bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 392: Delineating genomic heterogeneity in paired primary and metastatic colorectal cancer by massively parallel sequencing.
Authors: Brannon Angela Rose , Vakiani Efsevia , Scott Sasinya N. , Sylvester Brooke , Kania Krishan , Viale Agnes , Kemeny Nancy , Weiser Martin , Solit David B. , Berger Michael F.
Published: August 2014

Colorectal cancer (CRC) is the third most common cause of cancer and cancer death for men and women in the United States. While the genetic steps necessary for carcinogenesis are well-defined, the subsequent genetic alterations driving tumor evolution to metastasis are not well characterized. We have performed deep sequencing of 230 key cancer-associated genes in 60 patient-matched primary tumor, metastatic tumor, and normal samples from CRC patients. Genes were selected on the basis of recurrent and/or “actionable” mutations reported in human tumors. Our custom captured-based sequencing assay, termed IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), provided a median sequence coverage of 644-fold, allowing for the identification of low frequency genetic events involving target genes. Somatic mutations, insertions/deletions (indels), and copy number alterations were identified and compared in both primary and metastatic samples. The most frequently mutated genes have all been previously implicated in CRC: APC, TP53, KRAS, PIK3CA, and SMAD4. Most genetic events were identical between primary and metastatic tumors; however, we identified particular alterations specific to the metastatic samples. The majority of these alterations were members of two important proliferative pathways, further lending credence that they might play an important role in metastatic progression. For patients with metastasis-specific mutations, we have sampled multiple microdissected regions from primary tumors to quantify the extent of intratumor heterogeneity and ascertain whether these differences are caused by sampling biases. The identification of these genetic variations between primary and metastatic tumors could have significant impact on biomarker analysis as well as treatment decisions for CRC patients.

Citation Format: Angela Rose Brannon, Efsevia Vakiani, Sasinya N. Scott, Brooke Sylvester, Krishan Kania, Agnes Viale, Nancy Kemeny, Martin Weiser, David B. Solit, Michael F. Berger. Delineating genomic heterogeneity in paired primary and metastatic colorectal cancer by massively parallel sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 392. doi:10.1158/1538-7445.AM2013-392
Abstract 3920: In vivo tyrosinase reporter gene imaging with multispectral photoacoustic technology.
Authors: Heinmiller Andrew , Lakshman Minalini , Bates Dave , Needles Andrew , Theodoropoulos Catherine , Paproski Robert J. , Zemp Roger J.
Published: August 2014

Photoacoustic imaging is a powerful tool for assessing tumor vasculature and oxygen saturation as well as detecting contrast agents for molecular imaging. Another exciting possibility is to visualize gene expression by using a reporter which produces a photoacoustic contrast agent. Tyrosinase is such a reporter in that its expression results in the production of melanin, which gives a strong photoacoustic signal. Here we describe the use of a commercially available photoacoustic (PA) imaging system (Vevo LAZR, VisualSonics, Toronto) to image inducible gene expression in subcutaneous xenograft tumors using tyrosinase as a reporter gene.

The photoacoustic imaging system generated light from a tunable laser (680 - 970 nm) which was delivered through fiber optic bundles integrated into a linear array transducer (LZ-250, fc = 21 MHz), mounted to a linear stepper motor for 3D imaging. Animals (n=3) having MCF-7 xenograft tumors on either flank transfected with (+TYR) or without (-TYR) doxycycline-regulated tyrosinase were imaged before and one week after the induction of tyrosinase expression. 3D images of the tumors were acquired using multiple wavelengths (680, 750, 800, 850, 900 and 950nm) and 2D images were acquired across the entire wavelength range of the laser to generate absorption curves for blood and melanin. To confirm the presence of melanin and distinguish it from the endogenous blood signal, one animal was exsanguinated and the tumor was imaged again. Approximately 1mm-thick slices of the tumors were taken and photographed for visual confirmation of the presence of melanin.

Comparison of pre- and post-doxycycline images of +TYR tumors clearly showed enhanced contrast in the tumor which closely matched the absorption spectrum of melanin. This signal persisted upon exsanguination. -TYR tumors showed signal which corresponded with the spectra of oxy and deoxy hemoglobin and changed little over the course of the experiment. Visual inspection of the excised sliced tumors revealed pigmentation in the +TYR tumors which was absent in the -TYR tumors.

Here we have shown the ability of the Vevo LAZR photoacoustic imaging system to visualize inducible reporter gene expression in vivo. This has implications for assessing genetically controlled cellular processes and their response to anti-cancer drugs but also for monitoring gene therapy for cancer treatment non-invasively.

