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Chemoradiotherapy (CRT) represents a standard treatment for many human cancers, frequently combined with radical surgical resection. However, a considerable percentage of primary cancers are at least partially resistant to CRT, which represents a substantial clinical problem, because it exposes cancer patients to the potential side effects of both irradiation and chemotherapy. It is therefore exceedingly important to determine the molecular characteristics underlying CRT-resistance and to identify novel molecular targets that can be manipulated to re-sensitize resistant tumors to CRT. In this review, we highlight much of the recent evidence suggesting that the signal transducer and activator of transcription 3 (STAT3) plays a prominent role in mediating CRT-resistance, and we outline why inhibition of STAT3 holds great promise for future multimodal treatment concepts in oncology. peerReviewed

Increased activity of signal transducer and activator of transcription 3 (STAT3) is common in human malignancies, including colorectal cancers (CRCs). We have recently reported that STAT3 gene expression correlates with resistance of CRC cell lines to 5-fluorouracil (5-FU)-based chemoradiotherapy (CT/RT). This is of considerable clinical importance, because a large proportion of rectal cancers are resistant to preoperative multimodal treatment. To test whether STAT3 contributes to CT/RT-resistance, we first confirmed that STAT3 protein expression correlated positively with increasing resistance. While STAT3 was not constitutively active, stimulation with interleukin-6 (IL-6) resulted in remarkably higher expression levels of phosphorylated STAT3 in CT/RT-resistant cell lines. A similar result was observed when we determined IL-6-induced expression levels of phosphorylated STAT3 following irradiation. Next, STAT3 was inhibited in SW480 and SW837 using siRNA, shRNA and the small-molecule inhibitor STATTIC. Successful silencing and inhibition of phosphorylation was confirmed using Western blot analysis and a luciferase reporter assay. RNAi-mediated silencing as well as STATTIC treatment resulted in significantly decreased clonogenic survival following exposure to 3 µM of 5-FU and irradiation in a dose-dependent manner, with dose-modifying factors of 1.3-2.5 at a surviving fraction of 0.37. Finally, STAT3 inhibition led to a profound CT/RT-sensitization in a subcutaneous xenograft model, with a significantly delayed tumor regrowth in STATTIC-treated mice compared with control animals. These results highlight a potential role of STAT3 in mediating treatment resistance and provide first proof of concept that STAT3 represents a promising novel molecular target for sensitizing resistant rectal cancers to CT/RT.

Background: In recent years remarkable ethnic/racial differences in the frequency of prostate cancer (CaP) driver gene alterations have emerged. Along the lines of ethnic differences in CaP genomes, oncogenic activation of ERG proto-oncogene and deletion of the PTEN tumor suppressor gene are most widely studied. These findings underscore distinct biology of prostate cancer in different ethnicities and races. We have reported the cumulative analyses of 435 patients (whole genome sequencing (WGS): 14, FISH evaluations: 101, SNP array: 320) comparing CaP genomes of African American (AA) and Caucasian American (CA) patients. An AA CaP genome-associated deletion was identified and mapped to the Limbic System-Associated Membrane Protein (LSAMP) gene locus. Further examination of the data indicated the AA CaP-genome associated deletion of the Chromodomain Helicase DNA Binding Protein 1(CHD1) gene. The goal of the current study is the comparative assessment of frequencies and prognostic associations of CaP genome alterations in the context of racial/ethnic differences. Methods: A prostate tumor tissue microarray (TMA) of benign and tumor foci (500+ cores) was generated by sampling 2-3 cores representing the range of cell morphology in matched cohort of 42 AA and 59 CA patients with up to 20 years of follow-up. Comparative evaluation of frequencies and prognostic associations of ERG oncoprotein by immunohistochemistry and the deletion of LSAMP, CHD1, and PTEN genes by FISH was performed. ERG frequencies were further assessed in index tumors of Chinese CaPs (N=100) and were compared to ERG frequencies in index tumors of patients from the United States equal-access military health-care system (N=336 AA and N=594 CA). Results: Higher frequencies of recurrent deletions of CHD1 (29% AA vs. 10% CA p=0.017) and LSAMP (26% AA vs. 7% CA, p=0.006) were identified in AA CaP compared to CA CaPs. These deletions were associated with significantly higher risk of rapid biochemical recurrence. In contrast, PTEN deletions (15% AA vs. 63% CA) and the frequency of ERG positive CaPs (26% AA vs. 64% CA) were more frequent among CA CaPs. Comparative evaluation of ERG frequencies in CaPs (N=1,292) underscores highest ERG frequency among CA patients (49.3%) followed by AA (23.2%) and Chinese (22%) men. Conclusions: In light of distinct biology of CaPs in ethnically/racially diverse CaP patient populations, there is a need for developing broadly applicable diagnostic, prognostic marker panels and therapeutic approaches. Higher frequency of CHD1 deletion in CaPs of AA patients may provide new therapeutic opportunities due to the sensitivity of CHD1 deletion harboring tumors to PARP inhibitors and platinum agents. Acknowledgments: This research was supported by the CPDR-USU program HU0001-10-2-0002, the National Cancer Institute R01CA162383 grant, the EDRN/NCI ACN12011-001-0 grant, the Snyder Medical Foundation, the Otto Mønsteds Foundation, the Mazzone Foundation, the Breast Cancer Research Foundation, the Novo Nordisk Foundation, and grants by MTA-TKI643/2012, KTIA_NAP_13-2014-0021. Citation Format: Albert Dobi, Gyorgy Petrovics, Hua Li, Denise Young, Shyh-Han Tan, Yongmei Chen, Qingyu Xiao, Yidi Sun, Hong Li, Yixue Li, Yuan Ji, Jun Hou, Wendy Wang, Jacob Kagan, Guo-Ping Zhao, Sudhir Srivastava, Reinhard Ebner, Inger L. Rosner, Jennifer Cullen, Matthew Freedman, Isabell Sesterhenn, Zoltan Szallasi, Shiv Srivastava. Distinct genomic alterations in prostate cancer of African American men [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A018.

