Renal Cell Carcinoma Tumor Model Research Articles

ANGPTL4 suppresses clear cell renal cell carcinoma via inhibition of lysosomal acid lipase.

Renal cell carcinoma (RCC), the most common form of kidney cancer, is a heterogeneous disease with clear cell RCC (ccRCC) being the most prevalent and aggressive subtype. While most ccRCC tumors have elevated expression of angiopoietin-like 4 (ANGPTL4), in our study we identified a significant subset of patients whose cancers show no increase in ANGPTL4 expression. These patients have a worse prognosis compared to the patients with high expression of ANGPTL4. These ANGPTL4-low cancers are characterized by the increased frequency of wild-type Von Hippel-Lindau (wt VHL), a gene that is commonly mutated in ccRCC, and an enrichment for genes associated with lipid metabolism. Using RCC tumor models with wild type VHL, we demonstrate that ANGPTL4 behaves as a tumor suppressor. The loss of ANGPTL4 in ccRCC cell lines results in increased tumor growth and colony formation in a lysosomal acid lipase (LAL)-dependent manner, a phenotype rescued by the expression of N-terminus ANGPTL4. At the mechanistic level, the loss of ANGPTL4 increases lysosomal acid lipase activity in ccRCC cells. This data suggests that ANGPTL4 enacts its tumor-suppressive effects in ccRCC by regulating LAL activity. Importantly, the identified patient cohort with low ANGPTL4 expression may exhibit increased reliance on lipid metabolism, which can be a point of target for future therapy.

PERK Inhibition by HC-5404 Sensitizes Renal Cell Carcinoma Tumor Models to Antiangiogenic Tyrosine Kinase Inhibitors.

Tumors activate protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK, also called EIF2AK3) in response to hypoxia and nutrient deprivation as a stress-mitigation strategy. Here, we tested the hypothesis that inhibiting PERK with HC-5404 enhances the antitumor efficacy of standard-of-care VEGF receptor tyrosine kinase inhibitors (VEGFR-TKI). HC-5404 was characterized as a potent and selective PERK inhibitor, with favorable in vivo properties. Multiple renal cell carcinoma (RCC) tumor models were then cotreated with both HC-5404 and VEGFR-TKI in vivo, measuring tumor volume across time and evaluating tumor response by protein analysis and IHC. VEGFR-TKI including axitinib, cabozantinib, lenvatinib, and sunitinib induce PERK activation in 786-O RCC xenografts. Cotreatment with HC-5404 inhibited PERK in tumors and significantly increased antitumor effects of VEGFR-TKI across multiple RCC models, resulting in tumor stasis or regression. Analysis of tumor sections revealed that HC-5404 enhanced the antiangiogenic effects of axitinib and lenvatinib by inhibiting both new vasculature and mature tumor blood vessels. Xenografts that progress on axitinib monotherapy remain sensitive to the combination treatment, resulting in ∼20% tumor regression in the combination group. When tested across a panel of 18 RCC patient-derived xenograft (PDX) models, the combination induced greater antitumor effects relative to monotherapies. In this single animal study, nine out of 18 models responded with ≥50% tumor regression from baseline in the combination group. By disrupting an adaptive stress response evoked by VEGFR-TKI, HC-5404 presents a clinical opportunity to improve the antitumor effects of well-established standard-of-care therapies in RCC.

Abstract B026: Combined inhibition of mammalian target of rapamycin (mTOR) and survivin augments radiation therapy in renal cell carcinoma

