bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1461: Zoledronate inhibits proliferation and invasion of inflammatory breast cancer cells
Authors: Hall Carolyn S. , Singh Balraj , Tai Karen , Irving LaTashia R. , Lucci Anthony
Published: February 2011

Introduction: Inflammatory breast cancer (IBC) is aggressive, as approximately one third of patients present with distant metastases. Bisphosphonates are often administered to breast cancer patients for cancer-induced osteolysis. Recent evidence suggests bisphophanates may also have anti-tumor properties. This study examined the effects of the bisphosphonate, zoledronate (ZA), on the proliferation and invasive capabilities of highly aggressive IBC cell lines.

Methods: The IBC cell line SUM149 was cultured utilizing both adherent and serum-free, non-adherent cancer stem cell enriching conditions. An MTT assay was used to quantify proliferation and mammosphere formation of cells. Adherent cell growth was measured at days 1-4 following 0, 0.1, 1, 5, 10, 50 and 100uM zelodronate treatment. Mammosphere formation was measured at 10 days following 0, 0.01, 0.1, 1, 10, and 100uM ZA treatment. Modified Boyden chambers were employed to assess the ability of both adherent and non-adherent cells to invade to SDF-1 (100ng/mL) following 24 hour 1uM ZA treatment. Protein expression was assessed using immunofluorescent staining, protein arrays and Western blotting.

Results: ZA treatment with <5uM did not inhibit SUM149 adherent cell growth at any of the measured time points. ZA concentrations ≥10uM significantly inhibited SUM149 adherent cell growth at day 4 (p≤0.05). SUM 149 mammosphere formation was significantly inhibited following 10uM ZA treatment (p≤0.001). However, significantly more SUM149 non-adherent cells invaded to SDF-1 compared to adherent cells (p≤0.001). SUM 149 non-adherent cell invasion was inhibited by 24 hour 1uM ZA treatment (p≤0.001). SUM 149 non-adherent cells expressed significantly increased levels of the epithelial-mesenchymal transition (EMT) associated proteins smooth muscle actin (SMA), fibronectin, and N-Cadherin. The increased expression of SMA, fibronectin, and N-Cadherin observed in non-adherent cells returned to levels comparable to adherent cells following 24 hour 1uM ZA treatment.

Conclusions: ZA inhibited the growth of IBC cells grown under adherent and non-adherent conditions. SUM 149 non-adherent cells had much greater invasive potential than adherent cells did, and their invasive capability was inhibited following low dose ZA treatment. Interestingly, ZA inhibited up-regulation of EMT associated proteins SMA, fibronectin, and N-Cadherin in SUM 149 non-adherent cells. These results indicate that some IBC cells grown under cancer stem cell enriching conditions have the potential to undergo EMT and invade. ZA may also inhibit metastases through novel mechanisms.

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 1461.
Abstract 1463: Pharmacological impact of epithelial-mesenchymal plasticity in the H358 lung tumor model
Authors: Argast Gretchen M. , Mercado Peter , O'Connor Matthew , Mulford Iain J. , Pachter Jonathan A. , Epstein David M. , Thomson Stuart , Kan Julie L.C.
Published: February 2011

The phenotypic changes associated with epithelial to mesenchymal transition (EMT) give tumor cells at least two attributes that increase metastatic efficiency. First, mesenchymal cells have lost cell-cell contacts and are more invasive and can therefore escape the primary tumor, whereas epithelial cells remain anchored by cell-cell contacts in the primary tumor. Second, mesenchymal cells in vitro are less sensitive than epithelial cells to agents that inhibit the EGFR pathway. This appears to translate into the clinic, since patients with tumors that express mesenchymal markers do not respond to EGFR targeting therapeutics as well as patients with more epithelial tumors. To determine whether we can control sensitivity to EGFR inhibitors by manipulating a cell's epithelial/mesenchymal status, we examined the role of EMT and its reversibility in tumor cell sensitivity to the EGFR inhibitor erlotinib. We studied in vitro models of EMT driven by Transforming Growth Factor β (TGFβ), Hepatocyte Growth Factor + Oncostatin M (HGF+OSM), Snail or Zeb1 in the H358 non-small cell lung carcinoma model. EMT driven by TGFβ or HGF+OSM caused a significant decrease in sensitivity to erlotinib, while EMT driven by induced expression of Snail or Zeb1 was much less effective, suggesting a change in drug sensitivity requires more input than these canonical EMT transcription factors. Once mesenchymal tumor cells have localized to a metastatic site, it is thought the tumor cells must undergo a mesenchymal to epithelial transition (MET) in order to form a cohesive tumor. We examined the reversibility of EMT in our models and the corresponding erlotinib sensitivity, both by withdrawal of ligand and by pharmacological inhibition of the signaling pathways downstream of the drivers. Using these approaches, we found EMT driven by HGF+OSM was reversible as evidenced by morphology and marker changes, and this reversion correlated with an increase in erlotinib sensitivity, comparable to the parental H358 cells. EMT driven by TGFβ was partially reversed by both ligand withdrawal and pharmacological inhibition of the receptor, and the extent of reversion in erlotinib sensitivity correlated with the extent of marker reversion. These results demonstrate that the extent of reversibility of EMT is dependent on the driver. Furthermore, it may be possible to drive some erlotinib-insensitive tumors back to a more erlotinib-sensitive state with pharmacological agents, thus improving patient response.

