Human Renal Cell Carcinoma Tumors Research Articles

Abstract 1127: Single cell network-based analyses reveal dose-dependent increase in myeloid suppressive cell sub-populations in response to radiation therapy

Abstract Background: More than half of all cancer patients undergo radiation therapy (RT) with curative or palliative intent. With the widespread use of immunotherapy to treat cancer, immune effects of radiation are an important consideration in design of sequential and combination therapies. While radiation has been found to deplete lymphocytes, less is known about its effects on the myeloid compartment, which includes both immune activating and suppressive populations. We hypothesized that a network-based analysis of single cell proteogenomic data generated from tumor-infiltrating immune cells can identify druggable proteins in pathways driving unwanted immunosuppressive changes with radiation, particularly among myeloid cells. Methods: Proteogenomic (CITE-Seq) analysis was conducted on CD45+ immune cells from unirradiated and irradiated orthotopic 4T1 murine mammary tumors three- and ten-days after tumor irradiation (8 Gy x 1 or 8 Gy x 3). Based on expression of select surface proteins, analysis of the CITE-Seq data resulted in eight distinct immune clusters, including three myeloid clusters — monocytes, macrophages, and granulocytes. Using VIPER (virtual inference of protein activity by enriched regulon), an algorithm that infers protein activities from weighted expression on each protein’s downstream targets, we identified distinct functional subpopulations within these eight clusters. Further profiling subclusters enriched by RT using the OncoTarget algorithm, we identified druggable proteins with significant inferred activity as potential targets of therapeutic interest. Results: As expected, lymphocytes were persistently depleted after RT on both radiotherapy schedules. However, three distinct subclusters of myeloid cells with immune-suppressive phenotype became enriched following tumor irradiation, with greater enrichment at the lower radiation dose of 8 Gy x 1: TAM2, PMN1, and PMN4. Surface protein expression enabled us to define the immunophenotype and isolate these three cell clusters from murine 4T1 tumors as well as human renal cell carcinoma tumors for further functional analyses. OncoTarget identified a number of druggable targets, including PARP1 for the PMN1 subcluster. Conclusion: Suppressive myeloid subpopulations induced by radiation were identified among the immune cells. Combining radiation therapy with inhibitors of these cell populations may provide therapeutic benefit over radiation therapy alone or in combination with checkpoint inhibitor immunotherapies. Future work will experimentally determine if inhibition of druggable protein targets in these myeloid subpopulations improves the overall efficacy of RT. Citation Format: Aparna Krishnan, Shruti Bansal, Samanta Sarti, Michael D. Kissner, Charles Karan, Andrea Califano, Aleksandar Z. Obradovic, Catherine S. Spina. Single cell network-based analyses reveal dose-dependent increase in myeloid suppressive cell sub-populations in response to radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1127.

Abstract 3601: Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells

Abstract Patients with late-stage kidney cancer have poor long-term survival rates. Our lab isolated a low affinity CD8 dependent TCR named BZ-4 from CTLs of a patient who had immune-mediated regression of renal cell carcinoma (RCC) following an allogeneic stem cell transplant and found this TCR recognized a human endogenous retrovirus type E (HERV-E) derived 10-mer peptide, CT-RCC-1. The BZ-4 clone belongs to the Vβ7.1 family and binds to CT-RCC-1 in an HLA-A11 dependent manner. Adoptive infusion of CD8+ BZ-4 transduced T cells is currently being explored in an NIH phase I trial (NCT03354390). Because CD4+ T cells have been shown to augment the anti-tumor effects of TCR-modified CD8+ T cells, we hypothesized that CD4+ T cells genetically engineered to co-express BZ-4 and CD8 would recognize and kill RCC tumors expressing the CT-RCC-1 and would improve the in vivo proliferation and tumor cytotoxicity of CD8+ BZ-4 T cells. CD4+ T cells isolated from healthy donor PBMCs were transfected via Sleeping Beauty electroporation with the control MART-1 reactive TCR (DMF5), BZ-4, or BZ-4 alongside the CD8 co-receptor (BZ-4-8). CD8+ T cells were similarly transfected with DMF5 or BZ-4. Following magnetic bead enrichment, 66% to 84% (mean 77%) of transfected T cells stained positive for Vβ7.1. TCR engineered T cells were subsequently tested for their ability to lyse a) luciferase transduced HLA A-11 expressing RCC tumors and b) a T2 cell line modified to express both HLA A-11 and luciferase that was peptide-pulsed with decreasing concentrations (5 × 10−1, 5 × 10−2, 5 × 10−3 & 5 × 10−4 ug/mL) of CT-RCC-1. CD4+ T cells expressing BZ-4 or BZ-4-8 as well as CD8+ T cells expressing BZ-4 or DMF5 were used either alone or in combination in cytotoxicity assays. For combined T cell groups, the total effector/target ratio was maintained at the same ratio as individual T cell groups. For T-2 targets, CD4+ BZ-4 T cells exhibited no cytolytic activity in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which were highly cytolytic to target cells. For RCC tumor targets, CD4+ BZ-4 T cells exhibited no cytolytic activity at 48, 72 and 96 hours in contrast to both CD8+ BZ-4 T cells and CD4+ BZ-4-8 T cells, which exhibited high levels of tumor killing at these timepoints. Remarkably, CD4+ BZ-4-8 T cells alone or in combination with CD8+ BZ-4 T cells demonstrated tumor cytotoxicity that was comparable to that observed with CD8+ BZ-4 T cells alone. In conclusion, these data show that CD4+ BZ-4 T cells transfected with the CD8 co-receptor acquire the ability to lyse RCC tumors at levels comparable to that observed in CD8+ BZ-4 T cells. Additional experiments are planned to evaluate whether mixtures of CD8+ BZ-4 T cells with CD4+ BZ-4-8 T cells will augment the ability of TCR modified T cells to proliferate in vivo and kill human RCC tumors compared to CD8+ BZ-4 T cells alone in tumor bearing NSG mice. Citation Format: Muna Igboko, Long Chen, Stefan Barisic, Elena Cherkasova, David Allan, Mala Chakraborty, Joseph Clara, Angie Parrizzi, Kyra Carney, Rosa Nadal Rios, Robert Reger, Richard Childs. Improving killing of renal cell carcinoma through the combined effects of TCR engineered CD4+ and CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3601.