Citation Format: Andrew Heinmiller, Minalini Lakshman, Dave Bates, Andrew Needles, Catherine Theodoropoulos, Robert J. Paproski, Roger J. Zemp. In vivo tyrosinase reporter gene imaging with multispectral photoacoustic technology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3920. doi:10.1158/1538-7445.AM2013-3920
Abstract 3928: In vivo ERK1/2 reporting demonstrates the association and timing of ERK1/2 pathway reactivation and acquired resistance to RAF inhibitors.
Authors: Basile Kevin J. , Abel Ethan V. , Dadpey Neda , Aplin Andrew E.
Published: August 2014

Patients with V600 mutant BRAF positive metastatic melanoma are now treated with the RAF inhibitor, vemurafenib (Zelboraf), as a first line therapy. Although most patients treated with vemurafenib have tumor shrinkage, other patients exhibit disease progression. Furthermore, most patients who initially respond to the drug eventually develop acquired resistance to vemurafenib. Disease progression while on vemurafenib frequently correlates with ERK1/2 pathway reactivation but previous studies have been unable to precisely monitor the temporal changes in ERK1/2 activity. Here, we establish a cell-based ERK1/2 reporter system that provides quantitative analysis of RAF inhibitor action in mutant BRAF melanoma cells in vitro and in vivo. This system was utilized to show that the RAF inhibitor, PLX4720, effectively targets the ERK1/2 pathway in vivo and that ERK1/2 reactivation is associated with tumor re-growth. Levels of ERK1/2 reactivation varied substantially between tumors and were associated with several different molecular changes. These data present a new system from monitoring in vivo efficacy of ERK1/2 pathway inhibitors and identifying mechanisms of in vivo acquired resistance.

Citation Format: Kevin J. Basile, Ethan V. Abel, Neda Dadpey, Andrew E. Aplin. In vivo ERK1/2 reporting demonstrates the association and timing of ERK1/2 pathway reactivation and acquired resistance to RAF inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2013-3928
Abstract 3932: High resolution imaging mass spectrometry reveals the breast cancer cell-related profiles of phosphatidylinositol unsaturation.
Authors: Kawashima Masahiro , Iwamoto Noriko , Kawaguchi-Sakita Nobuko , Sugimoto Masahiro , Ueno Takayuki , Terasawa Kazuya , Sato Taka-Aki , Tanaka Koichi , Shimizu Kazuharu , Toi Masakazu
Published: August 2014

Lipid metabolism is supposed to be altered in breast cancer, and several literatures have suggested the possible compositional change of phospholipids in breast cancer tissues and its correlations with the tumor grade, sex hormone receptor status and patient's prognosis. However, the breast cancer cell-related alteration of phospholipid profiles and its underlying metabolic change remain unclear. The aim of this study is to investigate the cancer cell-related phospholipid alteration by using high resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). We evaluated the spatial distributions and the proportions of phosphatidylinositols (PIs) in seven breast cancer tissues (three luminal cases and four non-luminal cases including one Her2 positive case) and one normal breast tissue. MS images of PIs and their mass spectra showed that the fatty acyl chain compositions of observed PIs remarkably changed in cancer cell region, compared with those in normal duct epithelium or surrounding stromal cells. PIs containing polyunsaturated fatty acid (PUFA) were the major components in normal duct epithelium. In five of seven cancer cases, PIs containing monounsaturated fatty acid (MUFA) were the most dominant in the observed PIs and specifically upregulated in cancer cells. In the rest of the cases, the MUFA upregulation was less obvious than the aforementioned five cases, and PIs containing PUFA still constituted a large percentage of observed PIs. In non-luminal cases, the MUFA upregulation in cancer cells was well correlated with the expression level of stearoyl CoA desaturase 1 (SCD1), a key enzyme in de novo MUFA synthesis. This observation suggested that the upregulation of de novo MUFA synthesis in cancer cells could contribute to the increase of PIs containing MUFA. In conclusion, high resolution MALDI-IMS revealed the breast cancer cell-related changes of PI profiles according to the degree of the fatty acyl chain unsaturation, which implied the existence of cancer cell-related PI remodeling process.

Citation Format: Masahiro Kawashima, Noriko Iwamoto, Nobuko Kawaguchi-Sakita, Masahiro Sugimoto, Takayuki Ueno, Kazuya Terasawa, Taka-Aki Sato, Koichi Tanaka, Kazuharu Shimizu, Masakazu Toi. High resolution imaging mass spectrometry reveals the breast cancer cell-related profiles of phosphatidylinositol unsaturation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3932. doi:10.1158/1538-7445.AM2013-3932
Abstract 3938: Role of Chi3l1/YKL-40 in the tumor progression and metastasis in the lung.
Authors: Lee Chun Geun , Ma Bing , Koo Ja Seok , Herbst Roy S. , Elias Jack A.
Published: August 2014