Background: Distinct biology of prostate cancer (CaP) among men of different races and ethnicities have been recently highlighted. Along these lines higher frequencies of the most common CaP gene alterations, ERG proto-oncogene activation and deletion of the PTEN tumor suppressor, have been reported among Caucasian Americans (CA) when compared to other racial/ethnic groups. We have reported the cumulative analyses of CaPs from 435 patients (whole genome sequencing (WGS), FISH evaluations and SNP arrays) that highlighted the recurrent deletion and disease progression association of the Limbic System-Associated Membrane Protein (LSAMP) in CaP genomes of African American (AA) men. Further examination of these data indicated the AA CaP genome associated recurrent deletion of the Chromodomain Helicase DNA Binding Protein 1(CHD1) gene. Methods: Combined evaluation of frequencies and prognostic associations of ERG oncoprotein by immunohistochemistry and deletions of LSAMP, CHD1 and PTEN genes by FISH were performed using a multi-core TMA (42 AA and 59 CA patients) with longitudinal follow up data (median 16 years). CaP and matched blood genomic DNA samples (N=60) from AA patients were analyzed by WGS. ERG frequencies were further evaluated in index tumors of Chinese CaPs (N=100) and were compared to ERG frequencies in index tumors of AA (N=336) and CA (N=594) patients. Results: Frequent deletions of CHD1 (29% AA vs. 10% CA p=0.017) and LSAMP (26% AA vs. 7% CA, p=0.006) were found in AA CaPs by FISH assay. Both of these deletions were associated with rapid disease progression. Evaluation of CaPs by WGS further highlighted the recurrent deletion of CHD1 among AA men. Comparative evaluation of ERG (AA, N=336; CA, N=594, and Asian N=100) underscored highest ERG frequencies among CA patients (49.3%) followed by AA (23.2%) and Chinese (22%) men. Conclusions: In light of distinct biology of CaPs in ethnically/racially diverse CaP patient populations there is a need for developing broadly applicable diagnostic, prognostic marker panels and therapeutic approaches. Recurrent CHD1 deletion in CaPs of AA patients may provide new therapeutic opportunities in light of recent reports suggesting that CaP patients harboring CHD1 deletion may benefit from PARP inhibitor or platinum agents therapies. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to I.L.R., the NCI/EDRN IAA ACN12011-001-0 to S.S. and by the NCI R01CA162383 to S.S. Citation Format: Albert Dobi, Gyorgy Petrovics, Shyh-Han Tan, Hua Li, Denise Young, Cara Schafer, Jesse Fox, Kevin Babcock, Heng-Cheng Hu, Gauthaman Sukumar, Yingjie Song, Lakshmi Ravindranath, Yongmei Chen, Joseph Cheng, Reinhard Ebner, Qingyu Xiao, Yidi Sun, Yixue Li, Yuan Ji, Jun Hou, Wendy Wang, Guo-Ping Zhao, Jacob Kagan, Sudhir Srivastava, Joel T. Moncur, Clifton L. Dalgard, Matthew Wilkerson, Inger L. Rosner, Jennifer Cullen, Matthew Freedman, Zoltan Szallasi, Isabell A. Sesterhenn, Shiv Srivastava. Racial/ethnic differences in prostate cancer genomic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5352.