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 1463.
Abstract 1469: Cyclin D1 plays a critical role in the response of mammary gland to estrogen and progesterone by regulating progesterone receptor expression
Authors: Yang Chuanwei , Chen Li , Li Cuiqi , Lynch Mary C. , Schmidt Emmett V.
Published: February 2011

Estrogen and progesterone are important hormones in normal female development, yet both have critical roles in breast carcinogenesis. Cyclin D1 is a breast cancer oncogene whose amplification is linked to poor prognosis in estrogen and progesterone receptor positive breast cancers. Here we show that cyclin D1 regulates progesterone receptor expression, consequently enhancing responses to estrogen and progesterone in breast development and carcinogenesis. Estrogen treatment of cyclin D1 transgenic mice caused mammary hyperplasias expressing increased progesterone receptors, which were stimulated by progesterone and blocked by a progesterone antagonist. Cyclin D1 regulated progesterone receptor expresion through a novel estrogen- and cyclin D1-responsive enhancer in DNA encoding part of the 3’ untranslated region of the progesterone receptor gene. Small inhibitory RNAs for cyclin D1 decreased progesterone receptor expression in human breast cancer cells, which sensitized them to progesterone-antagonists. Since estrogen and progesterone regulate cyclin D1, our results suggest that cyclin D1's participation in a feed-forward loop could contribute to increased breast cancer risks associated with estrogen and progesterone combinations. Additionally, its regulation of the progesterone receptor identifies a novel role for cyclin D1 in ovarian hormone control of breast development and breast carcinogenesis.

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 1469.
Abstract 1473: Fra-2 and JunB are overexpressed in precancerous bile ducts and primary/transplanted hamster cholangiocarcinomas with a strong positive correlation
Authors: Jeon Byung-Suk , Lee Byung-Woo , Yoon Byung-Il
Published: February 2011

The transciption factor complex AP-1 plays important roles in cell proliferation, differentiation and apoptosis of which dysregulation is closely associated with cancer development. Fra-2 and JunB, components of AP-1 family proteins, had been found overexpressed in certain of tumors, suggesting their possible implication in carcinogenesis. In the present study, we investigated differential expression patterns of those two AP-1family proteins during hamster cholangiocarcinogenesis and aggressive transplanted cholangiocarcinomas (ChCs). First, we prepared appropriate liver tissue samples with precancerous lesions and ChCs from hamster ChC model as well as normal liver. The transplanted ChCs were produced by syngenic transplantation of the primary ChCs. We then performed immunohistochemistry (IHC) for Fra-2 and JunB using ABC methods. The typical nuclear staining for Fra-2 and JunB was determined as positive immunoreactivity and the ratio of positive cells was evaluated by counting 800-3000 cells, followed by statistical analysis. The correlation of Fra-2 and JunB expression was analyzed by scattergram. Our IHC indicated significantly increased frequency of Fra-2 and JunB positive cells according to the tumor progression, i.e., respectively in the early dysplatic bile ducts (10∼20%) and late dysplastic bile ducts (40∼50%), primary ChC (40∼60%) and transplanted ChC (higher than 70% positivity), while normal and simple hyperplastic bile duct epithelia were negative or indicated very low labeling index for both Fra-2 and JunB (under 1% positivity). The expression of Fra-2 and JunB showed a strong correlation (r=0.93) and the immunoreactive cells for Fra-2 and JunB were mainly localized in the invading areas adjacent to normal surrounding tissue. In summary, we found in the present study the followings 1) Expression of Fra-2 and JunB progressively increased as bile duct cells become carcinogenic, 2) Fra-2 and JunB have a high correlation in the expression patterns in the biliary cell populations including cancers, 3) In the transplanted biliary cancers, overexpression of Fra-2 and JunB (especially JunB) were localized in the neoplastic cells which are very aggressive to invade into adjacent normal tissue. Based on our results, Fra-2 and JunB could play significant roles in cholangiocarcinogenesis, possibly by collaboratively participating in dysplastic changes and acquisition of tumorigenicity of candidate bile duct cells for ChC. Also, those two AP-1 family proteins could contribute to invasiveness of biliary cancer cells.

