Citations

Abstract MUC1 is an excellent target for immunotherapeutic strategies for cancers expressing Mucin 1 (breast, pancreas etc). MUC1 was recently selected by the NCI working group as the number 2 cancer vaccine target antigen, based on its immunogenicity, oncogenicity and wide-spread cell-surface expression. More than 90% of breast cancers over express MUC1, it stimulates both humoral and cellular immunity, its normal apical distribution is lost in cancer cells and aberrant glycosylation exposes peptide epitopes and novel carbohydrate antigens. Clinical trials using unglycosylated MUC1 epitopes have resulted in MUC1 specific cytotoxic T lymphocytes (CTLs) with limited effects on clinical response. Patients with breast tumors have exhibited spontaneous immune responses to MUC1 with the presence of T cells and antibodies specific for glycosylated MUC1. Because these natural immune responses are often not efficient in eradicating the tumor, it is necessary to specifically modulate these immune responses. Hence it is possible that better immunogenicity would be obtained using peptides more representative of the novel (glycosylated) forms of MUC1 as seen in cancer to which individuals may be less tolerant. The presentation of antigenic peptides by MHC class I molecules is crucial for the development of vaccines. MUC1 peptides from the VNTR region, which has five potential glycosylation sites, can be presented by a variety of class I molecules and can generate MUC1 specific CTLs. But these epitopes lack definite anchors and bind with low affinity compared to high affinity peptides. Hence, we developed several peptides from the MUC1 H-2Kb peptide (SAPDTRPAP) epitope with all possible modifications to improve their MHC and TCR binding efficiency. Preclinical studies were performed using the modified H-2Kb-restricted peptides along with the DC stimulants (CpG-ODN and GM-CSF) and foreign helper epitope (hepatitis B virus optimized I-Ab T helper peptide) in incomplete Freund's adjuvant in C57BL/6 mice transgenic for human MUC1. Peptides that were glycosylated at threonine and had methionine or leucine as the terminal amino acid induced a strong CTL response and were able to overcome tolerance in MUC1.Tg.mice. In the breast cancer model with the MUC1- expressing MMT cell line, the onset of tumor was delayed with peptide immunization and there was significant reduction in the tumor weight. Increased lytic activity of CD8+ CTLs and infiltration of T lymphocytes to the tumor site were observed with modified peptides as compared to the effect of MHC class I Kb and Db restricted native MUC1 peptides. Interestingly, the CTLs specific for glycosylated peptides were able to recognize both the Tn (GalNAc) and peptide epitopes. Thus, MUC1 peptides with carbohydrate at the appropriate position may be an optimal vaccine candidate because they can generate CTLs of dual specificity and high affinity. 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 2403.

Abstract During development, epithelial cells acquire mesenchymal characteristics through a process known as epithelial-mesenchymal transition (EMT). Substantial evidence now exists that EMT-related processes play a major role in tumor progression. EMT is a focus of interest in lung cancer as loss of E-cadherin expression confers increased resistance to several types of chemotherapy. In previous studies, we found that matrix metalloproteinase-3 (MMP3) induced EMT in mouse mammary epithelial cells through induction of Rac1b, an alternatively spliced isoform of Rac1. We now show that exposure to MMPs stimulates Rac1b and induction of EMT markers in human lung cancer cell lines, and that knockdown of Rac1b blocks MMP induced EMT. We also show that Rac1b expression levels in human lung tumor biopsies is predictive of patient outcome, and that Rac1b and MMP expression levels are correlated in these samples, suggesting a direct relationship. We also describe the generation of new transgenic mouse models for which MMP3 or Rac1b is inducibly expressed in lung epithelial cells; activation of kRAS in these mice reveals that expression of MMP3 or Rac1b promotes increased tumor number, size, and progression to adenocarcinoma as well as increased expression of EMT markers vimentin, sm-actin, col1a1, and Snail. We also show that expression of Rac1b in lung epithelial cells leads to spontaneous adenocarcinoma formation. These results demonstrate a direct linkage between induction of EMT and tumor progression, implicate Rac1b as a key mediator of these processes, and demonstrate that MMPs can directly stimulate all of these events. 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 2318.