The 18 glycosyl hydrolase gene family (GH18) contains chitinases and chitinase-like proteins (CLP) that lack enzyme activity. Chitinase 3-like-1 (Chi3l1; also called BRP-39 in mouse and YKL-40 in man), the prototypic CLP, is expressed in an exaggerated fashion in the serum and or tissues from patients with a variety of malignancies including breast, colon/rectum, ovary, melanoma, prostate, kidney, brain, bone, and lung. In many of these malignancies, there is a strong correlation between the levels of Chi3l1/YKL-40 and disease progression, prognosis and disease-free survival. However, the roles of YKL-40 in the development and progression of lung cancer has not been determined. To begin to address this issue, we generated Chi3l1/BRP-39 null mutant (Chi3l1-/-) and lung-specific overexpressing YKL-40 transgenic (YKL-40 Tg) mice and evaluated them in a number of settings. The studies demonstrated that Chi3l1/BRP-39/YKL-40 is an important regulator of cellular apoptosis, inflammation, angiogenesis and M2 macrophage differentiation. We also found that melanoma metastasis to the lung was significantly decreased in Chi3l1-/- mice and increased in YKL-40 Tg mice and that activation of the RIG-like helicase (RLH) innate immune pathway significantly decreased BRP-39 production and melanoma or breast cancer lung metastasis. Lastly, immunohistochemical (IHC) analysis demonstrated that, in mice with specific mutation of KRAS and/or p53, Chi3l1/BRP-39 is expressed in an exaggerated manner in normal peritumor tissues and lung cancer cells in early and late stages of lung cancer development, respectively. In combination, our preliminary studies demonstrate that Chi3l1/BRP-39/YKL-40 plays a critical role in the generation of a metastasis-permissive microenvironment and is dysregulated in the course of the development of primary lung cancers.

Citation Format: Chun Geun Lee, Bing Ma, Ja Seok Koo, Roy S. Herbst, Jack A. Elias. Role of Chi3l1/YKL-40 in the tumor progression and metastasis in the lung. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3938. doi:10.1158/1538-7445.AM2013-3938
Abstract 3939: The collagen receptor discoidin domain receptor 2 facilitates breast cancer metastasis by stabilizing Snail1 protein.
Authors: Zhang Kun , Corsa Callie , Prior Julie , Piwnica-Worms David , Longmore Gregory D.
Published: August 2014

Mammographic density is a leading risk factor for the development of breast cancer and these cancers are more invasive and metastatic. The increase in breast density is, in part, due to increased fibrillar collagen deposition. In an RNAi human kinome screen to identify post-transcriptional regulators of Snail1 protein stability we found the collagen receptor DDR2, but not related DDR1, to stabilize Snail1 protein as a result of collagen I activated DDR2 stimulation of ERK2 activity. This can occur independent of β1-integrin and TGFβ signals. DDR2 is not required for TGFβ-induced Epithelial Mesenchymal transition (EMT) or EMT-induced Snail1 transcription. Rather DDR2 expression, which is absent in normal breast epithelia, is induced during EMT as breast tumor cells assume invasive, mesenchymal features. DDR2-induced ERK2 activity stabilizes Snail1 protein level in two ways. First, ERK2 interacts with and phosphorylates Snail1 and second ERK2 inhibits GSK3b.

DDR2 was found to be critical for breast cancer cell invasion and migration in vitro, in 3D collagen gels, and breast cancer metastasis in vivo, in syngeneic xenograft models. A majority of human invasive ductal breast carcinomas express DDR2 protein and there is a strong association between nuclear Snail1 expression, DDR2 expression, and the absence of E-cadherin expression. The small group of breast cancer patients with tumors harboring increased DDR2 gene copy number has diminished survival. We propose that DDR2 acts in a positive feedback loop to maintain Snail1 protein level and activity in breast tumor cells at the leading, invasive edge where tumor cells that have undergone EMT and invaded through the basement membrane to encounter extracellular matrix collagen I. This facilitates the continued tumor cell invasion and migration through the collagen I-rich ECM, and thereby, contributes to breast cancer metastasis.

That the collagen I receptor DDR2 maintains the EMT-inducing Snail1 protein level and activity and contributes to breast cancer metastasis could explain the increase in metastasis of breast cancers in women with dense breasts. As such, DDR2 could be considered a novel RTK target for the treatment of breast cancer metastasis.

Citation Format: Kun Zhang, Callie Corsa, Julie Prior, David Piwnica-Worms, Gregory D. Longmore. The collagen receptor discoidin domain receptor 2 facilitates breast cancer metastasis by stabilizing Snail1 protein. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3939. doi:10.1158/1538-7445.AM2013-3939
Abstract 3944: Serine phosphorylation of vasodilator-stimulated phosphoprotein regulates the tumorigenic potential of colon cancer cells.
Authors: Ali Mehboob , Chervoneva Inna , Pitari Giovanni M.
Published: August 2014

Colorectal cancer is one of the most common and deadly cancers in developed nations. Molecular mechanisms precisely defining colorectal tumorigenesis and cancer progression remain unclear, an obstacle for innovative research applications. The vasodilator-stimulated phosphoprotein (VASP) is an actin-binding protein which critically regulates the cytoskeleton and its dependent functions, including cell shape, adhesion and migration. Of relevance, VASP Ser phosphorylation becomes deregulated in colorectal transformation and promotes invasive protrusion dynamics. Here, VASP Ser phosphorylation was selectively inhibited in colon carcinoma cells employing dominant negative mutants. Effects on cell tumorigenic potential were examined with two mouse models of colorectal cancer. Compared to control conditions, suppression of VASP Ser239 phosphorylation resulted in an enhanced cancer cell capability to establish tumor colonies in both the subcutaneous and peritoneal xenograft model. Increased tumorigenicity reflected, in part, hyper proliferative kinetics of cancer cells bearing VASP Ser239 mutants, which exhibited accelerated growth and DNA synthesis rates. In contrast, selective inhibition of VASP Ser157 slowed cell proliferation and reduced the in vivo tumorigenic potential of colon carcinoma cells, compared to respective controls. These observations support the suggestion that VASP Ser phosphorylation represents a fine signaling mechanism to control intestinal tumorigenesis and invasion, a novel paradigm for targeted anticancer strategies in patients with colorectal cancer.