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 1473.
Abstract 1488: Role of human leukocyte antigen-G (HLA-G) in cancer progression and susceptibility
Authors: Abbas Ata , Hussain Syed R. , Naqvi Hena , Fatima Rabab , Hasan Sikandar , Mahdi Farzana
Published: February 2011

Human Leukocyte antigen -G is an immunosuppressive protein with multiple functions. The 14bp insertion / deletion in exon-8 of HLA-G gene have been shown to play an important role in HLA-G mRNA stability as well as expression level; and can be correlated with its immunosuppressive function. The role of HLA-G in various cancers has already been demonstrated. Cancerous cells start expressing HLA-G which in turn provides a shield for various immune responses. In the present study we have tested the possibility of the role of 14bp insertion / deletion polymorphism of HLA-G in cancer progression and susceptibility. We have genotyped 59 cancer samples (26 benign and 33 malignant) and 79 matched controls for 14bp polymorphism of HLA-G. Data showed increase of +14bp/-14bp and −14bp/-14bp genotype with both benign and malignant cancer. We observed significantly higher frequency of +14bp/+14bp genotype in control (p=0.01). This is consistent with both benign and malignant tumors as well as cancer types showing possibility of protective effect of +14bp/+14bp genotype in various cancers. However, further studies will be required with large sample size to test this hypothesis. The +14bp/-14bp and −14bp/-14bp genotypes have already been shown to be associated with higher expression of HLA-G and its mRNA stability and might be providing favorable micro-environment for cancer progression. There is no published work till date showing the role of HLA-G 14bp polymorphism in various cancers. These preliminary data showed that HLA-G 14bp genotypes might be playing an important role in pathogenesis of various cancers.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 1488.
Abstract 1489: Basal cell carcinomas express functional indoleamine 2,3-dioxygenase (IDO) which may confer immunoprotection
Authors: Lo Blanche K. , Jalili Reza B. , Zloty David , Ghahary Aziz , Cowen Bryce , Shapiro Jerry , McElwee Kevin J.
Published: February 2011

Basal cell carcinoma (BCC) is the most common malignancy in humans worldwide. Besides genetic alterations, abrogation of host immune defense has been suggested to enable tumor growth. Cancer cells may express factors that confer resistance to inflammatory cell mediated activity and may actively suppress immune targeting mechanisms. BCCs can derive from hair follicle bulge region keratinocytes; a structure which exhibits elements of immune privilege (IP). As such, BCCs may also exhibit immunoprotective characteristics during tumorigenesis.

Indoleamine 2,3-dioxygenase (IDO) suppresses T lymphocyte proliferation via tryptophan catabolism in the kynurenine pathway. IDO is used as an immune subversion strategy by several types of cancer such as ovarian carcinomas. Expression of IDO has also been identified in hair follicle keratinocytes. Taken together, we hypothesized that IDO might have an impact on the putative IP of BCCs.

In a preliminary study, we identified increased expression of mRNA in BCCs for several IP related factors. In this study, real-time RT-PCR identified significant upregulation of IDO-1 and IDO-2 (12.5- and 19.1-fold change respectively) in human nodular BCCs (n=10) as compared to non-lesional skin epithelium tissues (n=10). Dual label immunohistochemistry revealed co-localization of IDO and keratin 17 (K17), which is a BCC keratinocyte marker, in human BCC tissues (n=5). Western blot detected IDO (42kDa) and a 30kDa IDO isoform in BCC tissues (n = 6). Activity of IDO was shown by significantly increased production of L-kynurenine in the conditioned medium from cell cultures. CXCL11 has recently been shown to be a supplement required for the survival and growth of primary human BCC cells in culture. Addition of CXCL11 to primary BCC cell cultures in increased concentrations promoted increased L-kynurenine in a dose dependent manner. However, there was no significant increase in L-kynurenine production by normal primary human keratinocytes under CXCL11 treatment.

In conclusion, of several differentially expressed genes associated with IP, mRNA for IDO was identified with significantly increased expression. The co-expression of the peptide with K17, as well as the IDO-mediated L-kynurenine production in human BCC cell cultures, provides further evidence of functional IDO synthesis by BCCs during their growth. The results suggest that BCCs may exhibit IP characteristics through IDO production and induction of a local collapse of such IP systems in BCC tumors may be a novel strategy for promoting BCC regression.

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 1489.
Abstract 1490: Overexpression of a calcium channel protein TRPM8 in prostate cancer cells enhances their anchorage-independent growth and resistance to paclitaxel-induced apoptosis
Authors: Yu Shan , Xu Zhenyu , Chan Franky L.
Published: February 2011

