Renal Tumor Xenografts Research Articles

Ultrasound localization microscopy of renal tumor xenografts in chicken embryo is correlated to hypoxia

Ultrasound localization microscopy (ULM) permits the reconstruction of super-resolved microvascular images at clinically relevant penetration depths, which can be potentially leveraged to provide non-invasive quantitative measures of tissue hemodynamics and hypoxic status. We demonstrate that ULM microbubble data processing methods, applied to images acquired with a Verasonics Vantage 256 system, can provide a non-invasive imaging surrogate biomarker of tissue oxygenation status. This technique was applied to evaluate the microvascular structure, vascular perfusion, and hypoxia of a renal cell carcinoma xenograft model grown in the chorioallantoic membrane of chicken embryos. Histological microvascular density was significantly correlated to ULM measures of intervessel distance (R = −0.92, CI95 = [−0.99,−0.42], p = 0.01). The Distance Metric, a measure of vascular tortuosity, was found to be significantly correlated to hypoxyprobe quantifications (R = 0.86, CI95 = [0.17, 0.99], p = 0.03). ULM, by providing non-invasive in vivo microvascular structural information, has the potential to be a crucial clinical imaging modality for the diagnosis and therapy monitoring of solid tumors.

Abstract 2478: miR-182-5p suppresses progression of renal cancer through cell cycle arrest by targeting lncRNA MALAT-1

Abstract Background: Renal cell carcinoma (RCC) is a leading cause of death, accounting for nearly 14,000 (~2.4%) deaths in the United States in 2017, with clear cell carcinoma (ccRCC) being the most common histologic subtype. New and precise disease progression biomarkers are needed for early detection and follow-up. Characterization of these new biomarkers offers a new approach for the identification of novel therapeutic targets and drugs for RCC treatment. MicroRNAs (miRNAs) act as either onco-miRs or tumor suppressors in cancer. Here we show a tumor-suppressor role for miR-182-5p in ccRCC and novel regulation of cell proliferation through MALAT-1. Methods: Profiling of miR-182-5p and MALAT-1 was performed in microdissected renal cancer tissues, matched adjacent normal regions and in human renal cancer cell lines by quantitative real-time PCR. To assess the functional significance of miR-182-5p in RCC, we overexpressed miR-182-5p/control miRNA (miR-CON) in RCC cell lines (ACHN, Caki-1) followed by functional assays. We also examined the therapeutic potential of synthetic miR-182-5p mimics in vivo using a renal cancer xenograft mouse model. We performed luciferase reporter assay and Ago2-RIP assay to investigate the interaction between miR-182-5p and MALAT-1. In addition, we assessed the effects of miR-182-5p overexpression and MALAT-1 downregulation on cell cycle progression in ccRCC cell lines. Results: Expression analyses in a cohort of renal cancer clinical specimens showed that miR-182-5p expression is frequently downregulated in ccRCC. We observed that overexpression of miR-182-5p inhibited cell proliferation, colony formation, apoptosis and led to G2/M arrest, supporting an antiproliferative role for this microRNA. Overexpression of miR-182-5p led to decreased expression of CDC20 and AURKA, drivers of the cell cycle mitotic phase. Also, overexpression of miR-182-5p directly lowered the expression of MALAT-1 and knockdown of MALAT-1 mimicked the effects of miR-182-5p overexpression. Downregulation of MALAT-1 led to upregulation of p53 that ultimately downregulated CDC20, AURKA. In vivo studies demonstrated that administration of miR-182-5p caused regression of established renal tumor xenografts. Conclusions: Collectively, these data suggest that miR-182-5p plays a tumor-suppressive role in ccRCC. These findings offer new insight into role of miR-182-5p in the inhibition of ccRCC tumor growth through MALAT-1 downregulation. This study may provide rationale for development of new strategies targeting MALAT-1 through miR-182-5p for treatment of ccRCC. Citation Format: Priyanka Kulkarni, Pritha Dasgupta, Shahana Majid, Varahram Shahryari, Marisa Shiina, Yutaka Hashimoto, Nadeem Bhat, Guoren Deng, Sharanjot Saini, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. miR-182-5p suppresses progression of renal cancer through cell cycle arrest by targeting lncRNA MALAT-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2478.