Abstract Activation of the Sonic Hedgehog (SHH) pathway and increased expression of the Gli1 transcription factor have been implicated in the tumorigenesis of both sporadic and syndromic medulloblastoma, a malignant pediatric brain tumor that arises from cerebellar progenitor cells. While SHH pathway activity is required for normal neural stem cell proliferation, molecular mechanisms of SHH pathway dysregulation and transformation of neural precursor cells remain poorly defined. In epithelial cells, Snail1 was found to be induced by Gli1 expression and is believed to promote Gli1-mediated transformation (Li et al. 2006), however the role of Snail1 in neural cells is not well understood. We have identified Snail1 as a putative target of Gli1 in neural stem cells. We hypothesize that Snail1 participates in transduction of SHH/Gli1 signaling in the nervous system and plays a role in proliferation of neuronal progenitor cells in the cerebellum. Mice haploinsufficient for Patched (Ptc) have constitutive activation of the SHH pathway and a subset develop medulloblastoma. Quantitative RT-PCR analyses of mRNA isolated from early postnatal untransformed cerebella show that Snail1 mRNA is increased in vivo in Ptc+/- mice versus wild-type (WT) siblings.. Enforced expression of Gli1 in cultured WT murine granule precursor cells (GPCs) induced Snail1 mRNA.. Enforced expression of Snail1 was also sufficient to induce proliferation of GCPs in the absence of SHH/Gli1 exposure. Given that activation of SHH/Gli1-pathway induces proliferation of GPCs and is associated with medulloblastoma formation, we hypothesized that Snail1 may play a role in the proliferation and transformation of neural progenitor cells. We have found that the stem-like cells generated from medulloblastomas in Ptc+/- mice express higher levels of Snail mRNA than tumor parenchyma. Quantitative RT-PCR analysis demonstrates that Snail1 mRNA is expressed at baseline and is dramatically induced in human medulloblastoma cell lines after exposure to SHH. Enforced expression of Snail1 further increased proliferation of medulloblastoma cells as demonstrated by EdU incorporation. Analysis of potential Snail1 targets in human medulloblastoma cells reveals that β-catenin and N-Myc protein are induced by Snail1 overexpression. N-Myc is induced by SHH in GPCs (Kenney et al. 2003) and is known to induce proliferation in GPCs during both normal cerebellar development and medulloblastoma formation. Together these results give further insight into the mechanism of SHH-induced proliferation of neural progenitor and suggest that Snail1 may contribute to malignant transformation of GCPs through N-Myc. These data establish Snail1 as a target of SHH/Gli1 pathway activity in both normal and neoplastic cells derived from cerebellum. Ongoing experiments will further elucidate how this novel proliferative process affects transformation of neural 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 7.

Abstract Esophageal adenocarcinoma (EAC) is thought to arise from the metaplastic condition known as Barrett's esophagus (BE), which is caused by chronic inflammation due to gastroesophageal reflux disease. Since the molecular mechanisms underlying the pathogenesis of EAC are unknown, understanding EAC development mechanisms and their regulation is crucial for the treatment of EAC. Comparative genomic studies of evolutionarily similar and dissimilar species have been suggested as a means of studying the evolutionary biology in the species. Thus, we performed a cross-species comparison of gene expression patterns in human and rat tissues, uncovering the evolutionarily conserved gene expression patterns during EAC progression across both species. Using a non-carcinogenic rat model that progresses from normal esophagus to BE to EAC, we were able to identify the biological mechanisms of EAC development. We analyzed gene expression data from rat specimens (15 NE, 18 BE, and 30 EAC) as well as from human tissues (28NE, 72BE, and 75EAC). Using the presence of orthologous genes in both microarray platforms and various statistical tests, we uncovered a number of genes whose expression patterns were well conserved during progression from NE to BE in humans and rats. The expression of 869 genes was commonly altered when NE progressed to BE in both humans and rats (P<0.005). One of the most significantly conserved signaling pathways during the progression from NE to BE was IL-6-mediated E2F transcription factor1 (E2F1). The increased E2F1 induced activation of CCNA2, CCNB2, CCND1, CCNE 1 and 2, CDC 2 and 6, CASP3, CASP6, CASP8, CASP9, and Rad 51, which are important genes for the regulation of cell cycle, apoptosis, and DNA repair. These up-regulated genes may consequently cause abnormal cell cycle regulation and induce dysplasia and metaplasia of normal esophagus tissue during NE to BE progression. During BE to EAC progression in both species, 541 gene expressions were altered (P<0.005). An increased TGF-β/RAS/SPI1 signaling pathway significantly increased the expression of SPARC and SPP1 during progression from BE to EAC. Increased SPP1 and SPARC activated MMP9, CCL2, CCL4, COL1A1, COL3A1, and SNAL1, which mostly function in invasion, cell adhesion, and migration. Intriguingly, SPP1 and SPARC already had been discovered in our previous study (Kim et al.,) as markers of poor prognosis for EAC patients, strongly indicating that SPP1 and SPARC are also genetic determinants for progression to EAC. In conclusion, systems-level characterization of gene expression data from humans and a rodent model shows evolutionarily conserved genes and pathways during EAC progression, and these genes and pathways could become reliable biomarkers for early detection, or therapeutic targets for the treatment of EAC patients. 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 2005.