The non-voltaged-gated transient receptor potential protein melastatin member 8 (TRPM8) has been characterized to function as a cold- and menthol-activated calcium-permeable channel protein, which it was originally cloned in a prostate-specific substracted cDNA library and shown to be up-regulated in benign prostatic hyperplasia and prostate cancer as compared to normal prostatic epithelial cells. Recent studies in cultured prostate cancer cells and clinical prostate cancer tissues show that TRPM8 is an androgen receptor-regulated calcium channel which is required for the survival and androgen-dependent growth of prostate cancer cells. Based on this evidence, it is believed that TRPM8 may play an important role in the advanced growth of prostate cancer. However, the prostate-specific function of TRPM8 and the role of TRPM8-regulated calcium-influx in prostate function and its carcinogenesis remain unclear. Our preliminary studies showed that TRPM8 was significantly over-expressed in an androgen-independent and metastatic prostate cancer cell line C4-2B as compared to its parental line LNCaP and also in two animal models of metastatic prostate cancers (a transgenic mouse model of prostate cancer-TGMAP and xenograft model of C4-2B). In order to elucidate the functional significance of TRPM8 in prostate cancer cell growth, we over-expressed TRPM8 in an androgen-sensitive prostate cancer cell line LNCaP by lentiviral transduction and generated stable TRPM8 clones for phenotypic growth studies. Our findings showed that stable ectopic TRPM8 expression induced morphological changes in LNCaP cells with reduction of filopodial processes and also suppressed their in vitro cell proliferation as shown by MTT assay. However, stable LNCaP-TRPM8 clones demonstrated significantly enhanced colony formation efficiencies in soft agar assay. Moreover, stable TRPM8 expression also enhanced the in vivo tumor growth of LNCaP cells in immunodeficient SCID mice and resistance to paclitaxel-induced apoptosis, as compared to their parental line. The results of our present study suggest that altered calcium-homeostasis as disturbed by TRPM8 overexpression in prostate cancer cells could enhance their in vitro anchorage-independent and in vivo tumor growth, and also resistance to paclitaxel-induced apoptosis, while the involved calcium-dependent signaling pathways still remain to be elucidated. (Study supported by a RGC Competitive Earmarked Research Grant project code 461007)

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 1490.
Abstract 1494: Characterization of a novel pro-tumorigenic TGF-β RII mutation from oral squamous cell carcinoma
Authors: Son Hwakyung , Park Iha , Che Z.M. , Kim J
Published: February 2011

TGF-β signaling plays various roles in tumor progression. Tumor suppressive TGF- β signaling is involved in early steps of carcinogenesis through loss of function which leads to deregulated proliferation. On the contrary, TGF- β signaling could be pro-tumorigenic in later stages of carcinogenesis by promoting epithelial-mesenchymal transition (EMT). Most of the reported TGF- β receptor-II mutations were loss of tumor suppressor function mutations and no pro-tumorigenic TGF- β receptor-II mutation has been identified in human tumors. This study aimed to analyze a novel mutation of TGF- β receptor-II found in metastatic lymph node of oral squamous cell carcinoma. This mutant converts amino acid 227 from I to T and amino acid 236 from N to D of TGF- β receptor-II. Promoter assay using 3TP-lux reporter showed that 227/236 double mutation significantly increased both basal and TGF- β-induced luciferase activity as compared to wild type in DR-26, TGF- β receptor-II-defective mink lung epithelial cells. Stable expression of 227/236 mutant enhanced migration and invasion as well as cell growth of HSC-2 cells, human oral squamous cancer cell line whose TGF- β signaling is not growth inhibitory. In 227/236 mutant transfectant, E-cadherin expression is reduced whereas snail expression has not been changed significantly. In orthotopic xenograft model to monitor the tumor growth, 227/236 mutant transfectant formed significantly larger tumors as compared to wild type transfectant. But, unexpectedly, there was no difference in the expression pattern of involucrin and E-cadherin in these tumor tissues, suggesting that this mutation did not affect EMT in vivo model system. In the study we present a novel gain-of-function mutation of TGF- β receptor II, which increased TGF- β signaling and thereby induced tumor growth without affecting EMT in HSC-2 oral squamous cancer cells.

This work was supported by a grant from the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic research promotion fund KRF-2005-005-J05901).

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 1494.
Abstract 1496: Oxidatively induced DNA base damage in colorectal carcinoma
Authors: Kirkali Guldal , Keles Didem , Canda Aras Emre , Terzi Cem , Jaruga Pawel , Dizdaroglu Miral , Oktay Gulgun
Published: February 2011

Molecular pathways that play a role in the development of colorectal cancer involve multiple genetic changes in cancer-related genes that may be caused by overproduction of reactive oxygen species (ROS) including free radicals. Various types of ROS are capable of damaging DNA and other biomolecules. Increased formation of ROS can contribute to genetic instability and promote the development of malignancy. These facts led us to hypothesize that oxidatively induced DNA damage may accumulate in colorectal cancer patients. To test this hypothesis, we investigated oxidatively induced DNA damage in colon and rectum tissues of cancer patients in comparison with their normal paired tissues. DNA was isolated from 51 colorectal cancer tissues. The levels of oxidatively induced DNA lesions such as 4,6-diamino-5-formamidopyrimidine (FapyAde), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 8-hydroxyguanine (8-OH-Gua) and (5′S)-8,5′-cyclo-2′-deoxyadenosine (S-cdA) were measured by gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution mass spectrometry. We found that the level of 8-OH-Gua, FapyAde and FapyGua were significantly lower in cancer tissues than those in paired normal tissues (p=0.002, p=0.019 and p=0.005, respectively), whereas the S-cdA levels showed no significant difference between both tissues. These DNA base damages correlated with clinical and pathological data such as tumor location, tumor stage, gender and perineural invasion. The low levels of DNA base damage in tumor tissues when compared to their normal counterparts suggest an increase in DNA repair capacity in malignant tumors that may contribute to the resistance to therapeutic agents. This finding may lead to development of new therapeutic approaches.