Tumoral CD105 promotes immunosuppression, metastasis, and angiogenesis in renal cell carcinoma.

CD105 (endoglin) is a transmembrane protein that functions as a TGF-beta coreceptor and is highly expressed on endothelial cells. Unsurprisingly, preclinical and clinical evidence strongly suggests that CD105 is an important contributor to tumor angiogenesis and tumor progression. Emerging evidence suggests that CD105 is also expressed by tumor cells themselves in certain cancers such as renal cell carcinoma (RCC). In human RCC tumor cells, CD105 expression is associated with stem cell-like properties and contributes to the malignant phenotype in vitro and in xenograft models. However, as a regulator of TGF-beta signaling, there is a striking lack of evidence for the role of tumor-expressed CD105 in the anti-tumor immune response and the tumor microenvironment. In this study, we report that tumor cell-expressed CD105 potentiates both the in vitro and in vivo tumorigenic potential of RCC in a syngeneic murine RCC tumor model. Importantly, we find that tumor cell-expressed CD105 sculpts the tumor microenvironment by enhancing the recruitment of immunosuppressive cell types and inhibiting the polyfunctionality of tumor-infiltrating CD4+ and CD8+ T cells. Finally, while CD105 expression by endothelial cells is a well-established contributor to tumor angiogenesis, we also find that tumor cell-expressed CD105 significantly contributes to tumor angiogenesis in RCC.

TFEB Mediates Immune Evasion and Resistance to mTOR Inhibition of Renal Cell Carcinoma via Induction of PD-L1.

Despite the FDA approval of mTOR inhibitors (mTORi) for the treatment of renal cell carcinoma (RCC), the benefits are relatively modest and the few responders usually develop resistance. We investigated whether the resistance to mTORi is due to upregulation of PD-L1 and the underlying molecular mechanism. The effects of transcription factor EB (TFEB) on RCC proliferation, apoptosis, and migration were evaluated. Correlation of TFEB with PD-L1 expression, as well as effects of mTOR inhibition on TFEB and PD-L1 expression, was assessed in human primary clear cell RCCs. The regulation of TFEB on PD-L1 was assessed by chromatin immunoprecipitation and luciferase reporter assay. The therapeutic efficacies of mTORi plus PD-L1 blockade were evaluated in a mouse model. The function of tumor-infiltrating CD8+ T cells was analyzed by flow cytometry. TFEB did not affect tumor cell proliferation, apoptosis, and migration. We found a positive correlation between TFEB and PD-L1 expression in RCC tumor tissues, primary tumor cells, and RCC cells. TFEB bound to PD-L1 promoter in RCCs and inhibition of mTOR led to enhanced TFEB nuclear translocation and PD-L1 expression. Simultaneous inhibition of mTOR and blockade of PD-L1 enhanced CD8+ cytolytic function and tumor suppression in a xenografted mouse model of RCC. These data revealed that TFEB mediates resistance to mTOR inhibition via induction of PD-L1 in human primary RCC tumors, RCC cells, and murine xenograft model. Our data provide a strong rationale to target mTOR and PD-L1 jointly as a novel immunotherapeutic approach for RCC treatment.

Targeting the vasopressin type-2 receptor for renal cell carcinoma therapy.