Comparative Response of SC CAKI-1 Renal Tumor to Treatment with Doxorubicin HCl and Doxorubicin Orotate

Background: Doxorubicin (DOX) is an effective treatment for many cancers across the age spectrum, but its therapeutic potential is limited because of dose-dependent relation to both progressive and irreversible cardiomyopathy leading to congestive heart failure. While decreases in cardiotoxicity have been reported with liposomal doxorubicin, the long-term cardiac effects are not known. Orotate salts of cytotoxic drugs have been shown to confer antitumor effects with a better safety profile than unconverted drug, and therefore may offer an improved approach to cancer treatment. Materials and Methods: Male, athymic NCr-nu/nu mice with subcutaneously implanted CAKI-1 human renal tumor xenografts were treated with DOX and its orotate salt (DOX-O) to evaluate antitumor activity, measured by median tumor mass doubling time and tumor weight. Nontumored male, athymic NCr-nu/nu mice were treated with DOX, DOX-O and liposomal doxorubicin formulations to determine DOX concentration in liver and heart; and to evaluate their effect on body weight. Non-tumored female, athymic NCr-nu/nu mice were treated with daunorubicin and daunorubicin orotate to evaluate tolerance. Results: DOX and DOX-O exhibited significant, similar levels of antitumor activity. Mice treated with DOX-O had a lower percentage body weight loss. In the animals treated with DOX, DOX-O, or liposomal doxorubicin, liposomal doxorubicin was associated with the lowest percentage of body weight loss, but the highest concentration of DOX in heart. In daunorubicin tolerance experiments, animals showed a better tolerance for daunorubicin orotate as measured by a smaller percentage change in body weight. Conclusions: DOX-O is effective as an antitumor therapy and may offer a less toxic alternative to DOX for maintaining therapy. The lower percentage of body weight loss in animals treated with DOX-O and daunorubicin orotate is a measure of improved tolerance and may translate into better patient outcomes.

CAMP-Dependent Cytosolic Mislocalization of p27kip-Cyclin D1 During Quinol-Thioether–Induced Tuberous Sclerosis Renal Cell Carcinoma

The loss of tuberin, the tuberous sclerosis-2 (Tsc-2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc-2(EK/+)) and in human metastatic renal cell carcinoma tissue. Signaling associated with cytoplasmic mislocalization of p27 in renal cancer is relatively unknown. Renal tumors derived from 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-treated Tsc-2(EK/+) rats, and null for tuberin, display elevated nuclear and cytosolic p27, with parallel increases in cytosolic cyclin D1 levels. Similar changes are observed in TGHQ-transformed renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), which, in addition to the cytoplasmic mislocalization of p27 and cyclin D1, exhibit high ERK, B-Raf, and Raf-1 kinase activity. Renal tumor xenografts, derived from subcutaneous injection of QTRRE cells into nude mice, also display increases in cytosolic mislocalization of p27 and cyclin D1. Dibutyryl cAMP and/or phosphodiesterase inhibitors (PIs; pentoxifylline or theophylline) increase Rap1B activation, B-Raf kinase activity, and cytosolic p27/cyclin D1 protein levels in QTRRE cells. Inhibition of Raf kinases with either sorafenib or B-Raf small interfering RNA (siRNA) caused a mitogen-activated protein kinase-mediated downregulation of p27. Moreover, decreases in cyclin D1 were also associated with p27 siRNA knockdown in QTRRE cells. Finally, theophylline-mediated increases in p27 and cyclin D1 were attenuated by sorafenib, which modulated Raf/MEK/ERK signaling. Collectively, these data suggest that the cAMP/Rap1B/B-Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27-cyclin D1 in tuberous sclerosis gene-regulated-renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27-cyclin D1 cytosolic stabilization during renal tumor formation.