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 1496.
Abstract 150: HPV and aberrant DNA methylation status in paired saliva and tumor samples in HNSCC
Authors: Havard Shaleta , Chen Kang Mei , Stephen Josena K. , Worsham Maria J.
Published: February 2011

To examine the utility of non-invasive detection of high risk HPV and aberrant DNA methylation in matched saliva and tumor DNA in squamous head and neck cancer (HNSCC). In a cohort of 35 primary HNSCC with matched tumor (cases) and saliva specimens, HPV status was initially determined using the Linear Array HPV Genotyping kit and confirmed by PCR for HPV subtypes 16, 31, 33 and 45. Aberrant methylation of 24 tumor suppressor genes was interrogated using the methylation specific multiplex ligation probe amplification (MS-MLPA) assay. Of the 35 cases, 20 were negative for HPV in the saliva and tumor, 9 were positive for HPV in both and in 6 cases HPV was positive in the tumor specimen but not detected in the corresponding saliva. In the 9 cases that were positive for HPV in the tumor and matched saliva, 6 were HPV 16 positive and 1 case each was positive for HPV 31, 33 and 45. The sensitivity and specificity for HPV in this cohort were 100% and 60%, respectively. Promoter hypermethylation frequency in tumor samples was 60% (21/35) as compared to 14% (5/35) in saliva. The most frequently methylated genes in tumors included RARB-10/21, APC- 9/21, TIMP3 and CDKN2B- 7/21, CDKN2A and IGSF4- 5/21 tumor samples. The most frequently methylated genes in saliva samples included TIMP3, ESR1 and IGSF4 (3/5 saliva samples), followed by APC, CDKN2B and DAPK1 (2/3). Two of the 35 tumor and saliva pairs had commonly methylated genes, APC in one pair, and TIMP3, CDKN2B, ESR1 and IGSF4 in the other. There was no association between HPV and methylation. PCR analysis of HPV DNA in salivary rinses allows for detection of HPV and promoter methylation status in HNSCC and is a feasible noninvasive alternative to more invasive procedures.

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 150.
Abstract 1501: Tumor cells engineered to codisplay on their surface 4-1BBL and LIGHT costimulatory proteins as a novel vaccine approach for cancer immunotherapy
Authors: Sharma Rajesh K. , Yolcu Esma S. , Elpek Kutlu G. , Shirwan Haval
Published: February 2011

Therapeutic vaccines based on whole tumor cells genetically modified to express various immunomodulatory molecules represent an important modality for the treatment of cancer. However, generation of genetically modified tumor cells as vaccine is not only cost, labor, and time intensive, but also has safety repercussions. As an alternative, we have recently developed the ProtEx™ technology that involves modification of the cell membrane with biotin and engineering of biotinylated cells with proteins of interest chimeric with core streptavidin. We herein tested if tumor cells can be engineered efficiently to simultaneously display on their surface two SA-chimeric costimulatory ligands, SA-4-1BBL and SA-LIGHT, and if vaccination with such engineered cells has better therapeutic efficacy than cells displaying single molecule in the TC-1 cervical cancer mouse model. The choice of 4-1BBL is due to pleiotropic immune activity of 4-1BBL to stimulate innate, adaptive immune responses as well as overcome various immune evasion mechanisms. The choice of LIGHT is due to its stimulatory effects on tumor stroma, which pose a physical barrier to the entry of effector immune cells to tumor site. Biotinylated TC-1 tumor cells codisplayed both chimeric costimulatory ligands on their surface with equal efficiency after incubation with equal molar of SA-4-1BBL and SA-LIGHT proteins. Vaccination with irradiated TC-1 tumor cells codisplaying both chimeric costimulatory ligands resulted in the generation of antigen specific T cell proliferative and killing responses, resulting in 100% efficacy in prophylactic and > 57% efficacy in therapeutic settings. In marked contrast, vaccination with TC-1 tumor cells displaying on their surface either of these molecules singly only delayed the tumor growth in the prophylactic setting. Vaccine efficacy was associated with effective generation of primary and memory T cell and Ab responses, enhanced CD8+ T cell infiltration into the tumor, IFN-γ production, and tumor-specific killing. In conclusion, engineering tumor cells in a rapid and effective manner to simultaneously display on their surface a collection of immunostimulatory molecules with distinct immune functions presents a novel approach with considerable potential for cancer immunotherapy.

Funded in parts by NIH 2R44 AI071618-02, R43 AI074176, R41 CA121665, KLCP, the W.M. Keck Foundation, and the Commonwealth of Kentucky Research Challenge Trust Fund.