Background: Radical paradigm shifts in traditional thinking is paramount to winning the war on cancer and understanding why this disease survives even the most brutal of toxic therapies. There is mounting evidence that biophysical signals are integral to the cycle of initiation, progression and death of cancer cells. Innovative technologies that manipulate this vulnerability in solid tumors could effectively be used to perturb only diseased cells and tissues. Not compromising normally functioning cells while controlling tumor progression, is the ultimate goal for evolving cancer therapeutics like Quantum Magnetic Resonance Therapy, headed promisingly in that direction. Methods: A patented, CE marked device, the CYTOTRON® delivers rotating, target-specific, modulated, safe Radio Frequencies in the presence of an integrated, instantaneous magnetic field. The presumed modulation of the transmembrane potential of tumor cells and downstream cellular signalling by RF for tissue degeneration in cancer underlies Rotational Field Quantum Magnetic Resonance platform technology. Whole body MRI for tissue proton density determinations was used to compute individualized dosimetry to target solitary or multiple regions of interest in the whole body, simultaneously. Exposure to QMRT was for 1 hour daily for 28 consecutive days. Quality of Life assessments, overall survival and tumor stability using RECIST v1.1 were followed up for 12 months. Results: Significant increase in life expectancy from the predicted to the actual mean (p=2.13 E-12), and improvements in Karnofsky Performance Scale scores (p=7.25 E-06) and Quality of Life scores (p=1.71 E-08 and p=1.91 E-06) were noted. Thirty six of 51 (71 %) terminally ill patients had stable disease one month after completion of QMRT or longer. Conclusions: Exposure to radiofrequency-mediated QMRT improved life expectancy and quality of life, along with arrest of tumor progression. This therapy can be safely positioned in a palliative care setting, transitioning to mainstream cancer care with more rigorous clinical validation.

Cell-based assays are more complex than cell-free test systems but still reflect a highly artificial cellular environment. Incorporation of organotypic 3-dimensional (3-D) culture systems into mainstream drug development processes is increasingly discussed but severely limited by complex methodological requirements. The objective of this study was to explore a panel of standard assays to provide an easy-handling, standardized protocol for rapid routine analysis of cell survival in multicellular tumor spheroid-based antitumor drug testing. Spheroids of 2 colon carcinoma cell lines were characterized for evaluation. One of the assay systems tested could reliably be used to determine cell viability in spheroids. The authors verified that the acid phosphatase assay (APH) is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for HT29 and HCT-116 spheroids up to diameters of 650 microm and 900 microm, consisting of 40,000 and 80,000 cells, respectively. Treatment of HT29 and HCT-116 cells with 5-fluorouracil, Irinotecan, and C-1311 revealed critically reduced drug efficacies in 3-D versus monolayer culture, which is discussed in light of literature data. The experimental protocol presented herein is a small but substantial contribution to the establishment of sophisticated 3-D in vitro systems in the antitumor drug screening scenario.

Progress in cancer treatment has been slow, and the outlook for curing cancer is only marginally different from the situation a decade ago. Paradoxically, although the pharmaceutical industry has stepped up costly discovery research and drug development, approvals are on the decline and pipelines are dwindling. In an effort to reduce the number of drug failures and curtail burgeoning R&D costs, drug companies are exploring the use of biomarkers to evaluate toxicity and efficacy earlier in the development process. Biomarkers hold promise for optimization in dosing, adverse event prediction, efficacy evaluation, lead prioritization, and mechanism-of-action profiling of drug candidates. Furthermore, clinicians can use biomarkers to monitor patient response in clinical trials. In this perspective article, the authors explore the applications of cancer-related expression biomarkers in drug discovery and discuss how this will impact the industry and benefit the patient.

BACKGROUND The understanding of the molecular biology of the prostate and the process of prostate carcinogenesis is brought forward by the identification and characterization of new genes specifically expressed in prostate tissue. The encoded proteins may, in addition, provide novel diagnostic and therapeutic tools in prostate carcinoma (PCa). Here, we identify the novel gene Dresden-transmembrane protein of the prostate (D-TMPP) that is overexpressed in human prostate and prostate cancer. METHODS Proceeding from a prostate-specific expressed sequence tag identified with an Affymetrix chip-based expression database, the full-length cDNA of the novel gene was isolated from prostate tissue. The potential protein-coding function of the open reading frame (ORF) was tested by in vitro transcription-coupled translation and recombinant expression in transfected prostate cancer cells. The expression pattern of D-TMPP in malignant and nonmalignant tissues and tumor cell lines was analyzed by hybridization of a radioactively labeled cDNA probe with a multiple tissue expression array and by a quantitative real-time PCR assay. RESULTS The D-TMPP-mRNA encodes a putative seven-span transmembrane protein of 883 amino acids and is selectively overexpressed in prostate tissue. D-TMPP represents the first cloned and characterized transcript of a family of eukaryotic genes. D-TMPP transcripts were detected in all analyzed pairs (n = 25) of malignant and nonmalignant prostate tissues. In the androgen-dependent PCa cell line LNCaP, D-TMPP was upregulated by methyltrienolone. CONCLUSIONS We describe the novel prostate-restricted molecule D-TMPP widely expressed in prostate cancer tissues. © 2005 Wiley-Liss, Inc.

