Melanie Spitzner

A gene expression signature for chemoradiosensitivity of colorectal cancer cells.

PURPOSE The standard treatment of patients with locally advanced rectal cancers comprises preoperative 5-fluorouracil-based chemoradiotherapy followed by standardized surgery. However, tumor response to multimodal treatment has varied greatly, ranging from complete resistance to complete pathologic regression. The prediction of the response is, therefore, an important clinical need. METHODS AND MATERIALS To establish in vitro models for studying the molecular basis of this heterogeneous tumor response, we exposed 12 colorectal cancer cell lines to 3 μM of 5-fluorouracil and 2 Gy of radiation. The differences in treatment sensitivity were then correlated with the pretherapeutic gene expression profiles of these cell lines. RESULTS We observed a heterogeneous response, with surviving fractions ranging from 0.28 to 0.81, closely recapitulating clinical reality. Using a linear model analysis, we identified 4,796 features whose expression levels correlated significantly with the sensitivity to chemoradiotherapy (Q <.05), including many genes involved in the mitogen-activated protein kinase signaling pathway or cell cycle genes. These data have suggested a potential relevance of the insulin and Wnt signaling pathways for treatment response, and we identified STAT3, RASSF1, DOK3, and ERBB2 as potential therapeutic targets. The microarray measurements were independently validated for a subset of these genes using real-time polymerase chain reactions. CONCLUSION We are the first to report a gene expression signature for the in vitro chemoradiosensitivity of colorectal cancer cells. We anticipate that this analysis will unveil molecular biomarkers predictive of the response of rectal cancers to chemoradiotherapy and enable the identification of genes that could serve as targets to sensitize a priori resistant primary tumors.

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.

Identification of a microRNA expression signature for chemoradiosensitivity of colorectal cancer cells, involving miRNAs-320a, -224, -132 and let7g

BACKGROUND AND PURPOSE Preoperative chemoradiotherapy (CRT) represents the standard treatment for locally advanced rectal cancer. Tumor response and progression vary considerably. MicroRNAs represent master regulators of gene expression, and may therefore contribute to this diversity. MATERIAL AND METHODS Genome-wide microRNA (miRNA) profiling was performed for 12 colorectal cancer (CRC) cell lines and an individual in vitro signature of chemoradiosensitivity was established. Functional relevance of selected miRNAs was established by transfecting miRNA-mimics into SW480 and SW837 cells. The prognostic value of selected miRNAs was assessed in 128 pretherapeutic patient biopsies. RESULTS Thirty-six miRNAs were identified to significantly correlate with sensitivity to CRT (Q < 0.05) including miR-320a and other miRNAs involved in the MAPK-, TGF- and Wnt-pathway. Transfection of selected miRNAs (let-7g, miR-132, miR-224, miR-320a) each induced a shift of sensitivity. High expression of let-7 g was associated with a good prognosis in rectal cancer patients (P = 0.03). CONCLUSIONS This is the first report of a miRNA expression signature for in vitro chemoradiosensitivity of CRC cell lines. Many of the identified miRNAs have not been linked to the response to CRT and may represent potential molecular targets to sensitize resistant cancers. If further validated, let7g expression may serve as predictive biomarker.

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.

Histone Chaperone SSRP1 is Essential for Wnt Signaling Pathway Activity During Osteoblast Differentiation

Cellular differentiation is accompanied by dramatic changes in chromatin structure which direct the activation of lineage‐specific transcriptional programs. Structure‐specific recognition protein‐1 (SSRP1) is a histone chaperone which is important for chromatin‐associated processes such as transcription, DNA replication and repair. Since the function of SSRP1 during cell differentiation remains unclear, we investigated its potential role in controlling lineage determination. Depletion of SSRP1 in human mesenchymal stem cells elicited lineage‐specific effects by increasing expression of adipocyte‐specific genes and decreasing the expression of osteoblast‐specific genes. Consistent with a role in controlling lineage specification, transcriptome‐wide RNA‐sequencing following SSRP1 depletion and the induction of osteoblast differentiation revealed a specific decrease in the expression of genes involved in biological processes related to osteoblast differentiation. Importantly, we observed a specific downregulation of target genes of the canonical Wnt signaling pathway, which was accompanied by decreased nuclear localization of active β‐catenin. Together our data uncover a previously unknown role for SSRP1 in promoting the activation of the Wnt signaling pathway activity during cellular differentiation. Stem Cells 2016;34:1369–1376