Abstract Allogeneic hematopoietic stem cell (HSC) transplantation is a promising area of therapy for various hematological malignancies like chronic/acute myeloid leukemia, myelodysplasia and lymphomas. Its effectiveness at inducing the graft versus tumor (GVT) effect is however, compromised by the graft versus host disease (GVHD) frequently observed in recipients post transplant. One of the strategies utilized in minimizing the effect of GVHD while still preserving GVT activity has been to target immunosuppressive cell types like Treg cells or myeloid derived suppressor cells (MDSCs), in an attempt to correct the balance between GVHD and GVT. MDSCs have been shown to be able to inhibit GVHD but not much is known about the ability of MDSCs to preserve GVT. We have previously shown that myeloid progenitors lacking Muc1 from Muc1 knockout (KO) C57BL/6 (H-2b) mice generate more effective MDSCs in vitro and in vivo. In this current study, we study the potential of using MDSCs generated in the absence of Muc1 to alleviate T cell expansion while preserving CTL activity. Our preliminary data shows that MDSCs generated from Muc1 KO C57BL/6 (H-2b) bone marrow are able to inhibit C57BL/6 (H-2b) T cell proliferation in an allogeneic mixed lymphocyte reaction, using Balb/c (H-2d) stimulators, more effectively than wildtype (WT) MDSCs. Interestingly, despite the reduced T cell proliferation in the presence of KO MDSCs, the CTL activity of these C57BL/6 T cells was still preserved, as demonstrated by their ability to lyse Balb/c A-20 leukemia cells. These results inidicate that co transplant of MDSCs generated in the absence of Muc1 can alleviate GVHD across fully mismatched major histocompatibility complex (MHC) barriers while still preserving the GVT ability of the allograft. This would also validate the clinical desirability of down regulating MUC1 in HSCs for more effective generation of MDSCs in a clinical allogeneic HSC transplantation setting. 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 1935.

Abstract Colon cancer is the result of a multi-step process involving the accumulation of genetic and epigenetic alterations leading to the transformation of normal colon epithelium to adenocarcinoma. Identification of characteristic genetic alterations is of critical importance to increase our understanding of the transformation process. We focused our analysis on the most common molecular sub-type of colon cancers, microsatellite stable (MSS) and CpG island methylator phenotype (CIMP)-negative, to identify characteristic copy number alteration (CNA) events and associated gene expression profiles using high-resolution genome-wide microarrays. DNA and RNA were extracted from 41 fresh-frozen paired colon tumors and adjacent normal tissue collected by the Colon Cancer Family Registry. Genomic profiles, such as CNAs and loss of heterozygosity (LOH), were identified with the Affymetrix Genome-Wide Human SNP 6.0 array for both tumor and adjacent normal tissue. Gene expression profiles of tumors and adjacent normal tissue were identified with the Affymetrix GeneChip Human Exon 1.0 ST array. Partek Genomics Suite software was used for analysis and data integration. We identified recurrent (>25%) CNAs in several chromosomal regions: gain in 1q, 7p, 7q, 8q, 13q, 20p, 20q, Xp, Xq and loss in 5q, 8p, 14q, 16p, 17p, 18p, and 18q. In addition, based on the allele information from the SNP arrays, we found a subset of these recurrent CNAs were associated with LOH. Preliminary results from the integration of CNA and gene expression data in tumors indicate that a subset of these recurrent CNAs is associated with the disruption of gene expression. For example, we observed recurrent copy number gain in 8q24.21, the genomic region containing the c-MYC gene, and associated up-regulation of c-MYC gene expression in tumors compared to adjacent normal tissue. In another example, recurrent copy number loss at 5q22.2 was associated with down-regulation of APC gene expression; loss of function of this tumor suppressor is associated with both hereditary and sporadic colon cancer. This genome-wide characterization of both genomic alterations and gene expression may help identify key genes and pathways that are disrupted in this common molecular subtype of colon cancer. We will present detailed results from this integrated and comprehensive genetic profiling of MSS and CIMP-negative colon cancers. 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 2145.