Abstract Purpose: Although renal cell carcinoma (RCC) is relatively more resistant to radiation therapy (RT) compared to other cancer indications, recent advances in single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) made it possible to achieve better outcome and reduce treatment related toxicities. In addition, with studies demonstrating that certain therapeutic agents including chemotherapies can act as radiosensitizers, the idea of combining radiotherapy with those agents are being actively pursued. Among them, mammalian target of rapamycin (mTOR) inhibitor everolimus (E) and survivin inhibitor YM155 (Y) are of special interest since they target multiple RT-resistance mechanisms simultaneously. Additionally, YM155 has been shown to help in overcoming resistance to mTOR inhibitors in various cancer including RCC. Hence, the present study aims to evaluate the efficacy of a tumor-targeted liposomal formulation combining everolimus and YM155 (EY-L) in sensitizing RCC tumors towards radiation. Experimental Design: Previously we developed a tumor-targeted liposomal formulation and used it successfully to deliver multiple drugs for the treatment of RCC in both immune-deficient and immune-competent mouse models. Here we developed a similar formulation combining everolimus and YM155 (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further evaluated the efficacy of EY-L in sensitizing RCC cell lines and tumors towards radiation in vitro using clonogenic assay and in vivo in RCC xenografts in immunodeficient mice and murine RCC models in immunocompetent mice. Results: EY-L (2 mg/kg E, 1.4 mg/kg Y, twice a week intravenous administration) was better than E-L or Y-L, the corresponding single drug loaded formulations in containing the primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. Furthermore, EY-L leads to significantly higher sensitization of RCC cells towards radiation in vitro than E-L or Y-L. Finally, EY-L was able to sensitize RCC tumors towards radiation therapy in both RCC xenograft and murine RCC model. Conclusion: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy using in vitro clonogenic assay and in vivo radiosensitization studies in multiple RCC tumor models. Citation Format: Krishnendu Pal, Vijay S. Madamsetty, Hari K. Rachamala, Shamit K. Dutta, Enfeng Wang, Debabrata Mukhopadhyay. Combined inhibition of mammalian target of rapamycin (mTOR) and survivin augments radiation therapy in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B026.

Abstract 4010: Inhibition of PERK by HC-5404 sensitizes clear cell renal cell carcinoma tumor models to anti-angiogenic tyrosine kinase inhibitors

Abstract Anti-angiogenic agents form the backbone of standard of care for advanced clear cell renal cell carcinoma (ccRCC), but their clinical impact is limited by primary and secondary resistance mechanisms that remain a critical problem. Furthermore, the approvals for VEGFR-targeting receptor tyrosine kinase inhibitors (VEGFR-TKIs), cabozantinib in second-line, and tivozanib in third-line RCC patients were based on modest objective response rates and median progression-free survival. There is an urgent need for novel mechanisms that target adaptive tumor responses that drive resistance to these agents, as well as combination drug partners that improve outcomes for patients. As part of their mechanism, VEGFR-TKIs induce oxygen- and nutrient-deprivation that drives ER stress. Tumors can evade deleterious ER stress by activating PERK branch of the integrated stress response, which arrests global translation and restores homeostasis. We hypothesized that inhibiting PERK would enhance the anti-tumor activity of VEGFR-TKIs in vivo and tested this using HC-5404, a potent and selective PERK inhibitor currently in Ph1 clinical testing (NCT04834778). Here, we present preclinical evidence that supports combining HC-5404 with VEGFR-TKIs in ccRCC. We demonstrate that axitinib, cabozantinib, lenvatinib, and sunitinib all activate PERK in 786-O ccRCC xenografts in a dose-responsive manner. The addition of HC-5404 significantly enhanced the tumor growth inhibition (TGI) of VEGFR-TKIs across multiple ccRCC tumor models, resulting in tumor stasis or regression in combination groups. Expression profiling and IHC analysis of tumor sections revealed that HC-5404 enhanced the anti-angiogenic effects of axitinib and lenvatinib in 786-O tumors, highlighting the protective role of PERK in response to anti-angiogenics. To evaluate whether the combination treatments could benefit a diverse patient population, sensitivity to HC-5404 and axitinib was evaluated across a panel of patient-derived xenograft (PDX) models. This experiment confirmed widespread responsiveness to the combination treatment that in some cases achieved >50% tumor regression. As tumor progression on VEGFR-TKIs limits the success of these agents in the clinic, we evaluated the effect of adding HC-5404 to tumors that have previously progressed on axitinib. In this study, 786-O xenografts were treated with axitinib for 2-weeks and non-responders were rerandomized into groups of either single agent or combination of HC-5404 and axitinib. The combination treatment significantly improved TGI relative to either monotherapy, resulting in tumor regression of ~20%. Taken together, these findings highlight that by disrupting an adaptive stress response evoked by VEGFR-TKIs, HC-5404 presents a clinical opportunity to enhance the anti-tumor effects of well-established standard of care therapies in ccRCC. Citation Format: Michael E. Stokes, Veronica Calvo, Crissy Dudgeon, Sho Fujisawa, Sharon Huang, Leyi Shen, Nupur Ballal, Joe McGinley, David Liu, Mark J. Mulvihill, Alan C. Rigby, Nandita Bose, Eric S. Lightcap, David Surguladze. Inhibition of PERK by HC-5404 sensitizes clear cell renal cell carcinoma tumor models to anti-angiogenic tyrosine kinase inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4010.