The use of molecular targets in novel strategies of tumor treatment largely depends on the identification of proteins with a tumor- or tissue-restricted expression. We identified the novel protein D-GPCR that is selectively overexpressed in human prostate cancer and prostate and belongs to the subfamily of odorant-like orphan G protein-coupled receptors. Quantification of D-GPCR transcripts in different human tissues by real-time PCR demonstrated 27-fold overexpression in prostate compared to skeletal muscle, the organ with second highest transcript numbers in males. Investigation of tumor/normal cDNA pairs obtained from 241 cancer patients including four prostate tumors confirmed the preferential expression in prostate. When comparing the mean transcript level of 15 prostate cancer tissues to their non-tumorous counterparts, D-GPCR was almost 6-fold upregulated. Coupled in vitro transcription and translation of D-GPCR cDNA produced a protein band of approximately 28 kDa. Recombinant, His-tagged protein was expressed in transfected HEK293 cells and gave rise to a 30 kDa band specifically detected by anti-His antibody. These data provide the basis for future studies evaluating the diagnostic potential of D-GPCR and its utility as a novel target in immunotherapy of prostate cancer.

By screening a transcriptome database for expressed sequence tags that are specifically expressed in mammary gland and breast carcinoma, we identified a new human cytochrome P450 (CYP), termed CYP4Z1. The cDNA was cloned from the breast carcinoma line SK-BR-3 and codes for a protein of 505 amino acids. Moreover, a transcribed pseudogene CYP4Z2P that codes for a truncated CYP protein (340 amino acids) with 96% identity to CYP4Z1 was found in SK-BR-3. CYP4Z1 and CYP4Z2P genes consisting of 12 exons are localized in head-to-head orientation on chromosome 1p33. Tissue-specific expression was investigated using real-time reverse transcription PCR with normalized cDNA from 18 different human tissues. CYP4Z1 mRNA was preferentially detected in breast carcinoma tissue and mammary gland, whereas only marginal expression was found in all other tested tissues. Investigation of cDNA pairs from tumor/normal tissues obtained from 241 patients, including 50 breast carcinomas, confirmed the breast-restricted expression and showed a clear overexpression in 52% of breast cancer samples. The expression profile of CYP4Z2P was similar to that of CYP4Z1 with preference in breast carcinoma and mammary gland but a lower expression level in general. Immunoblot analyses with a specific antiserum for CYP4Z1 clearly demonstrated protein expression in mammary gland and breast carcinoma tissue specimens as well as in CYP4Z1-transduced cell lines. Confocal laser-scanning microscopy of MCF-7 cells transfected with a fluorescent fusion protein CYP4Z1-enhanced green fluorescent protein and a subcellular fractionation showed localization to the endoplasmic reticulum as an integral membrane protein concordant for microsomal CYP enzymes.

Identification of genes selectively expressed in tumors or individual tissues is a crucial prerequisite for molecular diagnosis and treatment of cancer by addressing molecular targets. By screening an expression database, we identified the novel gene D-PCa-2 (Dresden prostate carcinoma 2), which is highly overexpressed in normal prostate tissue and prostate carcinoma (PCa). The corresponding transcript contained an open reading frame of 453 nucleotides encoding a putative protein of 150 amino acids. A large part of exon 8 of the D-PCa-2 gene shows strong similarity to the high-mobility-group nucleosomal binding protein 2 (HMGN2) cDNA. The highly specific transcription of the D-PCa-2 gene in normal and malignant prostate tissues and in a few additional tumors was demonstrated by using multiple tissue dot blot, cancer profiling dot blot and real-time PCR analyses. Examination of 18 pairs of tumorous and nontumorous prostate tissues from PCa patients by quantitative RT-PCR revealed D-PCa-2 transcripts in all specimens. The potential usefulness of D-PCa-2 as a sensitive marker for metastatic prostate carcinoma cells in lymph nodes was demonstrated by the detection of one LNCaP cell in 1 x 10(5) normal lymph node cells using real-time RT-PCR. Examination of 22 lymph nodes from PCa patients either containing metastatic prostate cancer cells or diagnosed as cancer-free was in full concordance with histopathologic diagnoses. These results validate D-PCa-2 as a transcript with high tissue specificity and with a potential application in the diagnosis of PCa.

As part of a large scale effort to discover novel secreted proteins, a cDNA encoding a novel cytokine was identified. Alignments of the sequence of the new protein, designated IL-17B, suggest it to be a homolog of the recently described T cell-derived cytokine, IL-17. By Northern analysis, EST distribution and real-time quantitative polymerase chain reaction analysis, mRNA was detected in many cell types. A novel type I transmembrane protein, identified in an EST data base by homology to IL-17R, was found to bind specifically IL-17B, as determined by surface plasmon resonance analysis, flow cytometry, and co-immunoprecipitation experiments. Readily detectable transcription of IL-17BR was restricted to human kidney, pancreas, liver, brain, and intestines and only a few of the many cell lines tested. By using a rodent ortholog of IL-17BR as a probe, IL-17BR message was found to be drastically up-regulated during intestinal inflammation elicited by indomethacin treatment in rats. In addition, intraperitoneal injection of IL-17B purified from Chinese hamster ovary cells caused marked neutrophil migration in normal mice, in a specific and dose-dependent manner. Together these results suggest that IL-17B may be a novel proinflammatory cytokine acting on a restricted set of target cell types. They also demonstrate the strength of genomic approaches in the unraveling of novel biological pathways.