Abstract 3981: miR-708 induces apoptosis and suppresses tumorigenicity in renal cell carcinomas

Abstract MicroRNAs (miRNAs) constitute an evolutionarily conserved class of small non-coding RNAs that primarily regulate gene expression at the post-transcriptional level via sequence-specific interaction with the 3’-untranslated regions of cognate mRNA targets. Recent evidence indicates that dysregulated miRNA expression leads to tumorigenesis and miRNAs have been implicated to function as either tumor suppressors or oncogenes. The main objective of the present study was to identify miRNA genes that regulate renal cell carcinomas (RCC). Towards this, we performed microRNA expression profiling in normal immortalized renal cell line (HK2) and primary renal cancer cell lines (A498 and Caki2) and identified that miR-708 expression is significantly attenuated in renal cancer cell lines. This attenuation was validated by RT-PCR and in situ hybridization analysis of miR-708 in RCC tissue specimens. Further, trichostatin A, a pharmacological inhibitor of histone deacetylases, strongly increased miR-708 levels in cell lines from renal cancer suggesting that epigenetic silencing may lead to miR-708 downregulation in RCC. To evaluate the functional significance of decreased miR-708 expression, miR-708 was reintroduced in renal cancer cell lines followed by functional assays. Overexpression of miR-708 in the A498 cell line leads to significant decreases in cell growth, clonability, invasiveness and migratory properties. Importantly, miR-708 reexpression leads to dramatic induction of apoptosis in A498 cells suggesting that restoration of miR-708 expression in A498 cells suppressed tumorigenecity in vitro by primarily affecting the apoptotic program. In vivo, intratumoral delivery of miR-708 led to a regression of tumor growth in established renal tumor xenografts. Further, analysis of putative miR-708 targets showed that miR-708 targets survivin/BIRC5, a small inhibitor of apoptosis (IAP) protein that plays a pivotal role in renal cancer progression. In conclusion, our data suggests a crucial tumor suppressive role for miR-708 in RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3981. doi:10.1158/1538-7445.AM2011-3981

Abstract 2498: BNC105 is a tubulin polymerization inhibitor that exhibits vascular disruptive activity in renal cancer and causes upregulation in HIF1alpha, HIF2alpha and mTOR

Abstract BNC105 is a tubulin polymerization inhibitor. This agent selectively disrupts the vasculature in tumors, and in addition acts as a direct cytotoxic against cancer cells. BNC105 has demonstrated single agent efficacy in a number of cell lines and tumor models, including breast, colon, prostate, brain and lung cancer. We have investigated the activity of BNC105 in renal cancer cell lines and renal tumor xenografts. BNC105 exhibits cytotoxic activity with EC50 values in the range of 1-20nM. A number of other chemotherapeutic and targeted agents exhibit activity that is comparable or considerably lower than BNC105. Furthermore, we investigated the activity of BNC105 in disrupting the vasculature of tumors arising from human renal cancer cell lines. Our investigations included the renal cell lines Caki-1 and A498. Caki-1 cells express the wild type of the VHL gene, whereas A498 cells expresses a non-functional mutant form of VHL. In addition A498 cells do not express HIF1alpha. Somatic mutation of the VHL gene is the most frequent genetic alteration observed in renal cell carcinoma leading to inappropriate accumulation of HIF1alpha and HIF2alpha. Single dose BNC105 treatment of mice bearing tumor xenografts arising from these cell lines resulted in substantial tumor vascular disruption giving rise to large areas of tumor necrosis. Immunohistochemistry analysis revealed that expression of HIF1alpha was dramatically increased following BNC105 treatment in tumors arising from the renal cell line Caki-1. HIF1alpha expression in A498 tumors was restricted to endothelial cells and did not change following BNC105 treatment. HIF2alpha expression was found to be upregulated in cells surrounding areas of BNC105 induced necrosis in both Caki-1 and A498 tumors. These findings suggest that vascular disruption and necrosis caused by BNC105 lead to an angiogenic rebound effect that may contribute to tumor recovery from the vascular disruption insult. Given the key role of mTOR signaling in upregulation of HIF we examined the expression of phosphorylated mTOR in renal tumor xenografts following treatment with BNC105. Expression of phosphorylated mTOR was distinctly upregulated in viable cell zones surrounding tumor VDA-induced necrotic areas in both Caki-1 and A498 tumors. These findings suggest that tumor recovery from the necrotic insult of BNC105 is mediated through upregulation of mTOR activity. The potential benefit of combining BNC105 with mTOR inhibitors on the growth of renal tumors is currently being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2498.