Arginine vasopressin (AVP) and its type-2 receptor (V2R) play an essential role in the regulation of salt and water homeostasis by the kidneys. V2R activation also stimulates proliferation of renal cell carcinoma (RCC) cell lines in vitro. The current studies investigated V2R expression and activity in human RCC tumors, and its role in RCC tumor growth. Examination of the cancer genome atlas (TCGA) database, and analysis of human RCC tumor tissue microarrays, cDNA arrays and tumor biopsy samples demonstrated V2R expression and activity in clear cell RCC (ccRCC). In vitro, V2R antagonists OPC31260 and Tolvaptan, or V2R gene silencing reduced wound closure and cell viability of 786-O and Caki-1 human ccRCC cell lines. Similarly in mouse xenograft models, Tolvaptan and OPC31260 decreased RCC tumor growth by reducing cell proliferation and angiogenesis, while increasing apoptosis. In contrast, the V2R agonist dDAVP significantly increased tumor growth. High intracellular cAMP levels and ERK1/2 activation were observed in human ccRCC tumors. In mouse tumors and Caki-1 cells, V2R agonists reduced cAMP and ERK1/2 activation, while dDAVP treatment had the reverse effect. V2R gene silencing in Caki-1 cells also reduced cAMP and ERK1/2 activation. These results provide novel evidence for a pathogenic role of V2R signaling in ccRCC, and suggest that inhibitors of the AVP-V2R pathway, including the FDA approved drug Tolvaptan, could be utilized as novel ccRCC therapeutics.

Mediators of Inflammation-Driven Expansion, Trafficking, and Function of Tumor-Infiltrating MDSCs.

Myeloid-derived suppressor cells (MDSC) are induced by and accumulate within many histologically distinct solid tumors, where they promote disease by secreting angiogenic and immunosuppressive molecules. Although IL1β can drive the generation, accumulation, and functional capacity of MDSCs, the specific IL1β-induced inflammatory mediators contributing to these activities remain incompletely defined. Here, we identified IL1β-induced molecules that expand, mobilize, and modulate the accumulation and angiogenic and immunosuppressive potencies of polymorphonuclear (PMN)-MDSCs. Unlike parental CT26 tumors, which recruited primarily monocytic (M)-MDSCs by constitutively expressing GM-CSF- and CCR2-directed chemokines, IL1β-transfected CT26 produced higher G-CSF, multiple CXC chemokines, and vascular adhesion molecules required for mediating infiltration of PMN-MDSCs with increased angiogenic and immunosuppressive properties. Conversely, CT26 tumors transfected with IL1β-inducible molecules could mobilize PMN-MDSCs, but because they lacked the ability to upregulate IL1β-inducible CXCR2-directed chemokines or vascular adhesion molecules, additional PMN-MDSCs could not infiltrate tumors. IL1β-expressing CT26 increased angiogenic and immunosuppressive factors of tumor-infiltrating MDSCs, as did CT26 tumors individually transfected with G-CSF, Bv8, CXCL1, or CXCL5, demonstrating that mediators downstream of IL1β could also modulate MDSC functional activity. Translational relevance was indicated by the finding that the same growth factors, cytokines, chemokines, and adhesion molecules responsible for the mobilization and recruitment of PMN-MDSCs into inflammatory CT26 murine tumors were also coordinately upregulated with increasing IL1β expression in human renal cell carcinoma tumors. These studies demonstrated that IL1β stimulated the components of a multifaceted inflammatory program that produces, mobilizes, chemoattracts, activates, and mediates the infiltration of PMN-MDSCs into inflammatory tumors to promote tumor progression.

Elevated histone H3 acetylation and loss of the Sp1–HDAC1 complex de-repress the GM2-synthase gene in renal cell carcinoma

GM2-synthase produces sialic acid-containing glycosphingolipids called gangliosides, and its mRNA overexpression and the gangliosides it generates are linked to tumor progression, migration, and suppression of tumor-specific host immune responses. However, the mechanism underlying GM2-synthase de-repression in renal cell carcinoma (RCC) is poorly understood. Here, we demonstrate that higher GM2-synthase mRNA expression levels in various cancer cells and in human RCC tumors correlate with higher histone acetylation levels (H3K9, H3K14, or both) at region +38/+187 relative to the transcription start site (TSS) of the GM2-synthase gene than in normal kidney epithelial (NKE) cells or healthy adjacent tissues. An increase in GM2-synthase mRNA expression in cells treated with a histone deacetylase (HDAC) inhibitor was accompanied by increased histone acetylation levels at this promoter region. DNA methylation around the TSS was absent in both RCC cell lines and NKE cells. Of note, both the transcription factor Sp1 and corepressor HDAC1 associated with the +38/+187 region when the GM2-synthase gene was repressed in NKE and tumor-adjacent tissues, indicating plausible site-specific repressive roles of HDAC1 and Sp1 in GM2-synthase mRNA expression. Site-directed mutagenesis of the Sp1-binding site within the +38/+187 region relieved repressed luciferase activity of this region by limiting HDAC1 recruitment. Moreover, Sp1 or HDAC1 knock down increased GM2-synthase transcription, and butyrate-mediated activation of GM2-synthase mRNA expression in SK-RC-45 cells was accompanied by Sp1 and HDAC1 loss from the +38/+187 region. Taken together, we have identified an epigenetic mechanism for the de-repression of the GM2-synthase gene in RCC.

