and plasma membrane (PM) to facilitate the exchange of glycerophospholipids between the apposed membranes. We find in the differentiating adipocyte that the E-Syt3 carboxyl domain is cleaved by a multi-step mechanism that includes removing the C2C domain. Confocal and live-cell time-lapse studies show that truncated E-Syt3ΔC2C, as well as endogenous E-Syt3 and the coat protein PLIN1, target the LDs from an annular, single giant ER cisterna. Inhibition of the proteasome blocks the proteolytic cleavage of Esyt3 and E-Syt3ΔC2C and causes the E-Syt3ΔC2C retention in the giant cisterna. The Esyt3 and PLIN1 distributions and LDs biogenesis show that the primordial cisterna, as we call it, is the birth and nurturing site of LDs in the adipocyte. Isoproterenol-induced lipolysis results in loss of cytoplasmic LDs and reappearance of the primordial cisterna. Electron microscopy and 3D-electron tomography studies show that the primordial cisterna consists of a tightly packed network of varicose tubules with extensively blistered membranes. Rounds of homotypic fusions from nascent to mature LDs play a central role in LD growth. The knockdown of E-Syt3 inhibits LD biogenesis. The identification of the primordial cisterna, an organelle that substitutes the randomly scattered ER foci that mother the LDs in non-adipose cells, sets the stage for a better understanding of LD biogenesis in the adipocyte.     

转移性肾癌细胞因子疗法的研究进展

转移性肾癌(mRCC)对放疗、化疗均不敏感,虽然靶向治疗为转移性肾癌的治疗提供了新的治疗方案,但免疫疗法一直作为治疗转移性肾癌的基础疗法。在过去的20年中,研究者也一直在研究新的免疫疗法,研究方向趋向于研究各种细胞因子,其中最主要的有IFN-α和IL-2两种,二者可以明显提高患者的生存时间。但是转移性肾癌的细胞因子疗法仍需进一步优化,本文总结了使用细胞因子治疗转移性肾癌的Ⅲ期临床试验,以期为转移性肾癌细胞因子疗法的合理选择提供参考。