MAX.3 is a monoclonal antibody that preferentially reacts with mature macrophages (MAC), monocyte-derived dendritic cells, megakaryocytes and platelets. In this study, we describe the characterization, purification and identification of the MAX.3 antigen. Immunoprecipitation and SDS/PAGE revealed different molecular masses of MAX.3 antigen in MAC (60-90 kDa) and platelets (58-64 kDa), whereas a similar size (45 kDa) was observed in both cell types after digestion with N-glycosidase F. Lectin affinity and sequential treatment with different glycosidases suggests complex type glycosylation of MAX.3 antigen in MAC and hybrid type glycosylation in platelets. Amino acid sequencing led to the identification of a corresponding cDNA clone and showed its identity to the sequence of the CD84 antigen, a member of the CD2 family of cell surface molecules. MAX.3/CD84 was further studied by immunohistochemistry and a variable expression was found on tissue MAC, confirming this antigen to be mainly a marker for MAC in situ.

One of the universal characteristics of the long bones and spines of middle-age and older mammals is a loss in bone mass (osteopenia). In humans, if this bone loss is severe enough, it results in osteoporosis, a skeletal disorder characterized by a markedly increased incidence of fractures with sequelae that may include pain, loss of mobility, and in the event of hip fracture, even death within a relatively few months of injury. An important contributing factor to the development of osteopororsis appears to be a diminution in the number and activity of osteoblasts responsible for synthesizing new bone matrix. The findings in the present and other similar studies suggest that this reduction in osteoblast number and activity is due to an age-related diminution in the size and osteogenic potential of the bone marrow osteoblast progenitor cell (OPC or CFU-f) compartment. We previously postulated that these regressive changes in the OPC/CFU-f compartment occurred in old animals because of a reduction in the amount and/or activity of TGF-β1, an autocrine growth factor important in the promotion of OPC/CFU-f proliferation and differentiation. In support of this hypothesis, we now report that (1) the osteogenic capacity of the bone marrow of 24-month-old BALB/c mice, as assessed in vivo, is markedly reduced relative to that of 3–4-month-old animals, (2) that the matrix of the long bones of old mice contains significantly less TGF-β than that of young mice, (3) that OPC's/CFU-f's isolated from old mice produce less TGF-β in vitro than those recovered from young mice, and (4) that OPC's/CFU-f's from old mice express significantly more TGF-β receptor (Types I, II, and III) than those of young animals and that such cells are more responsive in vitro to exogenous recombinant TGF-β1. We also find that colony number and proliferative activity of OPC's/CFU-f's of young mice and old mice, respectively, are significantly reduced when incubated in the presence of neutralizing TGF-β1 antibody. Collectively, these data are consistent with the hypothesis that in old male mice the reduction in the synthesis and, perhaps, availability from the bone matrix of TGF-β1 contributes to a diminution in the size and development potential of the bone marrow osteoprogenitor pool. J. Cell. Biochem. 70:478–488. © 1998 Wiley-Liss, Inc.

INTRODUCTION: Disproportionately higher rates of prostate cancer (CaP) incidence and mortality have been reported among African American (AA) men. Although oncogenic TMPRSS2-ERG gene fusion and deletion of the PTEN tumor suppressor gene are established cancer driver gene alterations in CaP, they are known to be more prevalent among men of European ancestry. By utilizing carefully annotated specimens, this study focused on the discovery of recurrent genomic alterations in CaP of AA men in comparison to Caucasian Americans (CA). METHODS: Genomic DNA from clinically localized primary prostate tumors (Gleason 6 or 7 with primary pattern 3) and matched peripheral blood lymphocytes of seven AA and seven CA patients, were analyzed by paired-end sequencing on Illumina Genome Analyzer IIx to a depth of 30x. Following alignment to reference genome, somatic alterations on tumor DNA that include single nucleotide variants (SNVs), insertion and deletions (Indels), structural variations, copy number variations, and inter- and intra-chromosomal translocations of tumor DNA sequence were identified. To confirm prevalent genomic deletions we performed FISH analysis on a tissue microarray constructed from 42 AA and 59 CA tumor and normal samples of an independent cohort. Frequently deleted loci were further validated by analysis of TCGA CaP SNP array data from 41 AA and 279 CA prostate tumors. RESULTS: A comparative evaluation of whole genome sequences of AA and CA CaP revealed a prevalent deletion of the LSAMP locus of chromosome 3q13.31 in AA CaP. These observations were confirmed by SNP array and FISH assays in independent cohorts of specimens. AA CaP patients with LSAMP deletion showed rapid disease progression. In contrast to higher frequency of LSAMP deletion, significantly lower frequencies of PTEN and ERG alterations were noted in CaP of AA men. Furthermore, CaP genomes of AA men displayed a higher rate of inter-chromosomal rearrangements than those from CA men. CONCLUSIONS: We highlight distinct features of AA and CA CaP genomes including common CaP driver genes (TMPRSS2- ERG, PTEN) and define a novel recurrent deletion of the LSAMP locus. This study underscores the need for careful evaluations of cancer genomes in underrepresented populations in the global context with implications for precision medicine strategies. Citation Format: Albert Dobi, Gyorgy Petrovics, Hua Li, Shyh-Han Tan, Tanja Stümpel, Denise Young, Shilpa Katta, Qiyuan Li, Kai Ying, Bernward Klocke, Lakshmi Ravindranath, Indu Kohaar, Yongmei Chen, Dezső Ribli, Korbinian Grote, Hau Zou, Joseph Cheng, Clifton L. Dalgard, Shimin Zhang, István Csabai, Jacob Kagan, David Takeda, Massimo Loda, Sudhir Srivastava, Matthias Scherf, Martin Seifert, Timo Gaiser, David G. McLeod, Zoltan Szallasi, Reinhard Ebner, Thomas Werner, Isabell A. Sesterhenn, Matthew Freedman, Shiv Srivastava. LSAMP gene deletion is associated with rapid disease progression in prostate cancer of African American men. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 140.