Citation Format: Mehboob Ali, Inna Chervoneva, Giovanni M. Pitari. Serine phosphorylation of vasodilator-stimulated phosphoprotein regulates the tumorigenic potential of colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2013-3944
Abstract 3950: Extracellular protons promote the metastasis of breast cancer via activation of the proton-sensing receptor G-protein coupled receptor 4.
Authors: Lee Yu Jin , Heo Kyun , Park Soo-Ah , Noh Dong-Young , Kim Kyong-Tai , Ryu Sung Ho , Suh Pann-Ghill Suh
Published: August 2014

Acidosis of the tumor microenvironment is a typical malignant phenotype that enhances tumorigenesis and tumor progression induced by altered cellular signaling and transcriptional regulation. However, the underlying mechanism by which tumor cells sense and response the acidic environment remains unclear. In this study, we found that expression of G-protein coupled receptor (GPR4) was upregulated in breast cancer cells and tissues from patients with breast cancer compared to normal breast cells and tissues. In a xenograft mouse model, short hairpin RNA (shRNA)-mediated silencing of endogenous GPR4 significantly reduced distant metastases to several lymph nodes and the lung compared to control shRNA. In addition, endogenous GPR4 expression in breast cancer cells enhanced tumor cell migration and invasion in an acidic environment and induced the activation of RhoA and extracellular signal-regulated kinase (ERK) pathway. Acidosis/GPR4 activation also upregulated the expression of matrix metalloproteinase-9 (MMP9) and vascular endothelial growth factor (VEGF) in breast cancer cells. These findings highlight a novel mechanism by which acidosis-mediated tumor aggressiveness is acquired as a critical step for distant metastasis in breast cancer.

Citation Format: Yu Jin Lee, Kyun Heo, Soo-Ah Park, Dong-Young Noh, Kyong-Tai Kim, Sung Ho Ryu, Pann-Ghill Suh Suh. Extracellular protons promote the metastasis of breast cancer via activation of the proton-sensing receptor G-protein coupled receptor 4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3950. doi:10.1158/1538-7445.AM2013-3950
Abstract 3953: The omental microenvironment promotes ovarian cancer metastasis and enhances CD-133 mediated self-assembly of ovarian cancer spheroids.
Authors: Brewer Molly A. , Sun Guigin , Anderson Matthew , Meng Fanliang , Zhong Mei
Published: August 2014

Objective: Recent evidence suggests that the human omentum functions as a niche that promotes ovarian cancer metastasis. We investigated the role of the omentum in sequestration and enhancement of ovarian cancer stem cells.

Materials and Methods: To further explore this unique phenomenon, we examined the ability of cells isolated from ovarian cancer implants at distinct anatomic sites to create multicellular aggregates known as spheroids. In ovarian and other cancers, spheroids promote a pluripotent phenotype and play a critical role in metastasis.

Results: We found that ovarian cancer cells recovered from omentum express higher levels of the pluripotency-associated antigen CD133 and create larger numbers of more rapidly growing spheroids than cells recovered from the tubo-ovarian complex. Although CD133+ or CD44+ ovarian cancer cells are equally capable of creating large spheroids, spheroids self-assembled from CD133+ ovarian cancer cells are significantly more tumorigenic in vivo than CD44+, CD133−CD44− or unsorted cells. We also found that spheroids self-assembled from CD133+ ovarian cancer cells migrate to both the ovary and omentum of SCID mice and that CD133+ ovarian cancer cells induce intraperitoneal xenografts when inoculated subcutaneously. In contrast, our data indicate that spheroids self-assembled from CD44+ cells preferentially induce tumor adjacent to their inoculation site. Targeting CD133 expression inhibits the in vivo tumorigencity of spheroids assembled from A2780 ovarian cancer cells.

Conclusions: In addition to delineating novel pathways by which the omental microenvironment promotes ovarian cancer metastasis, these findings establish a new paradigm for understanding how ovarian cancer stem cells can be potentially maintained or regenerated within the peritoneal cavity. Strategies to target the omental niche have enormous potential to improve ovarian cancer outcomes.