Leukocyte‑associated immunoglobulin‑like receptor1 promotes tumorigenesis in RCC.

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults, responsible for approximately 90‑95% of cases. We previously reported a novel method that enables direct extraction of extracellular vesicles (EVs) from surgically resected viable tissues, yielding what we term tissue‑exudative extracellular vesicles (Te‑EVs). Quantitative LC/MS analysis identified 3,871 proteins in Te‑EVs, among which leukocyte‑associated immunoglobulin‑like receptor 1 (LAIR1) was highly enriched in tumor Te‑EVs. In the present study, we found that LAIR1 was significantly upregulated in clinical specimens of human RCC tumor tissues compared to that noted in adjacent non‑cancerous renal tissues as determined by quantitative PCR analysis. LAIR1 overexpression resulted in accelerated cell proliferation and tumor growth in RCC cells. Moreover, knockdown of LAIR1 using siRNA significantly inhibited cell proliferation in RCC cells. Mechanistically, LAIR1 upregulated the phosphorylation status of Akt, which in turn increased cell proliferation in RCC cells. In clinical RCC specimens, RCC patients with high LAIR1 mRNA expression showed poor progression‑free survival compared to those with low LAIR1 expression. These findings indicate that LAIR1 promotes tumorigenesis in RCC.

Abstract 94: Association of xCT overexpression with RTKI resistance and metastases in clear cell renal cell carcinoma

Abstract Background: Cystine/glutamate exchanger xCT is a catalytic component of system xc- involved in transport of ‘conditionally indispensable’ amino acid cystine. Cystine transport is a rate limiting step for the synthesis of glutathione, a major intracellular redox regulator. Recently, we and other groups reported the overexpression of xCT and its association with drug resistance in several human cancers including bladder, glioma, breast, and colon. There are no studies to show the xCT expression in clear cell renal cell carcinoma (ccRCC) and its association with tyrosine kinase inhibitors resistance and metastasis. In the current study we have evaluated xCT expression in human ccRCC tumors arranged in tissue microarray (TMA) and determined its role in receptor tyrosine kinase inhibitor (RTKI) resistance and metastases using the patient derived tumor xenografts (PDX) models and TKI resistance ccRCC cells. Methods: Human Renal cell carcinoma tumor nephrectomy specimens arranged in tissue microarray (TMA) were used to determine xCT expression by immunohistochemistry. Patient derived tumor xenografts (PDX) of primary, metastasis, and sunitinib resistance were used to determine the role of xCT in RCC. To understand the molecular alterations associated with RTKI resistance, we have generated sunitinib resistance 786 OR ccRCC cells and performed RNAseq analysis. To determine the xCT inhibition effect on ccRCC tumor metastases, sulfasalazine, an inhibitor of xCT was used to treat metastatic ccRCC tumor xenografts transplanted in SCID mice. Results: Immunohistochemical evaluation of xCT in RCC TMA revealed that 70 % (19 out of 27) of the tumors express different levels of xCT. Association with tumor response to RTKI will be presented. RTKI less responsive PDX RP-R-02 was developed using the dose escalation treatment strategy and was found to have an upregulation of xCT when the tumors become less responsive to sunitinib. RNAseq analysis revealed differential gene expression of various pathway genes including lysosome biogenesis and function such as SLC7A11, HPS4, HPS5, CTSB, IFI30, PPT1, SCPEP1, TPP1, ATP6AP1L, MCOLN1, PRKAG2, and VPS18 in sunitinib resistant 786 OR cells compared to parental cells supports the role of lysosomes function in RTKI drug resistance. Furthermore, xCT inhibitor sulfasalazine treatment significantly decreased the metastasis lung nodules of RP-R-02LM in SCID mice demonstrated the xCT role in RCC tumor metastasis. Conclusions: We found preliminary evidence that overexpression of xCT may be associated with RTKI resistance in ccRCC. These results suggest that targeting the xCT in ccRCC may reverse the resistance and enhance the efficacy of RTKI. Additional studies using larger numbers of ccRCC tumors are required to identify xCT as a potential predictive biomarker for response/resistance to RTKI in ccRCC patients. Citation Format: Sreenivasulu Chintala, Remi Adelaiye-Ogala, Ashley Orillion, Sreevani Arisa, May Elbanna, Nur P. Damayanti, Roberto Pili. Association of xCT overexpression with RTKI resistance and metastases in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 94. doi:10.1158/1538-7445.AM2017-94

The anticancer effects of supinoxin (RX-5902) in renal cell cancer.