A considerable percentage of rectal cancers are resistant to standard preoperative chemoradiotherapy. Because patients with a priori-resistant tumors do not benefit from multimodal treatment, understanding and overcoming this resistance remains of utmost clinical importance. We recently reported overexpression of the Wnt transcription factor TCF4, also known as TCF7L2, in rectal cancers that were resistant to 5-fluorouracil-based chemoradiotherapy. Because Wnt signaling has not been associated with treatment response, we aimed to investigate whether TCF4 mediates chemoradioresistance. RNA interference-mediated silencing of TCF4 was employed in three colorectal cancer (CRC) cell lines, and sensitivity to (chemo-) radiotherapy was assessed using a standard colony formation assay. Silencing of TCF4 caused a significant sensitization of CRC cells to clinically relevant doses of X-rays. This effect was restricted to tumor cells with high T cell factor (TCF) reporter activity, presumably in a β-catenin-independent manner. Radiosensitization was the consequence of (i) a transcriptional deregulation of Wnt/TCF4 target genes, (ii) a silencing-induced G(2)/M phase arrest, (iii) an impaired ability to adequately halt cell cycle progression after radiation and (iv) a compromised DNA double strand break repair as assessed by γH2AX staining. Taken together, our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance. Moreover, they suggest that TCF4 is a promising molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy.

Transforming growth factor-beta (TGF-beta) affects cellular proliferation, differentiation, and interaction with the extracellular matrix primarily through interaction with the type I and type II TGF-beta receptors. The type II receptors for TGF-beta and activin contain putative serine-threonine kinase domains. A murine serine-threonine kinase receptor, Tsk 7L, was cloned that shared a conserved extracellular domain with the type II TGF-beta receptor. Overexpression of Tsk 7L alone did not increase cell surface binding of TGF-beta, but coexpression with the type II TGF-beta receptor caused TGF-beta to bind to Tsk 7L, which had the size of the type I TGF-beta receptor. Overexpression of Tsk 7L inhibited binding of TGF-beta to the type II receptor in a dominant negative fashion. Combinatorial interactions and stoichiometric ratios between the type I and II receptors may therefore determine the extent of TGF-beta binding and the resulting biological activities.

Malignant glioma comprises the majority of primary human brain tumors with 16,800 new cases reported each year in the USA. Its prognosis remains dismal despite numerous attempts to improve conventional therapeutic modalities. Therefore, much effort is devoted to the exploration of alternative forms of treatment such as immunotherapy. The identification of potential target structures highly overexpressed in brain tumors is a crucial prerequisite for the activation of the immune defense against malignant glioma cells. By screening an expression database for genes highly expressed in glioblastoma multiforme (GBM), we identified the Pit-Oct-Unc (POU) cooperating transcription factor SOX11 that is known to be crucially involved in brain development. Analysis of the expression pattern of SOX11 in different normal adult and fetal tissues by multiple tissue dot blot and by a highly sensitive quantitative PCR assay confirmed the selective overexpression of SOX11 in fetal brain tissue. Examination of tissue specimens obtained from malignant gliomas and from normal brain by quantitative real-time PCR (Q-RT-PCR) revealed upregulation of SOX11 in almost all tumor samples (15/16) as compared to the pooled normal brain. Seventy-five percent of the tumor samples (12/16) showed a 5- to more than 600-fold overexpression. We conclude that, after downregulation of SOX11 in the adult brain, its expression is reactivated during tumorigenesis and that SOX11 therefore represents a promising novel molecular target for adjuvant therapy of malignant gliomas.

