Reinhard Ebner

Identification of SOX2 as a novel glioma-associated antigen and potential target for T cell-based immunotherapy.

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.

Identification of a Novel Mammary-Restricted Cytochrome P450, CYP4Z1, with Overexpression in Breast Carcinoma

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.

Silencing of the Wnt transcription factor TCF4 sensitizes colorectal cancer cells to (chemo-) radiotherapy.

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.

STAT3 inhibition sensitizes colorectal cancer to chemoradiotherapy in vitro and in vivo.

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.

STAT3: A Novel Molecular Mediator of Resistance to Chemoradiotherapy

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.

A genomic strategy for the functional validation of colorectal cancer genes identifies potential therapeutic targets

Genes that are highly overexpressed in tumor cells can be required for tumor cell survival and have the potential to be selective therapeutic targets. In an attempt to identify such targets, we combined a functional genomics and a systems biology approach to assess the consequences of RNAi‐mediated silencing of overexpressed genes that were selected from 140 gene expression profiles from colorectal cancers (CRCs) and matched normal mucosa. In order to identify credible models for in‐depth functional analysis, we first confirmed the overexpression of these genes in 25 different CRC cell lines. We then identified five candidate genes that profoundly reduced the viability of CRC cell lines when silenced with either siRNAs or short‐hairpin RNAs (shRNAs), i.e., HMGA1, TACSTD2, RRM2, RPS2 and NOL5A. These genes were further studied by systematic analysis of comprehensive gene expression profiles generated following siRNA‐mediated silencing. Exploration of these RNAi‐specific gene expression signatures allowed the identification of the functional space in which the five genes operate and showed enrichment for cancer‐specific signaling pathways, some known to be involved in CRC. By comparing the expression of the RNAi signature genes with their respective expression levels in an independent set of primary rectal carcinomas, we could recapitulate these defined RNAi signatures, therefore, establishing the biological relevance of our observations. This strategy identified the signaling pathways that are affected by the prominent oncogenes HMGA1 and TACSTD2, established a yet unknown link between RRM2 and PLK1 and identified RPS2 and NOL5A as promising potential therapeutic targets in CRC.

Quantum Magnetic Resonance Therapy: Targeting Biophysical Cancer Vulnerabilities to Effectively Treat and Palliate

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.

Expression biomarkers for clinical efficacy and outcome prediction in cancer.

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.

D-PCa-2: A novel transcript highly overexpressed in human prostate and prostate cancer

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 × 105 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.

Abstract 140: LSAMP gene deletion is associated with rapid disease progression in prostate cancer of African American men

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 Stumpel, 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, Istvan 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.