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 1501.
Abstract 1503: PSMA Aptamer-targeted siRNAs selectively Eehance prostate cancer radiation sensitivity
Authors: Ni Xiaohua , Zhang Yonggang , Ribas Judit , DeWeese Theodore L. , Lupold Shawn E.
Published: February 2011

The application of dose escalated radiation therapy for localized prostate cancer has a clear therapeutic benefit; however, its clinical use is hampered by the increased risk of damage to surrounding normal tissues. One attractive alternative to elevated doses is radiation sensitizing agents. Prostate cancer cells can be made radiosensitive by targeted reductions of DNA repair proteins via RNA interference. However, without tissue-selective targeting, these agents will also sensitize surrounding tissue, leading to increased adverse effects. Here we describe a strategy where prostate-targeted RNA interference is used to selectively radio-sensitize prostate cancer cells. A high throughput siRNA screen was completed to identify DNA-repair pathway targets for prostate cancer radio-sensitization. Candidate siRNAs were then conjugated to PSMA-targeted RNA aptamers for selective delivery of siRNAs to Prostate cancer cells. In vitro and in vivo data support PSMA and aptamer-specific targeting and gene knock-down. These studies support continued development of this technology as a path to enhance radiotherapeutic outcomes for men with Prostate cancer and it is the first report of targeted, prostate-specific siRNAs for radio-sensitization.

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 1503.
Abstract 1504: Inhibition of RNA-dependent protein kinase (PKR) enhances radiation sensitivity in lung cancer cells
Authors: Pataer Abujiang , Lu Xiaolin , Roth Jack A. , Swisher Stephen G.
Published: February 2011

We have previously demonstrated that radiation resistance in MEFs is mediated in part through RNA-dependent protein kinase (PKR) possibly through upregulation of AKT and NFk-B. The role of PKR in cancer cells has not been evaluated. In this study, we show that the transduction of MEFs and lung cancer cells with adenoviral wt PKR results in nuclear translocation of PKR, activation of AKT1/2 and increased radiation resistance. The transduction of lung cancer cells or MEFs with a dominant negative adenoviral PKR vector blocks nuclear translocation of PKR, inhibits AKT activation and leads to the reversal of radiation resistance. Plasmid transduction of MEFs and lung cancer cells with nuclear targeted wt PKR vectors also results in AKT1/2 activation and increased radiation resistance. This effect is selectively abrogated by plasmid transduction of dominant negative PKR vectors which inhibit AKT1/2 activation and restore radiation sensitivity. These findings suggest a novel role for PKR in lung cancer cells as a mediator of radiation resistance possibly through translocation of the protein product to the nucleus and activation of AKT1/2.

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 1504.
Abstract 1506: Prodrug targeted gene therapy for colorectal cancer
Authors: Kusumanchi Praveen , Zhang Yonghua , Chakka Padma , Jayaram Nagesh H. , Khan Rehana , Antony Asok C. , Jayaram Hiremagalur N.
Published: February 2011

In the US colorectal cancer is the second and third most common cause of cancer death in men and women, respectively. The drawback of conventional therapy is its non-specificity and a median survival of only 10-15 months with the present combinations. Our studies with human colorectal cancer cell lines demonstrate a relationship between human nicotinamide mononucleotide adenylyltransferase-2 (hNMNAT-2) expression and cell kill with tiazofurin (TR) and benzamide riboside (BR). Thus, the purpose of the present study is to examine whether over-expressing hNMNAT-2 selectively confers sensitivity to TR and BR in human colorectal cancer cells that show resistance to TR and BR therapy.

TR and BR are pro-drugs that are metabolized by the action of NMNAT to its active form, TAD (thiazole-4-carboxamide adenine dinucleotide) and BAD (Benzamide adenine dinucleotide), respectively. TAD/BAD potently inhibit inosine-5’-monophosphate dehydrogenase (IMPDH) causing reduced guanylate synthesis resulting in cancer cell death. We achieved 2- to 5-fold increase in expression of hNMNAT-2 by transfecting colorectal cancer cells (Caco-2 and HT-29) with pTracerNMNAT-2 as demonstrated by Western blot and FACS. hNMNAT-2 transfected colorectal cancer cells exhibited a 3- to 7-fold increase in tumor-cell kill with TR and BR. hNMNAT-2 transfected cells did not exhibit modulation of either NAD levels or IMPDH expression.

Folate receptor alpha (FR-α) is overexpressed on the colorectal cancer cell surface to sequester available folate to promote cancer cell proliferation. Primary colorectal carcinomas which exhibit high FR-α overexpression also correlated with decreased 5-year survival. We demonstrated an upregulation of FR-α expression in human colorectal cancer cells (up to 10-fold) by adaptation to growth in physiological low folate medium resulting in 2- to 5-fold increase in colorectal cancer cell-kill with TR or BR encapsulated in folate-tethered liposomes. To translate in vitro studies to in vivo applicability, we are utilizing adeno-associated virus (AAV-2) to transduce hNMNAT-2 gene into colorectal cancers implanted in athymic mice. To achieve this goal, we engineered hNMNAT-2 gene into AAV-2 vector to increase efficiency of gene transfer, and encapsulated in folate-tethered liposomes to target colorectal cancer cells. The potential significance of these new proof-of principle studies is that they will set the stage for the next level of studies that could culminate in a new form of pro-drug targeted gene therapy for colorectal cancer with enhanced tumor cell-kill with little toxicity.