Abstract In pancreatic cancer both chemotherapy and radiotherapy have shown deficient results in treating the disease, indicating a fundamental need for more efficient therapies. Most pancreatic cancer patients present with an advanced form of the disease that is incurable by surgery, and will receive palliative treatment in the form of surgery, chemotherapy, radiotherapy and symptom control. There are few standard chemotherapeutics for the treatment of pancreatic cancer, although Gemcitabine and 5-Fluorouracil (5-FU) are the most widely used. In search for specific nutritional, and/or pharmacological agents to target pancreatic cancer cells/tissues we identified a series of nutritional supplements known as polyunsaturated fatty acids (PUFAs) that have an inhibitory effect on cancer cells. However, Gamma-Linolenic-Acid (GLA), a member of the ω-6 family of PUFAs exerts selective cytotoxic effects on pancreatic cancer cells without affecting normal epithelial cells. The goal of our study was to determine whether GLA could sensitize the targeted pancreatic cancer cells to chemotherapeutic agents (Gemcitabine or 5-FU). Therefore, pancreatic cancer cells were treated initially with GLA followed by each of the chemotherapeutic agents separately: Our results demonstrated that: 1) GLA induced growth inhibition and apoptosis of pancreatic cancer cells (Panc-1 and BxPC3); 2) the combination of GLA and Gemcitabine or GLA and 5-FU: a) promote a synergistic inhibitory effect on the growth of pancreatic cancer cells; b) inhibition of anchorage-independent growth; and 4) inhibition of morphogenesis in 3-D matrigel culture. These results show that GLA increases the sensitivity of pancreatic cancer cells to chemotherapy through a synergistic effect with Gemcitabine or 5-FU, implying that GLA could be used as a supportive drug for the pancreatic cancer patients receiving the current chemotherapeutic regimens. 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 209.

Abstract Malignant dispersion of glioma cells is a nemesis to clinical management, provoking the need for a better understanding of the underlying molecular drivers of invasion. The changes in signal transduction and consequent alterations in activation/de-activation of Transcription Factors (TFs) in glioma cells were investigated coincident to migration-engagement or impaired FAK kinase function by small molecule inhibition (PF-562,271). Earlier work showed that FAK and Pyk2 affect proliferation and migration in reciprocal ways in glioma cells (Lipinski CA, et al 2008; Loftus JC, et al 2009). The interaction of FAK and Pyk2 with specific downstream signaling effectors and the resultant activation of disparate signaling pathways is likely to script a signaling “signature” that will reveal to what extent glioma cell malignant behaviors are dependent on these kinases. Inhibition of FAK leads to diminution of signaling by pSrc, pJnk, and pNFkB, and variable activation of pAkt, and pp38. Of the 20 TFs assayed (Marligen, Inc.), the activation state of 7 TFs showed significant and consistent patterns of change across 6 primary GBM cell strains following FAK inhibition. Across a panel of 6 human GBM xenograft lines SP1, p53, E2F1-5, and NFκB evidenced strong activation (heightened nuclear localization), while AR (androgen receptor), Myc-Max, and CREB activation levels dropped by more than 2-fold following PF-562,271 treatment. Separate studies of established glioma cell lines under conditions of sparse plating (migration) and of dense crowded plating (migration-constrained), showed that p53, E2F1-5, and NFκB were standouts for activation accompanying migration, also associated by strong reduced activation of AR, Myc-Max, and CREB. (Because glioma cells following FAK knockdown or FAK inhibition show reduced entry into cell cycle, the signaling linkage between FAK and Myc-Max activation and also between FAK signaling and CREB activation suggest mechanistic ties to phenotype. E2F1 is a transcription factor that regulates genes involved in cell proliferation and cell survival and has even been suggested to fit a paradigm of oncogene addiction in glioma cells. Our findings demonstrate phenotypic responses in glioma cells downstream of FAK signaling linked to the activation or inactivation of specific transcription factors. If these patterns of TF activation state, and the signaling pathways upstream of that activation, associate (or account for) patterns of glioma cell invasion or proliferation in situ, they will suggest points of vulnerability in the underlying pathological processes of this disease. Supported by NIH NS042262 (Berens) and NIH CA103956 (Loftus). 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 1250.