Citation Format: Molly A. Brewer, Guigin Sun, Matthew Anderson, Fanliang Meng, Mei Zhong. The omental microenvironment promotes ovarian cancer metastasis and enhances CD-133 mediated self-assembly of ovarian cancer spheroids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3953. doi:10.1158/1538-7445.AM2013-3953
Abstract 3960: Whole body irradiation extends the duration of antitumor immunity toward established prostate tumors by adoptively transferred CD8+ T cells in TRAMP mice.
Authors: Ward-Kavanagh Lindsay , Cooper Timothy , Schell Todd D.
Published: August 2014

Adoptive T cell transfer following whole body irradiation (WBI) can induce dramatic tumor regression in a subset of metastatic melanoma patients. We previously found that this approach also eliminates established autochthonous SV40 T-antigen induced murine brain tumors. However, the broader utility of this immunotherapy approach has not been systematically evaluated in solid tumors of different tissue origin. Here, we assessed the efficacy of applying WBI with adoptive T cell transfer to an SV40 T-antigen driven murine model of prostate cancer (TRAMP) previously shown to rapidly tolerize tumor-specific CD8+ T cells. TRAMP mice bearing established prostate tumors were treated with 475cGy WBI followed by adoptive transfer of T-antigen-specific T cell receptor transgenic CD8+ T cells. Accumulation, phenotype and function of the transferred T cells were monitored by flow cytometry, while therapeutic impact on mouse prostate tumors was assessed by histological scoring. Transferred T cells proliferated in both irradiated and non-irradiated TRAMP mice, but accumulated to significantly higher levels and persisted longer in the secondary lymphoid organs and prostate of irradiated mice. WBI also significantly increased the proportion of T cells that produced the effector molecule granzyme B. Prostate-infiltrating T cells in both treatment groups exhibited an effector differentiated phenotype, although neither developed a significant population of IFNγ-producing cells. Gross observations revealed that the urogenital tract in irradiated TRAMP mice was significantly smaller than that of non-irradiated mice two weeks after transfer, corresponding with a modest but statistically significant drop in pathological score. By three weeks after transfer, when prostate-infiltrating T cells had dramatically contracted in irradiated TRAMP mice, cumulative histological scores were generally equivalent although only mice in the non-irradiated treatment group had progressed to adenocarcinoma. Our results suggest that the addition of irradiation extends the persistence of functional tumor-specific T cells in the highly tolerogenic microenvironment of the TRAMP prostate. T cell accumulation was associated with a short-term delay in progression of established tumors.

Citation Format: Lindsay Ward-Kavanagh, Timothy Cooper, Todd D. Schell. Whole body irradiation extends the duration of antitumor immunity toward established prostate tumors by adoptively transferred CD8+ T cells in TRAMP mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3960. doi:10.1158/1538-7445.AM2013-3960
Abstract 3967: ErbB2/HER2-specific natural killer cells for adoptive immunotherapy of glioblastoma.
Authors: Zhang Concong , Schönfeld Kurt , Burger Michael , Genßler Sabrina , Sahm Christiane , Brendel Christian , Naundorf Sonja , Odendahl Marcus , Köhl Ulrike , Tonn Torsten , Grez Manuel , Steinbach Joachim P. , Wels Winfried S.
Published: August 2014

In addition to primary natural killer (NK) cells, continuously growing cytotoxic cell lines such as NK-92 are being considered for adoptive cancer immunotherapy. High cytotoxicity of NK-92 has been shown against malignant cells of hematologic origin in preclinical studies, and general safety of infusion of NK-92 cells has been established in phase I clinical trials. To enhance their therapeutic utility, we genetically modified NK-92 cells to express chimeric antigen receptors (CAR) specific for different tumor-associated surface antigens including ErbB2 (HER2). Such CAR were composed of a tumor-specific scFv antibody fragment fused via hinge and transmembrane domains to intracellular signaling proteins such as CD3 zeta chain or a composite CD28-CD3 zeta fusion molecule.

Glioblastoma multiforme (GBM) is the most common and severe intracranial malignant tumor in humans. Despite aggressive therapy, recurrence of GBM is very frequent, and the median survival of GBM patients is only 12 to 15 months. Since enhanced ErbB2 expression was found in up to 80% of GBM cases, adoptive ErbB2-targeted immunotherapy may represent a more efficient alternative to standard therapy. For development towards clinical applications, here we generated a lentiviral second generation CAR construct (5.28.z) specific for the ErbB2 antigen, and established GMP-compliant protocols for transduction and expansion of NK-92 cells. An ErbB2-specific single cell clone (NK-92/5.28.z) was isolated, which showed high and selective cytotoxicity towards different established ErbB2-expressing glioblastoma cells and tumor cells of various other origins in vitro, as well as specific tumor homing in murine in vivo models. Treatment with NK-92/5.28.z cells also resulted in marked inhibition of the growth of subcutaneous glioblastoma xenografts in NOD/SCID γc KO mice. Ongoing work now focuses on evaluating the feasibility and efficacy of intracranial application of NK-92/5.28.z cells in orthotopic xenograft models of ErbB2-positive glioblastoma cells as a basis for further development of these cells as an adoptive immunotherapy for glioblastoma patients.