524 Background: DEAD box RNA helicase DDX5/p68 may play several important roles in cancer. In particular, phosphorylated p68 has been shown to be associated with cell transformation, epithelial mesenchymal transition (EMT), and cell migration, deeming it a promising target for novel anti-cancer therapeutics. We have previously shown that Supinoxin (RX-5902) interacts with phosphorylated p68 on Tyr593, interfering with the phosphorylated p68-β-catenin signaling pathway. Methods: In this study, we first demonstrated anti-proliferative effects in ten renal cancer cell lines with a high level of sensitivity to Supinoxin (IC50 of 39 nM); TK-10 was the only resistant cell line in this study. We also sought to examine the tumor growth inhibition (TGI), tumor growth delay (TGD), and survival benefits of Supinoxin in the human renal cell carcinoma tumor (Caki-1) xenograft mouse model, using two different dosing schemas: weekly dosing at 20-160 mg/kg for 4 weeks, or 50-70 mg/kg daily (5 days on/2 days off) for 3 weeks. Results: Weekly dosing of Supinoxin at 160 mg/kg resulted in a 75% TGD (P < 0.001). Daily administration of Supinoxin resulted in dose-dependent TGI (80 and 96%; Day 21) and TGD (68 and 104%, P < 0.001), and extended the overall survival of the animals at both doses (P < 0.0001). At the dose of 70 mg/kg daily, 6/10 animals demonstrated partial tumor regressions and 1/10 a complete tumor regressions. Supinoxin did not result in a reduction in body weight gain, treatment related deaths, or clinical observations in either of the dosing schemas. Sunitinib (60 mg/kg; daily for 21 days) resulted in TGD for both in vivo studies validating the Caki-1 model herein. Conclusions: These data support the potential therapeutic activity of Supinoxin in renal cell cancers. A Phase I study of Supinoxin on relapse/refractory solid tumors is ongoing (NCT02003092).

Abstract 5056: PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells

Abstract Dysregulation of vitamin D signaling and metabolism enzymes is a frequent change in many cancers and may confound approaches to vitamin -D based therapies. The mechanisms by which aberrant vitamin D signaling and metabolism lead to resistance to vitamin D action are poorly understood. The kidney plays a critical role in vitamin D synthesis and metabolism; in renal cell carcinoma (RCC) cells vitamin D metabolism is dysregulated. Recently, we discovered that an oncogenic protein kinase PIM-1 is involved in vitamin D-induced expression of 24-hydroxylase, a key catalytic enzyme of vitamin D encoded by CYP24A1 gene, in bladder and prostate cancer. To study the impact of PIM1 on vitamin D signaling and vitamin D-mediated anti-tumor activity in RCC, we analyzed 4 human renal cell carcinoma cell lines (Caki-1, ACHN, A498 and 786-O) and 25 samples of human RCC tumor tissue and 13 benign kidney tissues (including 10 matched tumor and benign tissue samples) by qRT-PCR, immunohistochemistry (IHC), Western blot analysis, MTT and clonogenic assays. VDR mRNA and protein were detected in all RCC cell lines. IHC showed that VDR was strongly expressed in the normal kidney tissues, but decreased in RCC tissues. As expected, VDR expression was significantly increased by treatment with 1,25-dihydroxyvitamin D3 (1,25D3) in RCC cell lines. MTT and clonogenic assays showed that 1,25D3 inhibited RCC cell growth (p<0.05). We also noted a significant increase of CYP24A1 expression induced by vitamin D3 (p<0.0001); and effect which would be expected to promote the catabolism of 1,25D3. qRT-PCR and Western blot analysis demonstrated that PIM-1 is expressed in RCC cells. PIM-1 inhibition by siRNA or PIM-1 inhibitor JP_11646 significantly reduced 1,25D3-induced CYP24A1 expression (p<0.05). Therefore, we hypothesized that targeting PIM-1 kinase activity with a PIM-1 inhibitor may be an effective strategy to reduce 1,25D3 metabolism, thereby enhancing vitamin D-mediated anti-tumor activity. Inhibition of PIM1 kinase activity by JP_11646 reduced CYP24A1 expression at the transcriptional level in RCC cells. We further evaluated the efficacy of the PIM1 inhibitor alone or in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP_11646 reduced cell growth in RCC cell lines in a dose-dependent manner as measured by MTT and clonogenic assays. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in RCC cells (p<0.05). This study indicates that PIM-1 is involved in the regulation of vitamin D signaling in RCC. Inhibition of PIM kinase activity combined with vitamin D may be an attractive new therapeutic strategy for RCC. Citation Format: Wei Luo, Yingyu Ma, Carmen Baldino, Justin Caserta, Swathi Ramakrishnan, Pili Roberto, Candace Johnson, Donald Trump. PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2015-5056

MAPKs' status at early stages of renal carcinogenesis and tumors induced by ferric nitrilotriacetate.