Chemoradiotherapy Resistance in Colorectal Cancer Cells is Mediated by Wnt/β-catenin Signaling

Activation of Wnt/β-catenin signaling plays a central role in the development and progression of colorectal cancer. The Wnt-transcription factor, TCF7L2, is overexpressed in primary rectal cancers that are resistant to chemoradiotherapy and TCF7L2 mediates resistance to chemoradiotherapy. However, it is unclear whether the resistance is mediated by a TCF7L2 inherent mechanism or Wnt/β-catenin signaling in general. Here, inhibition of β-catenin by siRNAs or a small-molecule inhibitor (XAV-939) resulted in sensitization of colorectal cancer cells to chemoradiotherapy. To investigate the potential role of Wnt/β-catenin signaling in controlling therapeutic responsiveness, nontumorigenic RPE-1 cells were stimulated with Wnt-3a, a physiologic ligand of Frizzled receptors, which increased resistance to chemoradiotherapy. This effect could be recapitulated by overexpression of a degradation-resistant mutant of β-catenin (S33Y), also boosting resistance of RPE-1 cells to chemoradiotherapy, which was, conversely, abrogated by siRNA-mediated silencing of β-catenin. Consistent with these findings, higher expression levels of active β-catenin were observed as well as increased TCF/LEF reporter activity in SW1463 cells that evolved radiation resistance due to repeated radiation treatment. Global gene expression profiling identified several altered pathways, including PPAR signaling and other metabolic pathways, associated with cellular response to radiation. In summary, aberrant activation of Wnt/β-catenin signaling not only regulates the development and progression of colorectal cancer, but also mediates resistance of rectal cancers to chemoradiotherapy. Implications: Targeting Wnt/β-catenin signaling or one of the downstream pathways represents a promising strategy to increase response to chemoradiotherapy. Mol Cancer Res; 15(11); 1481–90. ©2017 AACR.

Abstract 1653: A novel role for Wnt/β-catenin signaling in mediating resistance of colorectal cancer to chemoradiotherapy