Citation Format: Concong Zhang, Kurt Schönfeld, Michael Burger, Sabrina Genßler, Christiane Sahm, Christian Brendel, Sonja Naundorf, Marcus Odendahl, Ulrike Köhl, Torsten Tonn, Manuel Grez, Joachim P. Steinbach, Winfried S. Wels. ErbB2/HER2-specific natural killer cells for adoptive immunotherapy of glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3967. doi:10.1158/1538-7445.AM2013-3967
Abstract 3975: Regression of established tumors requires timely adoptive T cell transfer following whole body irradiation.
Authors: Cozza Eugene M. , Budgeon Lynn R. , Christensen Neil D. , Schell Todd D.
Published: August 2014

Adoptive immunotherapy with CD8+ T cells has achieved some success in treating established cancer. Response rates are increased by preconditioning the patient with lymphodepleting whole body irradiation (WBI) prior to T cell transfer, including an increased fraction of complete responses. Using a mouse model of autochthonous brain cancer, we previously demonstrated that adoptive transfer of tumor-specific T cells following WBI promotes complete regression of established tumors and long-term survival. However, the mechanisms required to achieve complete responses remain to be defined. Here we investigate kinetic requirements for adoptive T cell transfer to achieve tumor control following WBI. To test the hypothesis that adoptive transfer early after irradiation is required for maximum long-term tumor control, we delayed adoptive transfer of T cells to tumor-bearing mice at varying intervals following irradiation. We show that delay of adoptive transfer did not affect initial T cell priming or the timing of initial appearance of activated T cells at the tumor site. However, we observed a modest decrease in T cell accumulation at the earliest appearance of cells in the brain (5 days post-transfer). Tumors were completely eliminated within 10 days in mice that received T cell transfer one day following WBI, while delayed transfer abrogated this effect. When adoptive transfer was delayed by one week following WBI, incomplete tumor regression was observed at 10 days post-transfer. Although mice in this group lived significantly longer than control animals, they eventually succumbed to lethal tumor recurrence. Our data suggest that lymphodepleting doses of WBI promote a prolonged window for effective T cell priming against the endogenous tumor antigen, but that optimal therapeutic potential requires T cell transfer early after irradiation conditioning. Supported by research grant CA02500 from the National Cancer Institute/National Institutes of Health.

Citation Format: Eugene M. Cozza, Lynn R. Budgeon, Neil D. Christensen, Todd D. Schell. Regression of established tumors requires timely adoptive T cell transfer following whole body irradiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3975. doi:10.1158/1538-7445.AM2013-3975
Abstract 398: Genome-wide comparison of matched canine osteosarcoma primary tumours and metastases by array comparative genomic hybridization.
Authors: Liu Jonathan H W , Shao Yang Washington , Molyneux Sam D. , Khokha Rama , Wood Geoffrey A.
Published: August 2014

Canine osteosarcoma is a common and highly aggressive cancer that shares many characteristics with human osteosarcoma and can serve as a model of this disease. We collected snap frozen normal muscle, primary appendicular skeleton tumour, and matched terminal lung metastases from 8 canine osteosarcoma patients. DNA was isolated from these samples and gene copy number aberrations were assessed using high-resolution oligonucleotide array comparative genomic hybridization (Agilent G3 Canine 180k array). Data analysis was performed using Agilent Genomic Workbench 5.0 analysis software and the Aberration Detection Method 2 and Circular Binary Segmentation algorithms were applied to identify aberrant chromosomal intervals with the log2 ratio threshold set to 0.5. As expected, many cases had copy number aberrations that contain genes commonly altered in canine and human osteosarcoma, including MYC, CDKN2A/CDKN2B, PTEN, p53, and RB. To assess the degree of genomic similarity of the 8 primary tumours to their corresponding metastasis, we used these same samples to generate 8 random pairings of unmatched primaries and metastases, as well as 8 random pairs of primary tumours and 8 random pairs of metastases. A set of 11,000 random 10 base pair segments spanning the entire genome was generated and a correlation analysis was conducted between pairs of samples using the log2 ratios for each segment. Matched samples showed significant correlation with the square of the correlation coefficient (R2) being 0.44 (p<0.05), whereas unmatched primaries and metastases, random pairs of primaries, and random pairs of metastases did not show any significant correlation (R2= 0.08, 0.08, and 0.06 respectively, p>0.05 for each). Thus, as a group, primary tumours are genomically more similar to their corresponding metastasis than to unmatched metastases, and are also more similar than primary tumours or metastases are to each other. However, the range of similarity between matched primaries and metastases was relatively wide, with half the cases showing very high similarity and half showing moderate similarity that was close to the upper range of unmatched samples. These results have implications for therapies that rely on analysis of primary tumours to derive strategies that target metastases.

Citation Format: Jonathan H W Liu, Yang Washington Shao, Sam D. Molyneux, Rama Khokha, Geoffrey A. Wood. Genome-wide comparison of matched canine osteosarcoma primary tumours and metastases by array comparative genomic hybridization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 398. doi:10.1158/1538-7445.AM2013-398
Abstract 3985: Sorafenib modulates macrophages to promote T helper type 1 immunity.
Authors: Sunay Melek M. , Leatherman James M. , Chen Gang , Emens Leisha A.
Published: August 2014