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.

A Listeria-based vaccine targeting ISG15 exerts anti-tumor efficacy in renal cell carcinoma.

Renal cell carcinoma (RCC) is the deadliest form of urological cancer and is projected to be the fourth most common neoplasm in the USA in males by 2040. In addition to the current poor prognosis with 5-year survival rates hardly reaching 15%, the prevalence of resistance to currently available systemic therapies has also established an urgent need to develop new treatment regimen(s) for advanced RCC. Interferon-stimulated gene 15 (ISG15) is the first identified ubiquitin-like modifier and has been intensively studied for its central role in innate immunity against intracellular pathogens. However, in this study, we identified ISG15 as a novel tumor-associated antigen and prognostic marker in RCC. Further, we therapeutically targeted elevated ISG15 expression by means of a Listeria monocytogenes (Lm)-based vaccine, designated Lm-LLO-ISG15, in both subcutaneous and orthotopic RCC mouse models. Treatment with Lm-LLO-ISG15 resulted in an influx of tumor-infiltrating effector T cells and significant anti-tumor efficacy in both subcutaneous and orthotopic RCC tumor models. Treatment with Lm-LLO-ISG15 also generated a robust interferon-gamma response and attracted a larger pool of polyfunctional T cells into the tumor microenvironment. Importantly, the therapeutic efficacy of Lm-LLO-ISG15 in RCC is comparable to that of anti-PD-1 and sunitinib, the current frontline therapies for RCC patients. Collectively, our work illustrates that targeting ISG15 in RCC with a CTL-based immunotherapy such as Lm-LLO-ISG15 is a promising and potentially translatable therapeutic strategy to enhance survival in RCC patients.

Growth differentiation factor 15 (GDF-15) inhibition to increase muscle mass and function in cancer cachexia.

e15633 Background: Almost half of cancer deaths are attributed to cancers most frequently associated with cachexia. Cachexia is a complex metabolic disease characterized by anorexia and unintentional weight loss. Skeletal muscle depletion has been recognized as a key feature of the disease, however muscle anabolic therapies have not been successful, suggesting that treatments that target multiple aspects of the disease will be most effective. Growth differentiation factor 15 (GDF-15) is a cytokine that induces anorexia and weight loss and is associated with cachexia in cancer patients. In preclinical cancer cachexia models, GDF-15 inhibition is sufficient to normalize food intake and body weight, including skeletal muscle mass. However, it remains to be determined whether the increased skeletal muscle mass also results in restoration of muscle function. Therefore, we examined the effect of GDF-15 inhibition on muscle mass and function in mouse models of cancer cachexia in comparison with myostatin inhibition, an established muscle anabolic pathway. Methods: Cachectic mouse tumor models were established with subcutaneous implantation of tumor cell lines reported to be GDF-15-dependent; mouse renal cell carcinoma (RENCA) and human ovarian cancer (TOV-21G) cell lines. Mice were treated with anti-GDF-15 (mAB2) or anti-myostatin (RK35) monoclonal antibodies and skeletal muscle function was assessed in vivo via maximum force, maximum rate of contraction and half relax time. In the RENCA tumor model, GDF-15 inhibition fully restored body weight and skeletal muscle mass whereas myostatin inhibition showed only a modest effect. Results: Consistent with the muscle mass improvement, GDF-15 inhibition dramatically increased functional muscle endpoints compared to the partial effect of myostatin inhibition. Interestingly, in the TOV-21G tumor model GDF-15 inhibition only partially restored body weight, however skeletal muscle mass and muscle function were completely normalized. Consistent with the functional assessment, GDF-15 inhibition in the RENCA tumor model decreased the expression of several catabolic genes (i.e. Trim63, Fbxo32, Myh7 and Myh2). The GDF-15 effect is likely to be secondary to the reversal of anorexia since wildtype mice pair-fed to Fc-GDF-15-treated mice demonstrated equivalent muscle mass loss. Conclusions: Taken together these data suggest that GDF-15 inhibition holds potential as an effective therapeutic approach to alleviate multiple aspects of cachexia.