GTI-2501, an antisense agent targeting R1, the large subunit of human ribonucleotide reductase, shows potent anti-tumor activity against a variety of tumors

GTI-2501 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of R1, the large subunit of ribonucleotide reductase (RNR). In vitro studies, have demonstrated that GTI-2501 decreases mRNA and protein levels of R1 in a sequence-specific and dose-dependent manner. Furthermore, GTI-2501 inhibits the growth of human lung, liver, ovary, brain, melanoma, breast and pancreatic tumor cells in colony forming assays. In vivo studies have shown that GTI-2501 significantly inhibits growth of human colon, pancreas, lung, breast, renal, ovarian, melanoma, brain glioblastoma-astrocytoma, and prostatic tumors in CD-1 nude, Balb/c nude and/or SCID mice. GTI-2501 treatment caused total regression of human breast and renal tumor xenografts in mice. These effects are not observed with a scrambled control oligonucleotide containing the same base content but not complementary to R1. GTI-2501 specifically inhibits metastasis of human melanoma cells to the lungs in CD-1 athymic nude mice and prolongs the survival of mice bearing human lymphoma. Taken together these results suggest that an antisense mechanism of action is responsible for growth inhibition in vitro and in vivo and that GTI-2501 can act as a selective and specific anti-tumor agent.

Verotoxin Induces Rapid Elimination of Human Renal Tumor Xenografts in SCID Mice

Verotoxins (VTs) are subunit toxins produced by enteropathogenic Escherichia coli. The VT receptor glycolipid Gb3, which mediates the cytotoxicity of VTs, has been reported to be elevated on the surface of several tumor cell lines. In this study the effect of VT1 as an antineoplastic agent was assessed using various human urological cancer cell lines. The expression of Gb3 on human cancer cell lines originating from renal cell carcinoma (ACHN, A-704, CAKI-1 and CAKI- 2), prostate cancer (LNCaP and PC3) and testicular tumor (2102Ep) were examined by FACScan (Becton Dickinson, Sunnyvale, California). These cell lines were cultured with various concentrations of VT1 and subjected to microculture tetrazolium dye assay for determination of cell viability. Furthermore, ACHN cells were inoculated into the backs of SCID mice and intratumor injection of VT1 was performed. Pathological samples were examined by hematoxylin and eosin staining as well as by TUNEL assay. The growth of ACHN, CAKI-1, A-704, 2102Ep and LNCaP but not CAKI-2 and PC3 was significantly inhibited by co-incubation with VT1, as determined by microculture tetrazolium dye assays, consistent with FACScan results for Gb3 expression. When mice bearing ACHN tumors were injected with VT1, rapid reduction in the size of subcutaneous tumors was observed with complete regression within 5 to 7 days. Pathological examination by the TUNEL method indicated that the cytotoxicity of VT1 was mediated by apoptosis. These results suggest that VTs could be candidates for antineoplastic agents against Gb3 expressing tumors for clinical use.