Multiple regulatory processes within the tumor microenvironment work in concert to limit local anti-tumor immunity, and it is likely that integrative cancer therapies that target multiple cellular components in the tumor will be required to optimize antitumor immunity. Sorafenib is a promiscuous small molecule tyrosine kinase inhibitor developed as an anti-angiogenesis agent. We have previously shown that Sorafenib treatment can shift bone-marrow derived macrophages activated with lipopolysaccharide (LPS) and prostaglandin E2 (PGE2) from the pro-tumorigenic IL-10 secreting phenotype to the anti-tumor IL-12 secreting phenotype. Here, we extend these studies to explore the effect of Sorafenib on tumor-associated macrophages (TAMs) in a murine model of HER2+ breast cancer. Sorafenib treatment effectively delays the outgrowth of HER-2-overexpressing tumors in FVB/N mice relative to vehicle-treated controls. Immunohistochemical analysis revealed increased infiltration of F480+ macrophages in Sorafenib-treated tumors. F480+ TAMs isolated from Sorafenib-treated tumors had greater levels of IL-12 gene expression, and secreted higher levels of monocyte chemotactic protein-1 (MCP-1); there was no difference in IL-10 gene expression. Furthermore, F480+ TAMs isolated from Sorafenib-treated tumors demonstrated a superior ability to stimulate CD4+ T cell proliferation, and intracellular cytokine staining of tumor infiltrating lymphocytes isolated from Sorafenib-treated tumors showed an increase in CD4+ IFNγ-secreting T cells. Taken together, these data suggest that Sorafenib may alter the activation state of TAMs to increase pro-immunogenic signals and promote T helper type 1 immunity in the tumor microenvironment.

Citation Format: Melek M. Sunay, James M. Leatherman, Gang Chen, Leisha A. Emens. Sorafenib modulates macrophages to promote T helper type 1 immunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3985. doi:10.1158/1538-7445.AM2013-3985
Abstract 3986: Protein-bound polysaccharide induces IL-1β via NLRP3 inflammasome activation.
Authors: Yang Yi , Inatsuka Carol , Disis Mary L. , Lu Hailing
Published: August 2014

Protein-bound polysaccharide-K (PSK) is a hot water extract from Trametes Versicolor mushroom. It is widely used in Asian countries for its immune enhancing and anti-cancer potential. We have recently reported that PSK activates TLR2 and has CD8 T cell- and NK cell- dependent anti-tumor activity. PSK stimulates the production of multiple pro-inflammatory cytokines, including IL-1β, the production of which requires both TLR and inflammasome activation. The current study was undertaken to investigate the mechanism by which PSK activates inflammasome and induces IL-1β. RT-PCR and Western blot analyses showed that PSK induces pro-IL-1β in a dose- and time- dependent manner. PSK activates caspase-1 and pre-incubation with z-VAD-FMK, a caspase inhibitor, abolishes PSK-induced IL-1β. Comparison of THP-1 cells and THP-1 cells deficient for NLRP3 demonstrated that PSK-induced IL-1β production requires NLRP3 inflammasome. PSK also up-regulates the expression of NLRP3 mRNA. Further experiments were performed to investigate whether potassium efflux, reactive oxygen species (ROS) generation, or lysosomal perturbation was involved in PSK-induced NLRP3 inflammasome activation. Results showed that CA-074-Me, a cathepsin B inhibitor, significantly decreased PSK-induced IL-1β, while the production of TNF-α was not affected. Phagocytosis or ROS generation was not required for PSK-induced NLRP3 inflammasome activation. Altogether, our results suggest that PSK-induced IL-1β production is a two step process. Step 1 requires TLR2-dependent induction of pro-IL-1β and upregulation of NLRP3 expression; Step 2 involves NLRP3 inflammasome activation in a cathepsin B-dependent manner. These results provide novel insights into the mechanisms of the immune stimulatory and anti-tumor effects of PSK.

Citation Format: Yi Yang, Carol Inatsuka, Mary L. Disis, Hailing Lu. Protein-bound polysaccharide induces IL-1β via NLRP3 inflammasome activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3986. doi:10.1158/1538-7445.AM2013-3986
Abstract 3987: Functional implication of TGFBR3 dysregulation and its clinical application in oral cancer.
Authors: Fang Wei-Yu , Hsiao Jen-Ren , Chang Jang-Yang , Tsai Sen-Tien , Hsu Li-Jin , Wu Li-Wha
Published: August 2014

TGF-β type III receptor (TGFBR3), a co-receptor for TGF-β family members, is required for the high-affinity binding of ligands to their receptors to begin their cellular function. More and more studies showed contrasting roles of TGFBR3, tumor suppressor or oncogene, depending on the cancer type. However, the exact role of TGFBR3 and the responsible mechanism in oral cancer remains to be characterized. By using immunohisotochemical staining and real-time PCR, we found a down-regulation of TGFBR3 in oral cancer tissues. In the same line of this observation, we also detected a significant decrease of soluble TGFBR3 (sTGFBR3) in the oral cancer patient sera when compared with normal sera. Using Kaplan Meyer survival curve analysis and log rank test, we found that low TGFBR3 expression patients tend to have poor clinical outcome than those with high expression. Genetic manipulation of TGFBR3 was used to examine the role of TGFBR3 in oral cancer cells. Although the alteration resulted in differential effects on cell proliferation, TGFBR3 knockdown significantly increased migration and Matrigel invasion. TGFBR3 overexpression had the opposite effect on cell migration and invasion. Xenograft tumorigenesis and metastasis will be used to confirm the effect observed in vitro. Since TGFB is frequently overxpressed in oral cancer cells, we will evaluate the therapeutic potential of using sTGFBR3 in treating oral cancer in mice. Our study should facilitate studying the role of TGFBR3 in oral cancer progression and metastasis, and evaluating the possibility of using TGFBR3 as a therapeutic target for oral cancer treatment.