Nucleobindin-2 enhances the epithelial-mesenchymal transition in renal cell carcinoma.

Nucleobindin 2 (NUCB-2) is a multifunctional protein that contains several functional domains and is associated with a wide variety of biological processes, such as food intake and energy homeostasis. NUCB-2 has been demonstrated to be associated with worse malignant outcomes and cell migration in breast and prostate cancer. However, to the best of our knowledge, its clinical and biological significance in renal cell carcinoma remains unknown. In the present study, tissue specimens from 68 patients with renal cell carcinoma and 10 normal controls were collected for NUCB-2 mRNA and protein assays. The NUCB-2 level in the patients with renal cell cancer was significantly increased compared with the normal control patients. NUCB-2-knockout in the renal cancer cell line SK-RC-52 inhibited migration and invasion. In addition, the expression levels of molecules associated with epithelial-mesenchymal transition (EMT), including E-cadherin, β-catenin, Slug and Twist, were affected by NUCB-2 suppression and the zinc finger E-box binding to homeobox 1 (ZEB1)-dependent pathway. The AMP-dependent protein kinase (AMPK)/target of rapamycin complex (mTORC) 1 signaling pathway participates in the regulation of NUCB-2-mediated metastasis and EMT. Suppression of NUCB-2 also inhibited tumor nodule formation in a murine renal cell carcinoma tumor model. In summary, NUCB-2 increased migration, invasion and EMT in renal cell carcinoma cells through the AMPK/TORC1/ZEB1 pathway in vitro and in vivo.

Polymeric nanoparticles encapsulating novel TLR7/8 agonists as immunostimulatory adjuvants for enhanced cancer immunotherapy

Cytotoxic T lymphocytes (CTLs) play a major role in cancer immunotherapy because of their ability to directly kill tumor cells and secrete tumor suppressive cytokines. Anticancer vaccines aim to provoke tumor-specific CTL responses, which require activation of antigen presenting cells (APCs) including dendritic cells (DCs) and macrophages. Therefore, a potent immunostimulatory adjuvant capable of activating APCs is an essential component of anticancer vaccines. In this study, we introduce novel TLR 7/8 bi-specific agonists that significantly enhance cytokine secretion compared to TLR7 mono-selective compounds. Encapsulation of these TLR 7/8 agonists in poly(lactide-co-glycolide) (PLGA) nanoparticles increased the co-stimulatory molecule expression and antigen presentation via MHC I by DCs compared to the soluble agonist. When administered subcutaneously, these nanoparticles migrated to draining lymph node and triggered DC activation and expansion. This lead to expansion of antigen-specific CD8 T cells and enhanced CTL response, which resulted in significant prophylactic and therapeutic efficacy in melanoma, bladder and renal cell carcinoma tumor models. Importantly, our studies demonstrate significant reductions in systemic metastasis with the nanoparticle vaccine. Our results suggest novel TLR 7/8 agonist-encapsulated nanoparticles are potent immunostimulatory adjuvants for cancer immunotherapy.

Detecting functional changes with [(18)F]FAZA in a renal cell carcinoma mouse model following sunitinib therapy.