Background: Preoperative chemoradiotherapy represents the standard treatment for patients with rectal cancer. However, the clinical response of individual tumors to multimodal treatment is not uniform, and ranges from complete response to complete resistance. Therefore, the identification of novel therapeutic targets whose modification could be harnessed to sensitize a priori resistant tumors is exceedingly important. Previously, we demonstrated that the Wnt transcription factor TCF7L2 (also known as TCF4) was overexpressed in primary rectal cancers that were resistant to chemoradiotherapy (CRT), and that TCF7L2 functionally mediates resistance of CRC cells to clinically relevant doses of ionizing radiation (IR). Methods: Using siRNAs we silenced CTNNB1 (β-catenin), another key-component of canonical Wnt-signaling, in colorectal cancer cell lines LS1034, SW480, and SW837. To asses influence on CRT, cells were exposed to 0, 1, 2, 4, 6 and 8 Gy of X-rays and 5-FU. Wnt- signaling was stimulated in retinal pigment epithelial cells (RPE) either by adding Wnt-3A, or overexpressing non degradable β-catenin (S33Y-mutated) and analyzed changes in CRT. Finally we repetitively irradiated SW1463 (68Gy) to establish an isogenic radio-resistant cell line and examined changes in protein expression. Results: Silencing of CTNNB1 resulted in (chemo-) radiation-sensitization of all three CRC-cell lines. To further investigate the potential role of Wnt/β-catenin signaling in controlling therapeutic responsiveness, non-tumorigenic RPE cells were stimulated with Wnt-3A, which significantly increased resistance to CRT. This effect could be recapitulated by overexpression of β-catenin (S33Y-mutated), resulting in a significantly increased resistance to CRT. The effect could be rescued by siRNA mediated knockdown of β-catenin. Consistent with these findings, we observed higher expression levels of active (unphosphorylated) β-catenin as well as increased TCF reporter activity in SW1463 cells that were rendered radiation-resistant due to repeated IR treatment. Conclusion: Together, these findings strongly support the interpretation that Wnt/β-catenin signaling plays a central role in mediating resistance of CRC cells to CRT. Hence, pathway inhibition may represent a promising strategy to increase therapeutic responsiveness to CRT, which represents the standard treatment for locally advanced rectal cancers. This would have considerable clinical implications. Citation Format: Georg Emons, Melanie Spitzner, Sebastian Reineke, Frank Kramer, Margret Rave-Fraenk, Jochen Gaedcke, Michael Ghadimi, Thomas Ried, Marian Grade. A novel role for Wnt/β-catenin signaling in mediating resistance of colorectal cancer to chemoradiotherapy. [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 1653.

Abstract 3946: Wnt/ß-catenin signaling mediates resistance of colorectal cancer cells to chemoradiotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Preoperative chemoradiotherapy represents the standard treatment for patients with rectal cancer. However, the clinical response of individual tumors to multimodal treatment is not uniform, and ranges from complete response to complete resistance. Therefore, the identification of novel therapeutic targets whose modification could be harnessed to sensitize a priori resistant tumors is exceedingly important. In this context, we have previously demonstrated that the Wnt transcription factor TCF4 was over-expressed in rectal cancers that were resistant to chemoradiotherapy. The aim of this study was to explore the functional relevance of TCF4 and Wnt/β-catenin signaling for mediating treatment resistance. Methods: Colorectal cancer cell lines SW837, SW480, and HT-29 were transfected with shRNAs targeting TCF4. After establishing stable single-cell clone (SCC) populations, selected clones were irradiated at 0, 1, 2, 4, 6 and 8 Gy of X-rays. To assess the putative Wnt/β-catenin dependency, β-catenin was inhibited in cancer cell lines using siRNAs, the tankyrase inhibitors XAV939 / JW55 and the porcupine inhibitor LGK 974. Furthermore Wnt/β-catenin signaling activity was stimulated in normal retinal pigment epithelial (RPE) cells through incubation with Wnt3a. Results: RNAi-mediated silencing of TCF4 led to a significant radiosensitization in SW837 and SW480, two cell lines with a high basal TCF4-activity, whereas no effect was observed in HT-29 a cell line with low activity. Inhibition of β-catenin similarly caused a pronounced sensitization of CRC cells to clinically relevant doses of X-rays. Finally, exogenous stimulation of canonical Wnt signaling with Wnt3a resulted in a significantly decreased sensitivity of RPE cells to radiotherapy, i.e. induced radioresistance. Conclusion: We have uncovered a novel role of Wnt/β-catenin signaling in mediating resistance to chemoradiotherapy. Moreover, these data suggest that targeting key components of this pathway may represent a potential therapeutic strategy to increase the proportion of patients that respond to chemoradiotherapy, with considerable clinical implications. Citation Format: Georg Emons, Melanie Spitzner, Sebastian Reineke, Janneke Moller, Emil Kendziorra, Tim Beissbarth, Margret Rave-Fraenk, Jochen Gaedcke, Thomas Ried, Michael Ghadimi, Marian Grade. Wnt/s-catenin signaling mediates resistance of colorectal cancer cells to chemoradiotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3946. doi:10.1158/1538-7445.AM2014-3946