Citation Format: Wei-Yu Fang, Jen-Ren Hsiao, Jang-Yang Chang, Sen-Tien Tsai, Li-Jin Hsu, Li-Wha Wu. Functional implication of TGFBR3 dysregulation and its clinical application in oral cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2013-3987
Abstract 3996: ErbB4 is critical for tumor suppressive HPP1/STAT signaling in colon cancer.
Authors: Elahi Abul , Clark Whalen , Hernandez Jonathan , Humphries Leigh Ann , Wang Jian , Ajidahun Abidemi , Shibata David
Published: August 2014

Background: HPP1 (also known as TMEFF2) encodes a cleavable transmembrane protein that is thought to be a tumor suppressive EGF-like ligand that activates STAT1 and 2-associated pathways. From phylogenetic analyses, it is postulated that HPP1 exerts its effects via erbB4. We sought to characterize the role of erbB4 in the context of the HPP1/STAT tumor suppressive signaling axis.

Methods: Transient knockdown of erbB4 was achieved using siRNA in stable HPP1-overexpressing HCT116 transfectants. The impact of erbB4 manipulation on STAT signaling was assessed by expression and activation analyses using RT-PCR and Western blotting. Subsequent alterations in cell proliferation and anchorage-independent growth were evaluated by MTT and soft agar assays, respectively.

Results: We have previously demonstrated that HPP1’s tumor suppressive effects are associated with activation of suppressive STAT1 and 2 and downregulation of oncogenic STAT3 and 5. Abrogation of erbB4 expression resulted in a reversal of this profile with a reduction in activated STAT1 and 2 and increased phosphorylation of STAT3 and 5. Moreover, inhibition of ErbB4 receptor in HPP1 over-expressing transfectants resulted in an attenuation of tumor suppressive behavior with increased cell proliferation (Optical Density: 0.12±0.11 vs. 0.35 ±0.040; p≤0.001) and colony formation in soft agar (385±25 vs. 66±30 colonies; p≤0.001) as compared to scrambled siRNA control transfectants.

Conclusion: The EGF-like domain of HPP1 via erbB4 receptor signaling is essential for its tumor suppressive effects. Knockdown of erbB4 elicits a reversal of HPP1-mediated effects on cell behavior and STAT-family activation status. Therapeutic targeting of the erbB family of receptors is of great interest and our findings may lead to a greater understanding of the complex nature of their associated signaling pathways.

Citation Format: Abul Elahi, Whalen Clark, Jonathan Hernandez, Leigh Ann Humphries, Jian Wang, Abidemi Ajidahun, David Shibata. ErbB4 is critical for tumor suppressive HPP1/STAT signaling in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3996. doi:10.1158/1538-7445.AM2013-3996
Abstract 4007: Different patterns of Akt and ERK feedback activation in response to allosteric and active-site mTOR inhibitors in comparison to metformin in pancreatic cancer cells.
Authors: Soares Heloisa P. , Yang Ni , Sinnett-Smith James , Kui Robert , Rozengurt Enrique
Published: August 2014

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feedback loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with allosteric mTOR inhibitors (rapamycin and everolimus) or active-site mTOR inhibitors (KU63794 and PP242) suppressed p70S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin and everolimus caused a striking increase in Akt phosphorylation at Ser473 while the active-site inhibitors of mTOR completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin and everolimus did not have any stimulatory effect on ERK activation. Exposure to the compound A66, a selective inhibitor of the 110α catalytic subunit of PI3K, did not prevent enhancement of ERK activation in response to exposure to either KU63794 or PP242, implying that KU63794 and PP242 enhance ERK via a novel PI3K-independent pathway. These results suggest that active-site inhibitors, in contrast to mTORC1 allosteric inhibitors, enhanced ERK through a PI3K-independent pathway. In contrast, metformin which potently inhibited the mTORC1/S6K axis did not cause over-stimulation of Akt phosphorylation on Ser473 and inhibited mitogen-stimulated ERK activation in PDAC cells. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited mTORC1/S6K. Metformin is emerging as a potential novel agent in cancer chemoprevention. We propose that the favorable effects of metformin on preventing over-activation of pro-oncogenic pathways, such as Akt and ERK, may contribute to its beneficial anticancer effects.

Citation Format: Heloisa P. Soares, Ni Yang, James Sinnett-Smith, Robert Kui, Enrique Rozengurt. Different patterns of Akt and ERK feedback activation in response to allosteric and active-site mTOR inhibitors in comparison to metformin in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4007. doi:10.1158/1538-7445.AM2013-4007