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 1506.
Abstract 1508: Development of a strategy to knock down SDF-1 using shRNA as a means to prevent pulmonary metastasis
Authors: Annan Anand C. , Preuss Meredith A. , Borovjagin Anton V. , Ugai Hideyo , Curiel David T.
Published: February 2011

Metastasis, a poor prognostic indicator causing increased mortality, occurs in an organ specific pattern with lungs and lymph nodes most commonly involved. Cancer cells expressing ‘CXC'-chemokine family Receptor-4 (CXCR4) are chemo-attracted to the pulmonary tissue expressing its cognate ligand Stromal Derived Factor-1 (SDF-1), which aid in formation of metastasis. In this regard, we propose to modulate this axis through knock down of SDF-1 specifically in pulmonary vasculature to prevent lung metastasis. Our laboratory has previously shown that human adenovirus (Ad), when injected via the tail vein into human Coxsackie Adenovirus Receptor (hCAR) transgenic mice, mice that ubiquitously express the hCAR native Ad receptor, transduces pulmonary vasculature preferentially. To this end, we plan to knock- down SDF-1 using shRNA against SDF-1 employing Ad as the vector to deliver them in vivo to achieve pulmonary targeting. To establish a pulmonary metastatic model in the “hCAR” mouse, we injected B16-F10 melanoma cells stably expressing CXCR4 (to cause metastasis) and firefly luciferase (to track tumor cells) via tail vein. Presence of lung metastasis was confirmed by histopathological analysis, immunohistochemistry for CXCR4 and luciferase assay of the tissues. In order to identify a shRNA to deliver to the pulmonary vasculature to decrease SDF-1, we evaluated several plasmid shRNAs for their ability to suppress SDF-1 expression at the mRNA level using qRT-PCR in vitro. From this experiment, we identified one shRNA which gave significant decrease in mRNA of SDF-1 measured by qRT-PCR (>70%). We created an Ad vector expressing this particular shRNA and measured its efficiency of inhibiting SDF-1 expression of approximately 75% for mRNA by qRT-PCR and approximately 90% for protein by Western Blot. Future studies include demonstrating the functional knock-down of SDF-1 in vitro by transwell chamber assay and subsequently analyzing the biological effect of knocking down SDF-1 in vivo in the pulmonary metastatic model established in “hCAR” mouse. In conclusion, therapies available for prevention of metastasis are limited despite different treatment modalities available for cancer. Our approach of targeted knock down of SDF-1 would reduce the metastatic burden to the lungs with potential for clinical translation into a therapeutic approach to prevent lung metastasis.

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 1508.
Abstract 1513: Pre-clinical evaluation of two oncolytic viral vectors expressing IFNβ in treatment of thoracic cancer: Vaccinia virus versus vesicular stomatitis virus
Authors: Wang Liang-Chuan Steve , Saloura Valia , Alexander Edward , Cheng Guanjun , Kapoor Veena , Vile Richard , Thorne Stephen H. , Isaacs Stuart N. , Albelda Steven M.
Published: February 2011

Rationale: Given recent success in delivering interferon-beta (IFNβ) using replication incompetent adenovirus in patients with malignant pleural effusions, we hypothesized that vectors that could replicate (inducing oncolysis) and express IFNβ at high level would be more effective. Both vaccinia virus (VV) and vesicular stomatitis (VSV) expressing mouse IFNβ have been constructed in a manner to improve their safety and efficacy as oncolytic vectors. In this study, we evaluated these two oncolytic vectors in different murine thoracic cancer models for clinical translation.

Experimental procedures: We compared the efficacy of VVIFNβ and VSVIFNβ to kill 3 mouse lung cancer and 4 mouse mesothelioma lines in vitro over a 5 day-period assessing cell viability by MTT assay. The amount of IFNβ produced by these two vectors were measured with ELISA. The viral replication in infected mice was assessed by measuring viral titers in both immunocompetent and immunodeficient mice bearing an IFN-non-responsive AB12 tumor, following intratumoral injection of either vector at 108 pfu/mouse. To assess their anti-tumor activity, the two vectors were also given at 108 pfu/mouse intratumorally in mice bearing two lung cancer tumors on their flanks.

Results: The replication of VSVIFNβ and its killing ability was limited in some of the tumor lines. This appeared to be due to an intact IFN response pathway. In contrast, VVIFNβ was able to kill all of the tumor lines. In culture, VSV replicates faster than VV, which resulted in faster cell killing (in susceptible cell lines), but with less IFNβ production. The amount of IFNβ production by VVIFNβ was comparable with AdIFNβ over a 5 day period. VVIFNβ-infected immunodeficient mice showed minimal or no toxicity when given virus intravenously, while all 5 mice infected with VSVIFNβ developed neurotoxicity within a week. In the TC-1 lung cancer model, VVIFNβ caused tumor regression, while VSVIFNβ and AdIFNβ were completely ineffective. In a second model using LKRM2, all 3 viral vectors were equivalent. The anti-tumor effect of VVIFNβ was shown to be mainly CD8 dependant, as depletion of CD8 with antibody dampened the tumor growth inhibition from 48% to 20%.