Abstract Background. MBL is defined as a monoclonal expansion of B lymphocytes (< 5 × 109/L) in the blood without associated lymphadenopathy or organomegaly; MBLs with a chronic lymphocytic leukemia (CLL) immunophenotype have been termed as CLL-like MBL. The prevalence of CLL-like MBL in the general population is 3.5-12% but increases with age; however, the frequency is even higher (∼13-18%) among unaffected relatives from CLL families (i.e., families with two or more CLL members). We and others have found that a small proportion of individuals with CLL-like MBL individuals and lymphocytosis will progress to CLL requiring treatment (∼1.1% per year). Because CLL and NHL have been shown to aggregate together within families, suggesting a shared pathogenesis, we evaluated the frequency of MBL in NHL families. Methods. Individuals were obtained from an ongoing study of NHL families (i.e., families with 2 or more NHL members). However, we limited to NHL families with at most 1 individual with CLL due to the known increased frequency of MBL in CLL families. Multi-color flow cytometry was performed on fresh peripheral blood samples. A CBC and differential count was simultaneously obtained using an automated blood cell counter. Five possible flow diagnoses were defined as: CLL-like MBL (CD5+CD20dim), atypical MBL (CD5+CD20bright), non-CLL like MBL (CD5−), and normal immunophenotype (NIPT). Medical records of NHL cases were reviewed to confirm disease. Results. A total of 30 individuals from 16 NHL families had available flow cytometry results. The mean age at flow analysis was 56 years (range 37− 83 years). We identified 5 individuals with MBL from 4 families: 3 of the 6 had CLL-like MBL, 1 had atypical MBL, and 1 had non-CLL like MBL. The remaining 25 individuals had a NIPT. Thus, the MBL rate in NHL families is 17% (5/30). Among the CD5+ MBL (both CLL-like and atypical MBL) individuals, all were female and the mean absolute lymphocyte count was 1.4 × 109/L. Of interest, 1 of the CLL-like MBL individuals is from a family where two members had monoclonal gammopathy of uncertain significance (MGUS). Conclusion: Our data show that MBLs of various phenotypes can be found at a higher prevalence in NHL families than that of the general population. Further, the frequency of MBL in NHL families is comparable to that of CLL families. Although more work is needed, our results suggest that MBL may be clustering within NHL families, and thus support a possible common inherited predisposition of NHL and CLL, with MBL signifying a shared early pathogenetic step. 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 1853.

Abstract Background: Obesity has been linked to NHL risk in some studies, and may be stronger for obesity in early adulthood (ages 18-20). There has only been limited evaluation of physical activity and risk of NHL, particularly physical activity earlier in life. The purpose of this study was to evaluate the association of body mass index (BMI), physical activity (PA), and NHL risk during adulthood and at age 18. Methods: Self-reported anthropometrics and PA were collected in a clinic-based study of 954 newly diagnosed NHL cases and 1146 controls enrolled at the Mayo Clinic from 2002-2008. PA variables included duration and frequency of mild, moderate, and strenuous PA two years prior to case diagnosis or control selection, strenuous activity at age 18 (any vs. none), and height and weight at both time periods. An overall PA index was estimated by weighting the reported duration and frequency of each PA intensity by the average energy requirement (defined in METs) to obtain an average MET-minutes/week for each participant. BMI was calculated as weight (kg) divided by height (m) squared. Odds Ratios (OR), 95% confidence intervals (CI), and test for trend were estimated using unconditional logistic regression adjusted for the age, gender, and residence. Continuous variables were divided into quartiles according to the distribution among the controls; results are reported for the highest vs. lowest category or quartile unless otherwise specified. Results: The mean age at diagnosis/enrollment was 61 for both cases and controls; 47% and 43% of the cases and controls, respectively, were female. The most common NHL subtypes were CLL/SLL (32%), follicular lymphoma (FL; 26%), and diffuse large B-cell lymphoma (DLBCL; 21%). BMI 2 years prior to diagnosis was not associated with NHL risk (OR=1.21, p-trend=0.13), while there was a positive association with BMI at age 18 (OR=1.40, p-trend=0.001). There was no association of the PA index with risk (OR=0.91, p-trend=0.29). Furthermore, frequency of walking (OR=0.71, p-trend 0.43), mild (OR=1.30, p-trend=0.64), moderate (OR=0.99, p-trend=0.75), and strenuous (OR=0.77, p-trend=0.20) PA were not associated with NHL risk when evaluated individually. However, there was a suggestive weak inverse association between strenuous activity at age 18 and NHL risk (OR=0.85, 95% CI 0.71-1.05). On exploratory analysis, the association of BMI at age 18 with NHL risk was specific to DLBCL (OR=1.84, p-trend=0.002) and CLL/SLL (OR=1.25, p-trend=0.044) but not FL; all other results were similar across these subtypes. Conclusions: We did not observe an association between adult BMI and NHL risk; however a higher BMI in early adulthood was associated with NHL risk, consistent with limited prior reports. We found little evidence to support an association between PA in either early adulthood or later in life and NHL risk. 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 1833.