Transforming growth factor-β (TGF-β? is thought to play a role in mesenchymal cell development and, specifically, in muscle differentiation, yet its precise role in the latter process remains unclear. TGF-β has been shown to both inhibit and induce myoblast maturation in vitro, depending on the culture conditions. Whether the type I or type II TGF-β receptor mediates the various TGF-β effects on myogenesis is not known. In the present study, C2C12 myoblasts were transfected with an expression vector for a truncated type II TGF-β receptor, which has been shown to act as a dominant negative inhibitor of type II receptor signaling. In contrast to the parental cells, the transfected clones did not efficiently form myotubes or induce expression of MyoD, myogenin and several other differentiation markers following incubation in low serum media. However, some muscle differentiation markers continued to be expressed in the transfected cells suggesting that at least two pathways are involved in muscle cell differentiation. These cells could still growth arrest in low serum media, showing that decreased proliferation can be dissociated from differentiation. Unlike several oncogenes known to block myogenic differentiation, expression of the truncated TGF-β receptor did not result in myoblast transformation. Injection of the parental or the transfected C2C12 cells into the limb muscle of nude mice revealed quantitative and qualitative differences in their behavior, and suggested that myoblasts expressing the truncated TGF-β receptor cannot fuse in vivo. Finally, retrovirusmediated expression of MyoD in the transfected cells restored their ability to form myotubes in vitro, indicating that inhibition of myoblast differentiation by the truncated TGF-β receptor may depend on decreased MyoD expression. We propose that TGF-β signaling through the type II receptor is required for several distinct aspects of myogenic differentiation and that TGF-β acts as a competence factor in this multistep process.

Rotational Field Quantum Magnetic Resonance (RFQMR) is a device-mediated novel therapeutic modality for cancer, currently in the clinical stage of development. It can also be used to deliver an electromagnetic force with strong, embedded, variable radio signals to induce Continuous Flow Focused Resonance Nano-permeabilization or CO-FORN concomitantly with drugs of choice for cancer treatment. Transient permeabilization of cells by Focused Resonance pulses induces a significant increase of anti-tumor drug concentration within the tumor and enables a practical solution to deliver valuable systemic chemotherapy and mitigate associated adverse effects. CO-FORN was administered to a refractory medullobalstoma patient who had previously received metronomic chemotherapy with Cyclophosphamide and VP16, significantly helping to reduce the associated cytotoxicity and myelosuppression commonly accompanying such treatment. The protocol allows for extremely accurate RFQMR dosing for therapeutic purposes. RFQMR can be applied as an extraneous source to permeate intractable tumors by creating molecular dimension-specific pore sizes. Targeted drug delivery is enabled thereby minimizing associated systemic drug toxicities. It has been shown that electroporation of cultured cells potentiates cytotoxicity of various chemotherapy agents and other targeted molecules by several hundred fold. This potentiation can be highly controlled using RFQMR by adjusting delivery parameters like molecular mass of the drug, peak plasma concentration, total tissue volume, cell membrane characteristics etc. Specific tumors can be resonated with high instantaneous magnetic fields and radio frequency (RF), followed by nano second signals which penetrate only resonating cells. The CO-FORN technique can potentiate antitumor effectiveness of drugs from 10 to 70 times and can be highly localized and used without implanting any electrodes, probes or particles into the body. Exposure to non-ionizing, non-thermal RFQMR does not cause uncontrollable destruction and entropy within the cell but more likely re-organizes related bio-structures. In principle, depending on the specific variations within the configured RFQMR, a predictable cell response can be expected. The technology allows for extremely accurate RFQMR dosing for therapeutic purposes. Of the several disease indications and applications that the device has been evaluated in extensive R&D efforts at the Center for Advanced Research & Development (CARD) in Bangalore India, the use of RFQMR to permeabilize tumors in conjunction with systemic chemotherapy, is an innovative application. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C102.