BackgroundThe multitargeting tyrosine kinase inhibitor (TKI) sunitinib is currently the first-line drug therapy for metastasizing renal cell carcinoma (RCC). TKIs have profound effects on tumor angiogenesis, leading to modifications of the tumor microenvironment. The goal of this study was to determine whether these treatment-induced changes can be detected with [18F]FAZA.MethodsThe present study utilized positron emission tomography (PET) to analyze tumor oxygenation status during and after sunitinib therapy in the murine Caki-1 RCC tumor model. Dynamic and static scans were performed, as well as ex vivo biodistributions at 3 h post injection (p.i.). Immunohistochemical analysis of tumor tissue was carried out for the quantification of pimonidazole binding and the hypoxia-associated factors CD-31, Ki-67, and Von Willebrand factor (VWF). In addition, in vitro cellular uptake studies were done to analyze the direct effects of sunitinib on the Caki-1 cells.ResultsDuring therapy with sunitinib (40 mg/kg/day), uptake of [18F]FAZA into Caki-1 mice decreased by 46 ± 5% (n = 4; 5 days) at 3 h post injection (p.i.) during the first study and 22 ± 5% (n = 8; 9 days) during the long-term study, indicating a decrease in the tumor's hypoxia level. However, when drug therapy was stopped, this effect was reversed completely, and the tumor [18F]FAZA uptake increased to 126 ± 6% (n = 6) of the control tumor uptake, indicative of an even higher level of tumor hypoxia compared to the therapy starting point. Sunitinib had no direct effect on [18F]FAZA uptake into Caki-1 cells in vitro.Conclusion[18F]FAZA PET could be used to monitor drug response during sunitinib therapy in RCC and may guide combination therapies based on the tumor's hypoxia status.

Inhibition of GSK-3 induces differentiation and impaired glucose metabolism in renal cancer.

Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase, is a key regulator of numerous cellular processes ranging from glycogen metabolism to cell-cycle regulation and proliferation. Consistent with its involvement in many pathways, it has also been implicated in the pathogenesis of various human diseases, including type II diabetes, Alzheimer disease, bipolar disorder, inflammation, and cancer. Consequently, it is recognized as an attractive target for the development of new drugs. In the present study, we investigated the effect of both pharmacologic and genetic inhibition of GSK-3 in two different renal cancer cell lines. We have shown potent antiproliferative activity of 9-ING-41, a maleimide-based GSK-3 inhibitor. The antiproliferative activity is most likely caused by G(0)-G(1) and G(2)-M phase arrest as evident from cell-cycle analysis. We have established that inhibition of GSK-3 imparted a differentiated phenotype in renal cancer cells. We have also shown that GSK-3 inhibition induced autophagy, likely as a result of imbalanced energy homeostasis caused by impaired glucose metabolism. In addition, we have demonstrated the antitumor activity of 9-ING-41 in two different subcutaneous xenograft renal cell carcinoma tumor models. To our knowledge, this is the first report describing autophagy induction due to GSK-3 inhibition in renal cancer cells.

617 DETECTING TUMOR HYPOXIA DURING SUNITINIB THERAPY IN A RENAL CELL CARCINOMA MOUSE MODEL USING POSITRON EMISSION TOMOGRAPHY