Renal cell carcinoma (RCC) is asymptomatic at early stages, and thus, initial diagnosis frequently occurs at advanced or even metastatic stages, leading to a high rate of mortality. Ferric nitrilotriacetate (FeNTA)-induced RCC model is a useful tool to analyze molecular events at different stages of the carcinogenesis process in vivo. MAPKs' alterations seem to play an important role in the development and maintenance of human RCC tumors. Based on the above, p38α/β/γ, JNK1/2, and ERK1/2 statuses were studied at early stages of FeNTA-induced renal carcinogenesis (1 and 2 months of carcinogen treatment) as well as in tumor tissue. MAPKs showed distinct response along carcinogenesis process, either as total proteins and/or as their phosphorylated forms. While the increase in total and phospho-p38α/β levels became lower as carcinogenesis progressed, p38γ overexpression grew. Instead, total JNK2 diminished, but JNK1 was elevated at all studied times, and p-JNK1 levels increased at early stages, but not in tumors. In contrast, p-JNK2 rose at 2 months of treatment and in tumor tissue. Increased levels of p-ERK1/2 were observed at all stages analyzed. Very interestingly, at 1 and 2 months of FeNTA treatment, no alterations in MAPKs were found in liver or lung, where no primary tumors are induced with the scheme of FeNTA administration followed here. In conclusion, MAPKs' behavior evolved differentially as renal carcinogenesis advanced, even among isoforms of the same family, but it did not change in other tissues. All this strongly suggests a role of these kinases in FeNTA-induced RCC tumor development and maintenance.

Molecular Profiling Reveals a Tumor-Promoting Phenotype of Monocytes and Macrophages in Human Cancer Progression

Monocytes and macrophages are major components of the tumor microenvironment, but their contributions to human cancer are poorly understood. Weused molecular profiling combined with functional assays to investigate the role of these cells in human renal cell carcinoma (RCC). Blood monocytes fromRCC patients displayed a tumor-promoting transcriptional profile that supported functions like angiogenesis and invasion. Induction of this protumor phenotype required an interleukin-1 receptor (IL-1R)-dependent mechanism. Indeed, targeting of IL-1-IL-1R axis in a human RCC xenograft model abrogated the protumor phenotype of tumor-associated macrophages (TAMs) and reduced tumor growth invivo. Supporting this, meta-analysis of gene expression from human RCC tumors showed IL1B expression to correlate with myelomonocytic markers, protumor genes, and tumor staging. Analyzing RCC patient tumors confirmed the protumor phenotype of TAMs. These data provide direct evidence for a tumor-promoting role of monocytes and macrophages in human cancer and indicate IL-1-IL-1R as a possible therapeutic target.

Abstract 1217: Development of a spontaneous in vivo cachexia model using the Champions TumorGraft™ platform

Abstract Cachexia is present in the majority of patients with advanced cancer progression and is not reversible through nutritional supplementation, leading to loss of skeletal muscle and adipose tissue. There are a lack of quality pre-clinical models for cachexia research. Previously, Champions Oncology reported a mouse cachexia model derived from a primary human renal cell carcinoma tumor implanted in immunocompromised mice. This model, CTG-0804, was developed using the innovative Champions TumorGraft™ platform, which preserves the biological properties of the original human tumor. The Champions TumorGraft platform demonstrated dramatic weight loss in these mice as the tumor grew. Plasma from these mice also showed elevated levels of human IL-6. We have now further characterized this TumorGraft model by evaluating standard of care (SOC) agents for renal cell carcinoma and anti-IL-6 compounds to determine the mechanism of cachexia. The SOC evaluation revealed that sorafenib, sunitinib, temsirolimus and bevacizumab as single agents provided a survival advantage by inhibiting tumor growth and preventing weight loss in these mice. Blockade of human IL-6 with tocilizumab or ALD518 did not inhibit tumor growth and weight loss still occurred, demonstrating the possibility of other pathways involved in the cachexia mechanism. In addition, the SOC data for sunitinib showed significant tumor growth inhibition, but no regression at the study end point. This correlated with the clinical profile, where sunitinib could not prevent metastasis and the patient eventually progressed. The TumorGraft model was molecularly characterized and mutation analysis revealed a KIT V530I mutation. This mutation has been reported in extra-abdominal aggressive fibromatosis (desmoid tumor) and acute myeloid leukemia patients that have responded to c-kit targeting therapies, such as imatanib and dasatinib. Further characterization of this TumorGraft is ongoing to include additional bioinformatics using gene expression and mutation pathway analyses. In summary, we have continued development of this cachexia model using the Champions TumorGraft platform, along with molecular evaluation, to produce a robust and well-characterized approach for cachexia investigation which can also be applied to other areas of drug development and cancer research. Citation Format: Nathan Anderson, Tin Khor, Andrew Feldhaus, Andreya Gatling, Katie Olson, John Latham, David Sidransky, Elizabeth M. Bruckheimer. Development of a spontaneous in vivo cachexia model using the Champions TumorGraft™ platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1217. doi:10.1158/1538-7445.AM2014-1217

Abstract B206: Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers.