Background: The clinical response of locally advanced rectal cancers to preoperative chemoradiotherapy is very heterogeneous. We recently profiled a series of responsive and resistant rectal carcinomas using gene expression microarrays and identified TCF7L2, the main downstream effector of the Wnt/β-catenin pathway, as over-expressed in the resistant tumors. The aim of this study was to explore the relevance of TCF7L2 for resistance to radiation and to study the underlining mechanisms. Methods: We transfected three microsatellite-stable colorectal cancer cell lines (SW837, SW480 and HT-29) with two different shRNA-vectors targeting TCF7L2 and a non-silencing. Next, we established single cell clone (SCC) populations, and after confirming the reduction of TCF7L2 protein levels by Western blotting, we irradiated the SCCs at 0, 1, 2, 4, 6 and 8 Gy and calculated survival fractions. To gain functional insight, we chose SW837 and HT-29 for further experiments. Using γH2AX staining we evaluated DNA double strand repair after irradiation at 2 Gy and analyzed cell cycle changes before and after radiation. Furthermore, we studied Wnt/β-catenin pathway activity using the TOPFLASH/FOPFLASH reporter assay. Results: RNAi-mediated silencing of TCF7L2 led to a pronounced reduction in TCF7L2 protein levels. In radiation experiments we observed a highly significant radiosensitization in SW837 and SW480 SCCs, whereas no effect was observed in HT-29 SCCs. Well-fitting, we observed significantly more γH2AX foci 24h after radiation in SW837 SCCs compared to HT-29 SCCs, pointing to impaired DNA damage repair in the SCCs from SW837. Furthermore, in SW837 SCCs but not in HT-29 SCCs we noticed a change in the cell cycle distribution towards radiosensitive cell cycle phases before radiation and compromised cell cycle control after radiation. Finally, using the TOPFLASH/FOPFLASH reporter assay we found SW837 wild-type cells to show high Wnt/β-catenin pathway activity, while HT-29 wild-type cells only showed very low activity. Conclusion: TCF7L2 was found to be over-expressed in resistant rectal carcinomas, and its RNAi-mediated silencing caused a significant radiosensitization, mediated by DNA damage repair deficiencies and impaired cell cycle control. Interestingly, radiosensitization effetcts were only observed in cell lines showing Wnt/β-catenin pathway activity. Thus, we have uncovered a novel role of Wnt/β-catenin signaling for mediating resistance to multimodal cancer therapy. Moreover, these data suggest that TCF7L2 is a potential molecular target for sensitizing Wnt/β-catenin-dependent tumor cells to radiation. 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 2490. doi:10.1158/1538-7445.AM2011-2490

Summary Sucrose-positive derivatives of Escherichia coli K-12, containing the plasmid pUR400, and of Kiebsieiia pneumoniae hydrolyse intracellular sucrose 6-phosphate by means of an invertase into D-glucose 6phosphate and free D-fructose. The latter is phosphorylated by an ATP-dependent fruclokinase (gene scrK of an ser reguion) to D-fructose 6-phosphate. The lack of ScrK does not cause any visible phenotype in wildtype strains of both organisms. Using genes and enzymes normally involved in D-arabinitoi metabolism from E. coli C and K. pneumoniae, derivatives of E. coii K-12 vi/ere constructed which allowed the identification of scr/C mutations on conventional indicator plates. Cloning and sequencing of scrK from sucrose ptasmid pUR400 and from the chromosome of K. pneumoniae revealed an open reading frame of 924 bp in both cases — the equivalent of a peptide containing 307 amino acid residues {M, 39 and 34 kDa, respectively, on sodium dodecyl sulphate gels). The sequences showed overall identity among each other (69% identical residues) and to a kinase from Vibrio alginoiyticus (57%) also involved in sucrose metabolism, iower overall identity (39%) to a Dribose-kinase from E. coli, and local similarity to prokaryotic, and eukaryotic phosphofructokinases at the putative ATP-binding sites.

Prognosis for patients suffering from malignant glioma has not substantially improved. Specific immunotherapy as a novel treatment concept critically depends on target antigens, which are highly overexpressed in the majority of gliomas, but the number of such antigens is still very limited. SOX2 was identified by screening an expression database for transcripts that are overexpressed in malignant glioma, but display minimal expression in normal tissues. Expression of SOX2 mRNA was further investigated in tumour and normal tissues by real-time PCR. Compared to cDNA from pooled normal brain, SOX2 was overexpressed in almost all (9 out of 10) malignant glioma samples, whereas expression in other, non-malignant tissues was almost negligible. SOX2 protein expression in glioma cell lines and tumour tissues was verified by Western blot and immunofluorescence. Immunohistochemistry demonstrated SOX2 protein expression in all malignant glioma tissues investigated ranging from 6 to 66% stained tumour cells. Human leucocyte antigen-A(*)0201-restricted SOX2-derived peptides were tested for the activation of glioma-reactive CD8+ cytotoxic T lymphocytes (CTLs). Specific CTLs were raised against the peptide TLMKKDKYTL and were capable of lysing glioma cells. The abundant and glioma-restricted overexpression of SOX2 and the generation of SOX2-specific and tumour-reactive CTLs may recommend this antigen as target for T-cell-based immunotherapy of glioma.

Herpes simplex virus (HSV) 1 and 2 infect activated T lymphocytes by attachment of the HSV envelope glycoprotein D (gD) to the cellular herpesvirus entry mediator (HVEM), an orphan member of the tumor necrosis factor receptor superfamily. Here, we demonstrate that HVEM binds two cellular ligands, secreted lymphotoxin α (LTα) and LIGHT, a new member of the TNF superfamily. LIGHT is a 29 kDa type II transmembrane protein produced by activated T cells that also engages the receptor for the LTαβ heterotrimer but does not form complexes with either LTα or LTβ. HSV1 gD inhibits the interaction of HVEM with LIGHT, and LIGHT and gD interfere with HVEM-dependent cell entry by HSV1. This characterizes herpesvirus gD as a membrane-bound viokine and establishes LIGHT-HVEM as integral components of the lymphotoxin cytokine-receptor system.8 These authors made equal contributions.