You have accessJournal of UrologyKidney Cancer: Basic Research (IV)1 Apr 2013617 DETECTING TUMOR HYPOXIA DURING SUNITINIB THERAPY IN A RENAL CELL CARCINOMA MOUSE MODEL USING POSITRON EMISSION TOMOGRAPHY David Chapman, John Mercer, Leonard Wiebe, Melinda Wuest, and Ronald Moore David ChapmanDavid Chapman Edmonton, Canada More articles by this author , John MercerJohn Mercer Edmonton, Canada More articles by this author , Leonard WiebeLeonard Wiebe Edmonton, Canada More articles by this author , Melinda WuestMelinda Wuest Edmonton, Canada More articles by this author , and Ronald MooreRonald Moore Edmonton, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.168AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sunitinib is currently the first line drug therapy for metastasizing renal cell carcinoma (RCC). It has been shown to have a profound effect on tumor angiogenesis leading to modifications of the tumor's microenvironment. Tumor hypoxia plays an important role in the metastatic potential of a solid tumor and its resistance to any chemotherapy. Therefore monitoring tumor hypoxia could potentially be used to detect and analyze therapeutic response. The present study utilized Positron-Emission Tomography (PET) to determine changes in tumor oxygenation during and following sunitinib therapy in a mouse RCC tumor model. METHODS Subcutaneous bilateral Caki-1 tumors were grown for 40 days in BALB/c nu/nu mice. Mice bearing tumors were sorted into 2 groups: a) receiving 40mg/kg/d sunitinib i.p. and b) vehicle control injections. PET imaging (3 h p.i.) utilizing the hypoxia radiotracer [18F]FAZA was performed after 4 days, 7 days, and 13 days of therapy, and again 12 days after 15 days of therapy. In vitro cell uptake experiments were conducted under hypoxic conditions to observe whether sunitinib would have a direct effect on uptake and intracellular trapping of [18F]FAZA. In addition, biodistribution of [18F]FAZA and immunohistochemical staining to detect pimonidazole adducts were determined. RESULTS PET experiments revealed a relatively low uptake level of [18F]FAZA into murine RCC tumors. After 7 days of sunitinib therapy (resulting in decreased tumor growth) the uptake (SUV) was significantly reduced: 0.24+/-0.02 in control versus 0.19+/-0.01 in sunitinib treated mice (n=8; p<0.05). Allowing a 12-day break following 15 days of sunitinib therapy resulted in an increased [18F]FAZA uptake into the treated mice: 0.18+/-0.01 (n=5, control) versus 0.23+/-0.01 (n=6; p<0.05, sunitinib). Biodistribution studies with [18F]FAZA and pimonidazole staining supported the reduced uptake during therapy and the increase again after the therapy was stopped. In vitro cell uptake experiments showed that the [18F]FAZA uptake of 0.25 ± 0.04 (normoxic) or of 2.46 ± 0.42 (hypoxic; n=9) into Caki-1 cells was not changed in the presence of 3 uM sunitinib indicating no direct sunitinib effect on the cellular uptake of the radiotracer. CONCLUSIONS Uptake of [18F]FAZA tended to decrease during therapy of sunitinib indicating a decrease in the tumor's hypoxia. However, when stopping drug therapy in tumor-bearing mice, this effect was reversed and tumor hypoxia was increased again. [18F]FAZA could potentially be used to monitor drug response to therapy with sunitinib in RCC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e252 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information David Chapman Edmonton, Canada More articles by this author John Mercer Edmonton, Canada More articles by this author Leonard Wiebe Edmonton, Canada More articles by this author Melinda Wuest Edmonton, Canada More articles by this author Ronald Moore Edmonton, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 3753: Potent anti-tumor activity of mTOR kinase inhibitor in combination with anti-angiogenic agents in preclinical models of renal cell cancer

Abstract Background: Multiple agents that inhibit angiogenesis and tumor cell growth via the VEGF and mTOR (TORC1) pathways have been approved for locally advanced or metastatic renal cell carcinoma (RCC) in recent years. Despite this progress, improving overall survival remains a significant clinical challenge for the treatment of this cancer type. The tumorigenesis of certain types of RCC is dominated by genes that play critical role in hypoxia response, including stimulation of neo-angiogenesis. Anti-angiogenic therapies target the tumor microenvironment but provide little direct impact to the tumor cell, allowing the potential for disease progression despite treatment. Approval of allosteric partial TORC1 inhibitors in RCC stimulated interest in pursuing TORC1/2 inhibitors, that block mTOR signaling more potently and comprehensively, alone and in combination with anti-angiogenic agents in preclinical models of RCC. Results: INK128 is an orally bioavailable, potent and selective ATP site mTOR kinase inhibitor (TORC1/2) that has entered clinical development. INK128 inhibited the phosphorylation of S6 and 4EBP1, downstream substrates of TORC1, and AKT and NRDG1, downstream substrate of TORC2 in RCC cell lines. TORC1/2 pathway inhibition correlated with potent and complete blockade of cell proliferation. In vivo, daily treatment of INK128 as single agent suppressed tumor growth in multiple mouse RCC xenograft models. Pharmacodynamic analysis demonstrated that INK128 inhibits both TORC1 and TORC2 pathways in vivo. INK128 did not alter the tumor-associated microvasculature density despite significant decrease of VEGF supply by lowering the capacity of protein translation of the tumor cells. This prompted us to explore the potential of the combination of INK128 with anti-angiogenic agents to maximize anti-tumor activity by targeting both tumor microenvironment and tumor cells directly. Co-treatment of INK128 with sorafenib resulted in much enhanced activity in RCC tumor models but did not achieve sufficient tolerability. We next employed an alternating dosing strategy of INK128 and sorafenib to improve tolerability. An optimized dose/schedule sequence of both agents caused sustained inhibition of tumor-associated angiogenesis, suppression of mTOR pathway resulting into marked blockade of tumor growth with significant induction of apoptosis and acceptable tolerability. Conclusion: The sequential, yet potent blockade of both mTOR and VEGF pathways obtained by combining INK128 with sorafenib acts synergistically in inducing cell death and tumor regression in preclinical renal cancer models. Adaptive combination of receptor tyrosine kinase-based anti-angiogenic agents with mTOR kinase inhibitors currently in development may offer a safe and effective therapeutic strategy for renal cancer patients who fail to respond to standard of care therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3753. doi:1538-7445.AM2012-3753