Abstract Protein kinases play key roles in defining the transformation of many solid tumors, including renal cell carcinoma (RCC), and have become attractive drug targets. The Ror-family receptor tyrosine kinases (RTKs) are transmembrane proteins with putative tyrosine kinase activities that play crucial roles during the development of various organs and tissues. One of these receptors, the RTK-like orphan receptor 2 (Ror2) is known as a developmentally regulated receptor that enhances tumor cell migration and tumor invasiveness. Recently, our lab reported on the expression of Ror2 in human RCC tumors and cell lines, and that its expression is correlated with invasive growth in culture. In mammals, Ror2 has been shown to act as a receptor or co-receptor for Wnt5a, a member of the Wnt family, inducing a noncanonical Wnt signaling cascade. We have found that Ror2 is expressed in various cell lines, including 786-O, HEK293, HeLa, SaOS2, and U2OS. Cell migration analysis using single cell tracking confirmed that Ror2 promotes cell migration, further enhanced by Wnt5a stimulation. Separately, we have shown that Ror2 expression correlates with enhanced canonical Wnt-signaling through an increased pool of downstream stable β-catenin in RCC and activation of canonical targets. However, the kinase activity of Ror2 has been controversial. Using 786-O Ror2 overexpressing cells (786-O/Ror2), we detected that Ror2 becomes phosphorylated upon Wnt5a treatment. Based on a report of antibody induced homodimerization of Ror2 necessary for stimulation, we treated 786-O/Ror2 cells with Ror2 antibody and verified a significantly enhanced phosphorylation. Based on these findings, we hypothesize that receptor dimerization via Wnt ligand engagement or antibody treatment is necessary for effective signal transduction. We have thus utilized the PathHunter Ror2 activity assay developed by DiscoveRX, to use blockade of dimerization as an assay for Ror2 targeted drug development. This system utilizes an EGFR/Ror2 chimera cell line that expresses the cytosolic portion of ROR2 containing the kinase domain tagged with a ProLink tag at the C-terminus and fused to the extracellular and transmembrane domains of EGFR. Receptor activation is mediated by EGF addition, which results in a dose-dependent increase in signal caused by complementation of the SH2 tagged with the complementary EA enzyme fragment binding to the phosphorylated receptor. Thus, activation reads out in dimerization and phosphorylation which in turn, results in enzyme fragment complementation in this assay. Our data using this EGFR/Ror2 chimera U2OS cell line show that stimulation of these cells with EGF induces phosphorylation to a great extent both by IP/western, and chimeric signal. We believe these tools are useful in screening compounds in search of Ror2 inhibitors for RCC or other cancer therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B206. Citation Format: Zufan Debebe, Melissa Porter, Emily E. Hull-Ryde, Neal Rasmussen, Adam Sendor, Jacqueline Norris-Drouin, Keefe Chan, James E. Bear, William P. Janzen, Kimryn Rathmell. Ror2 as a therapeutic target in renal cell carcinoma and other invasive cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B206.

Receptor Tyrosine Kinase-like Orphan Receptor 2 (Ror2) Expression Creates a Poised State of Wnt Signaling in Renal Cancer

Expression of the receptor tyrosine kinase-like orphan receptor 2 (Ror2) has been identified in an increasing array of tumor types and is known to play a role as an important mediator of Wnt signaling cascades. In this study, we aimed to clarify Ror2 interactions with the Wnt pathways within the context of renal cell carcinoma (RCC). An examination of Ror2 expression in primary human RCC tumors showed a significant correlation with several Wnt signaling genes, including the classical feedback target gene Axin2. We provide evidence that Ror2 expression results in a partially activated state for canonical Wnt signaling through an increased signaling pool of β-catenin, leading to an enhancement of downstream target genes following Wnt3a stimulation in both renal and renal carcinoma-derived cells. Additionally, inhibition of low-density lipoprotein receptor-related protein 6 (LRP6) with either siRNA or dickkopf decreased the response to Wnt3a stimulation, but no change was seen in the increased β-catenin pool associated with Ror2 expression, suggesting that LRP6 cofactor recruitment is necessary for a Wnt3a-induced signal but that it does not participate in the Ror2 effect on β-catenin signaling. These results highlight a new role for Ror2 in conveying a tonic signal to stabilize soluble β-catenin and create a poised state of enhanced responsiveness to Wnt3a exogenous signals in RCC.

Aberrant methylation and loss of CADM2 tumor suppressor expression is associated with human renal cell carcinoma tumor progression

Cell adhesion molecules (CADMs) comprise a protein family whose functions include maintenance of cell polarity and tumor suppression. In this report, we show that the CADM2 gene is repressed in human clear renal cell carcinoma by DNA promoter hypermethylation and/or loss of heterozygosity. Moreover, the loss of CADM2 expression is associated with a higher tumor pathology stage (p<0.05). The re-expression of CADM2 in the renal cancer cell line 786-O significantly suppressed tumor cell growth in vitro and in mouse xenografts by a G1 phase cell cycle arrest and the induction of apoptosis. Lentivirus-mediated CADM2 expression also significantly suppressed cancer cell anchorage-independent growth and invasion. Furthermore, the inhibition of endogenous CADM2 expression using siRNAs induced a tumorigenic phenotype in polarized non-tumorigenic MDCK cells. Thus, we conclude that CADM2 functions as a novel tumor suppressor and may serve as a potential therapeutic target for human renal cell carcinoma.