Enhanced antitumor activity of anti-VEGF antibody HuMV833 in combination with gemcitabine in human pancreatic and renal tumor xenografts

Enhanced antitumor activity of anti-VEGF antibody HuMV833 in combination with gemcitabine in human pancreatic and renal tumor xenografts

HLA-class-I and-class-II expression on renal tumor xenografts and the relation to sensitivity for α-IFN, γ-IFN and TNF

In this study we evaluated the usefulness of the histocompatibility leucocyte antigen (HLA) class-I and class-II expression on renal-cell carcinoma (RCC) xenografts as predictive markers for response to cytokine therapy. Eight different RCC xenografts growing in BALBC nu/nu mice were treated with 0.5 or 5.0 ng/g recombinant human alpha- or gamma-interferon (IFN), or 500 ng/g recombinant human tumor necrosis factor (TNF). Modulation of HLA class-I, -II expression was evaluated immunohistochemically using the monoclonal antibodies (MAbs) W6.32 and B8.11.2 and at the mRNA level using the plasmids pDP001 and DR alpha 120. HLA class-I expression in all lines was upregulated by alpha- and gamma-IFN and was highest in the high-IFN-dose-treated tumors. TNF also stimulated HLA-class-I expression and up-regulated class-I expression still further when combined with IFN. Highest up-regulation of HLA-class-I in all tumors was measured in the alpha-IFN-5.0/TNF-500-ng/g-treated mice, although this was not necessarily the treatment regimen resulting in the most pronounced effect on tumor growth. Hence, maximum upregulation of class-I antigens at a given regimen was not always indicative for the highest achievable anti-tumor effect. HLA-class-II expression which was present on only 3 of the untreated tumors was up-regulated by both alpha and gamma-IFN. TNF itself did not up-regulate class-II expression but enhanced the class-II expression on the alpha-IFN-treated tumors but not on the gamma-IFN-treated tumors. Irrespective of the basic expression level, inducibility of both HLA-class-I and -class-II antigens appear to be correlated to the direct effects on growth of renal-tumor xenografts towards alpha-IFN, gamma-IFN and TNF. Modulation of HLA antigens was studied in the nude mouse, hence T-cell-mediated effector mechanisms cannot explain the good correlation between inducibility and response. Nonetheless, our studies indicate that the extent of modulation of HLA-class-I and -II can serve as predictive marker for response to cytokine therapy, which may serve as a valuable criterion for inclusion of patients in cytokine treatment regimens.

In vitro antiproliferative efficacy of Interferon-alpha,-gamma and Tumor Necrosis Factor on two human renal tumor xenografts

The in vitro antitumor activity of recombinant alpha- and gamma-Interferon as well as recombinant Tumor Necrosis Factor alpha was tested on renal cell carcinoma xenografts using the double layer soft agar method. Using this assay, the effect of the drugs on the clonogenic potential of tumor cells in soft agar was determined and used as an indication for the antiproliferative capacity of these drugs. There appeared to be a differential response of the tested xenografts towards these drugs in a dose dependent way. However, when used in combination in most cases an additive or synergistic effect was observed. Even though again the response was differential, the combination of alpha-Interferon (10 ng/dish) and Tumor Necrosis Factor (100 ng/dish) resulted in a complete inhibition of colony formation in soft agar for both tumor lines. We conclude that there is a direct effect of alpha-Interferon, gamma-Interferon and Tumor Necrosis Factor on renal cell carcinoma xenografts. In combination drug tests the effect of alpha-Interferon and Tumor Necrosis Factor was strongly synergistic. The implication of these studies for in vivo use of these drugs remain to be established.

Effect of alpha- and gamma-interferon and tumor necrosis factor on colony formation of two human renal tumor xenografts in vitro.

The in vitro antitumor activity of alpha- and gamma-interferon as well as tumor necrosis factor was tested on two human renal cell carcinoma xenografts--RC-43 and NC-65. A dose-dependent inhibition of colony formation in soft agar was found for both tumor lines. NC-65, however, showed no sensitivity for gamma-interferon. Combination drug tests showed a synergistic effect on colony formation of both tumor lines. The strongest inhibition was shown by the combination of alpha-interferon and tumor necrosis factor. We conclude that both interferons as well as tumor necrosis factor show direct in vitro cytostatic effects against renal tumor direct in vitro cytostatic effects against renal tumor cells.