308 DETECTION OF FUNCTIONAL CHANGES IN RENAL CELL CARCINOMA WITH POSITRON EMISSION TOMOGRAPHY

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2012308 DETECTION OF FUNCTIONAL CHANGES IN RENAL CELL CARCINOMA WITH POSITRON EMISSION TOMOGRAPHY David W. Chapman, Melinda Wuest, Leonard I. Wiebe, and Ronald B. Moore David W. ChapmanDavid W. Chapman Edmonton, Canada More articles by this author , Melinda WuestMelinda Wuest Edmonton, Canada More articles by this author , Leonard I. WiebeLeonard I. Wiebe Edmonton, Canada More articles by this author , and Ronald B. MooreRonald B. Moore Edmonton, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.367AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Broad-spectrum tyrosine kinase inhibitors (TKIs) are clinically used for the therapy of metastasizing renal cell carcinoma (RCC). It has been shown in vivo that multitargeted TKIs such as sunitinib exert antiangiogenic effects directly on endothelial cells, but also by blocking compensatory changes in the activity of hypoxia-inducible factors (HIF), mainly HIF-1α. The present study investigated whether sunitinib-induced changes in the microenvironment of a mouse RCC tumor model can be detected utilizing positron-emission tomography (PET). METHODS Caki-1 tumors were grown for 4 weeks in Balb c/nu-nu mice after implantation subcutaneously or into the renal subcapsule. Both models were analyzed first with dynamic small animal PET with [18F]FDG and [18F]FAZA up to 3h post-injection (p.i.). In a second setup, mice bearing 2 subcutaneous Caki-1 tumors were sorted into 2 groups: a) receiving 40mg/kg/d sunitinib i.p. for 5 days prior to analysis with [18F]FAZA PET, b) vehicle control injections. Tumor uptake of [18F]FAZA and immunohistochemical tissue staining with pimonidazole HCl and CD-31 were determined. RESULTS Functional analysis of subcutaneous Caki-1 tumors with PET revealed a mean standardized uptake value (SUV) after 3h p.i. of 0.60±0.02 for [18F]FDG, which is indicative of glucose metabolism within the tumor, and 0.32±0.02 (n=6/3) for [18F]FAZA determining tumor's hypoxia status. Based on the clearance patterns, orthotopically grown tumors were not detectable with PET alone. The SUV3h for [18F]FAZA was significantly lower in sunitinib group compared to controls in the subcutaneous model: 0.23±0.02 vs. 0.42±0.05 (n=4/2). The time-activity curve indicated significant [18F]FAZA washout from treated tumors as compared to the control. This observation was confirmed by the biodistribution of [18F]FAZA, resulting in SUV3h of 0.29±0.03 in treated vs. 0.45±0.06 (n=8/4) in control mice. Pimonidazole binding was reduced by 9% in the treated tumors. CD31 binding (%) was reduced in sunitinib treated groups: 1.8±0.1% vs. 0.6±0.1% (n=80/4). CONCLUSIONS Reduced trapping of [18F]FAZA in this RCC tumor model, in vivo, indicates that TKI inhibition with mutitargeting sunitinib leads to changes in tumor oxygenation, which is functionally detectable with PET. Furthermore, sunitinib led to a significant reduction in vascular density. Monitoring early response to TKI therapy in RCC tumor patients could potentially utilize [18F]FAZA. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e125 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information David W. Chapman Edmonton, Canada More articles by this author Melinda Wuest Edmonton, Canada More articles by this author Leonard I. Wiebe Edmonton, Canada More articles by this author Ronald B. Moore Edmonton, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Progression of renal cell carcinoma is inhibited by genistein and radiation in an orthotopic model