Conclusions: Overall, VVIFNβ appears to be a better oncolytic vector than VSVIFNβ for a number of reasons: 1) it is safer, 2) it is able to replicate in vitro and in vivo in almost every cell line tested, 3) it induces longer and larger IFNβ production, and 4) it can potentially be given intravenously.

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 1513.
Abstract 1514: Oncolytic viral therapy for glioblastoma induces robust natural killer cell activation
Authors: Alvarez-Breckenridge Christopher A. , Yu Jianhua , Pradarelli Jason , Liu Tom , Kaur Balveen , Caligiuri Michael , Chiocca E. Antonio
Published: February 2011

Oncolytic viral (OV) therapy is a promising treatment being tested for safety and efficacy. Natural killer cells (NK) limit viral infections, and previous work suggests they may similarly attenuate virotherapy. We used flow cytometry to evaluate the temporal pattern of NK recruitment in intracranial tumors (U87DEGFR glioma) treated with the oncolytic herpes virus, rQnestin 34.5, or PBS. Quantification of NK recruitment into intracranial tumors treated with rQnestin34.5 or PBS revealed significant enrichment of these cells into the tumor hemisphere at early timepoints and continuing through 72 hours (2.2-fold, p=0.02) following infection. Moreover, NK cells recruited to the site of inoculation exhibited an activated phenotype, including enhanced CD69, CD62L, CD27, NKG2D, and CD127 staining compared to PBS treated mice. This robust NK response was demonstrated to be detrimental to OV efficacy through the enhanced survival of NK depleted mice treated with OV compared to OV treated mice containing NK cells (median survival 31.6 days compared to median survival 23.6 days, p=0.04). Since NK cells are recruited into the tumor microenvironment following infection, we postulated that OV infection of glioma cells was also activating NK cells. We measured granzyme B and IFNg production by NK cells in the presence of infected or uninfected glioma. Our results indicate that glioma mediated activation of NK cells was augmented by OV infection. Additionally, these findings were corroborated with increased NK killing of OV infected glioma compared to uninfected glioma. Due to the enhanced NK cytotoxic properties induced by OV infection, we proceeded to investigate the expression of NK activating ligands on the surface of infected glioma cells that could potentially mediate this effect. Focusing on ligands for NKG2D and the natural cytotoxicity receptors (NKp30, NKp44, and NKp46), viral infection of cultured gliomas was shown to upregulate ligand expression for NKG2D (24%, p=0.004), NKp46 (41%, p=0.01), and NKp30 (2.75-fold, p=0.001) while selectively downregulating ligand expression for NKp44 (2.8-fold, p=0.005). These findings suggest that viral infection of gliomas is altering the ligand signature for NK activation. Since NK cells possess both antiviral and antitumor properties, further work will investigate if virus induced NK activating signals can be selectively blocked in order to enhance viral efficacy while maintaining NK antitumor efficacy. This is the first study investigating the changes in NK cell activation and recruitment in gliomas upon oncolytic HSV-1 treatment.

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 1514.
Abstract 1516: In vivo tumor targeted delivery of siRNA by biodegradable nanoparticles
Authors: Woodle Martin C. , Scaria Puthupparampil , Liu Yijia , Spears Ryan , Zhang Betty Hongxue
Published: February 2011

We are developing synthetic, engineered, ElectroNanoPlex™ nanoparticles for tumor targeted in vivo delivery of siRNA, plasmids, and other nucleic acids. These nanoparticles employ biodegradable cationic polyamide macromolecules that form an electrostatic association with the anionic nucleic acid cargo, and provide a hydrophilic steric polymer coating on the surface of the nanoparticles. Conditions have been identified to form siRNA or plasmid carrying nanoparticles with a mean diameter of approximately 100 nm, which falls within the range appropriate for enhanced permeability and retention (EPR) localization in tumor tissue. We have successfully delivered nucleic acids in vivo to solid tumors using nanoparticles with the steric coating but without a ligand for specific targeting, suggesting a mechanism of action based on EPR targeting. These nanoparticles are being commercialized under the tradename InVivoPlex™ for tumor gene function research, and drug target validation, in murine xenograft tumor models. Compared to other in vivo delivery technologies these nanoparticles show vastly diminished expression of plasmid DNA in lung, spleen, liver and kidney. Also, plasmid DNA and siRNA oligonucleotides can be co-packaged and delivered together using these nanoparticles, permitting studies of simultaneous down-regulation and up-regulation of different genes. Active targeting to cell surface receptors or antigens may be employed by decorating the nanoparticle surface with peptides such as cyclic RGD or monoclonal antibodies. Studies of antibody-mediated tumor cell delivery by ligand decorated nanoparticles will be presented. These nanoparticle systems will advance tumor targeted research using siRNA and are expected to eventually enable therapeutic applications of siRNA, or other nucleic acid agents that are promising in light of their molecular biological properties but currently face daunting pharmaceutical delivery challenges.

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 1516.