TNF-α Induces Epithelial–Mesenchymal Transition of Renal Cell Carcinoma Cells via a GSK3β-Dependent Mechanism

TNF-α is a cytokine with antitumorigenic property. In contrast, low dose, chronic TNF-α production by tumor cells or stromal cells may promote tumor growth and metastasis. Serum levels of TNF-α are significantly elevated in renal cell carcinoma (RCC) patients. Here, we showed that TNF-α induced epithelial-mesenchymal transition (EMT) and promoted tumorigenicity of RCC by repressing E-cadherin, upregulating vimentin, activating MMP9, and invasion activities. In addition, TNF-α treatment inhibited glycogen synthase kinase 3β (GSK-3β) activity through serine-9 phosphorylation mediated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway in RCC cells. Inhibition of PI3K/AKT by LY294002 reactivated GSK-3β and suppressed the TNF-α-induced EMT of RCC cells. Inactivation of GSK-3β by LiCl significantly increased MMP9 activity and EMT of RCC cells. Activation of GSK-3β by transduction of constitutively active GSK-3β into RCC cells suppressed TNF-α-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient GSK-3β, in contrast, potentiated EMT, anchorage-independent growth and drastically enhanced tumorigenicity in vivo. Most importantly, a 15-fold inactivation of GSK-3β activity, 3-fold decrease of E-cadherin, and 2-fold increase of vimentin were observed in human RCC tumor tissues. These results indicated that inactivation of GSK-3β plays a pivotal role in the TNF-α-mediated tumorigenesis of RCC.

WWOX Protein Expression Varies Among RCC Histotypes and Downregulation of WWOX Protein Correlates with Less-Favorable Prognosis in Clear RCC

WWOX has been shown to be a candidate tumor suppressor gene in numerous human cancers. The objective of this study is to determine the expression of WWOX in human renal cell carcinoma tumor cells and its possible correlation with clinical outcome. The WWOX protein expressions of human renal cell carcinoma (RCC) tumor and of matched normal renal parenchyma were examined, and its correlation with clinical cancer-specific survival was investigated. Downregulation of WWOX only in clear cell type RCC was demonstrated in our results including immunohistochemistry, Western blot, and RT-PCR assay. For the remnant cell types of RCC, sample sizes were insufficient to draw any conclusion of WWOX protein expression. The decreased expression of WWOX in clear cell RCC tumor compared with matched normal renal parenchyma was also correlated with clinical cancer-specific survival (Kaplan-Meier, p=0.0482). We proved that the expression level of WWOX is downregulated in human clear cell RCC. Moreover, the decreased expression of WWOX in clear cell RCC tumor compared with matched normal renal parenchyma was also correlated with clinical cancer-specific survival. Since clear cell RCC is a special human cancer using unique molecular pathogenesis, further investigation will provide more linking intracellular signaling of WWOX and novel therapeutic targets of human renal cell carcinoma.

Direct and Differential Suppression of Myeloid-Derived Suppressor Cell Subsets by Sunitinib Is Compartmentally Constrained

The antiangiogenic drug sunitinib is a receptor tyrosine kinase inhibitor with significant, yet not curative, therapeutic effects in metastatic renal cell carcinoma (RCC). Sunitinib is also an immunomodulator, potently reversing myeloid-derived suppressor cell (MDSC) accumulation and T-cell inhibition in the blood even of nonresponder RCC patients. We observed that sunitinib similarly prevented MDSC accumulation and restored normal T-cell function to the spleens of tumor-bearing mice, independent of the capacity of sunitinib to inhibit tumor progression (RENCA>CT26>4T1). Both monocytic and neutrophilic splenic MDSC were highly repressible by sunitinib. In contrast, MDSC within the microenvironment of 4T1 tumors or human RCC tumors proved highly resistant to sunitinib and ambient T-cell function remained suppressed. Proteomic analyses comparing tumor to peripheral compartments showed that granulocyte macrophage colony-stimulating factor (GM-CSF) predicted sunitinib resistance and recombinant GM-CSF conferred sunitinib resistance to MDSC in vivo and in vitro. MDSC conditioning with GM-CSF uniquely inhibited signal transducers and activators of transcription (STAT3) and promoted STAT5 activation. STAT5ab(null/null) MDSC were rendered sensitive to sunitinib in the presence of GM-CSF in vitro. We conclude that compartment-dependent GM-CSF exposure in resistant tumors may account for the regionalized effect of sunitinib upon host MDSC modulation and hypothesize that ancillary strategies to decrease such regionalized escape will enhance the potency of sunitinib as an immunomodulator and a cancer therapy.