BackgroundWe have previously reported the potentiation of radiotherapy by the soy isoflavone genistein for prostate cancer using prostate tumor cells in vitro and orthotopic prostate tumor models in vivo. However, when genistein was used as single therapy in animal models, it promoted metastasis to regional para-aortic lymph nodes. To clarify whether these intriguing adverse effects of genistein are intrinsic to the orthotopic prostate tumor model, or these results could also be recapitulated in another model, we used the orthotopic metastatic KCI-18 renal cell carcinoma (RCC) model established in our laboratory.MethodsThe KCI-18 RCC cell line was generated from a patient with papillary renal cell carcinoma. Following orthotopic renal implantation of KCI-18 RCC cells and serial in vivo kidney passages in nude mice, we have established a reliable and predictable metastatic RCC tumor model. Mice bearing established kidney tumors were treated with genistein combined with kidney tumor irradiation. The effect of the therapy was assessed on the primary tumor and metastases to various organs.ResultsIn this experimental model, the karyotype and histological characteristics of the human primary tumor are preserved. Tumor cells metastasize from the primary renal tumor to the lungs, liver and mesentery mimicking the progression of RCC in humans. Treatment of established kidney tumors with genistein demonstrated a tendency to stimulate the growth of the primary kidney tumor and increase the incidence of metastasis to the mesentery lining the bowel. In contrast, when given in conjunction with kidney tumor irradiation, genistein significantly inhibited the growth and progression of established kidney tumors. These findings confirm the potentiation of radiotherapy by genistein in the orthotopic RCC model as previously shown in orthotopic models of prostate cancer.ConclusionOur studies in both RCC and prostate tumor models demonstrate that the combination of genistein with primary tumor irradiation is a more effective and safer therapeutic approach as the tumor growth and progression are inhibited both in the primary and metastatic sites.

Identification of differentially expressed genes in a renal cell carcinoma tumor model after endostatin-treatment

Endostatin is a cleavage product of collagen XVIII that has shown to inhibit tumor-angiogenesis in experimental tumor models. At present, the exact molecular mechanism of action of endostatin is not completely elucidated. In this study, we wanted to identify specific target genes of endostatin. For this purpose, the human renal cell carcinoma RC-9 was subcutaneously implanted in nude mice and treated with endostatin. Tumor growth was inhibited by endostatin after 4 days of treatment. Using immunohistochemistry and the hypoxia marker pimonidazole, we demonstrate disintegration of blood vessels and hypoxia and anoxia as a result of the treatment. Hereafter, we applied the polymerase chain reaction (PCR)-based subtractive suppression hybridization (SSH) method, together with the mirror orientation selection (MOS) technique to identify specifically induced and suppressed genes after endostatin-treatment. We found eight genes to be specifically induced and 11 to be suppressed by the endostatin-treatment. Among other genes, core binding factor a-1/osteoblast-specific factor-2 (cbfa1/osf2) was found to be specifically suppressed by endostatin. Unexpectedly, cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor, not only in the experimental RC-9 tumor model, but in sections of human breast cancer as well. Since an effect of antiangiogenic therapy on granulocytes has been reported before, this might lead to new insights in the role of granulocytes in antiangiogenic therapy in general. In conclusion, the SSH-PCR implemented with the MOS-technique is a powerful tool to identify differentially expressed genes. Using these techniques, we have identified several target genes of endostatin, of which cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor.