Pancreatic cancer (PC) is one of the most lethal cancers worldwide, associated with poor prognosis and restricted therapeutic options. Clostridium perfringens enterotoxin (CPE), is a pore-forming (oncoleaking) toxin, which binds to claudin-3 and -4 (Cldn3/4) causing selective cytotoxicity. Cldn3/4 are highly upregulated in PC and represent an effective target for oncoleaking therapy. We utilized a translation-optimized CPE vector (optCPE) for new suicide approach of PC in vitro and in cell lines (CDX) and patient-derived pancreatic cancer xenografts (PDX) in vivo. The study demonstrates selective toxicity in Cldn3/4 overexpressing PC cells by optCPE gene transfer, mediated by pore formation, activation of apoptotic/necrotic signaling in vitro, induction of necrosis and of bystander tumor cell killing in vivo. The optCPE non-viral intratumoral in vivo jet-injection gene therapy shows targeted antitumoral efficacy in different CDX and PDX PC models, leading to reduced tumor viability and induction of tumor necrosis, which is further enhanced if combined with chemotherapy. This selective oncoleaking suicide gene therapy improves therapeutic efficacy in pancreas carcinoma and will be of value for better local control, particularly of unresectable or therapy refractory PC.

Colorectal cancer (CRC) is the second-most common malignant disease worldwide, and metastasis is the main culprit of CRC-related death. Metachronous metastases remain to be an unpredictable, unpreventable, and fatal complication, and tracing the molecular chain of events that lead to metastasis would provide mechanistically linked biomarkers for the maintenance of remission in CRC patients after curative treatment. We hypothesized, that Metastasis-associated in colorectal cancer-1 (MACC1) induces a secretory phenotype to enforce metastasis in a paracrine manner, and found, that the cell-free culture medium of MACC1-expressing CRC cells induces migration. Stable isotope labeling by amino acids in cell culture mass spectrometry (SILAC-MS) of the medium revealed, that S100A4 is significantly enriched in the MACC1-specific secretome. Remarkably, both biomarkers correlate in expression data of independent cohorts as well as within CRC tumor sections. Furthermore, combined elevated transcript levels of the metastasis genes MACC1 and S100A4 in primary tumors and in blood plasma robustly identifies CRC patients at high risk for poor metastasis-free (MFS) and overall survival (OS). Mechanistically, MACC1 strengthens the interaction of β-catenin with TCF4, thus inducing S100A4 synthesis transcriptionally, resulting in elevated secretion to enforce cell motility and metastasis. In cell motility assays, S100A4 was indispensable for MACC1-induced migration, as shown via knock-out and pharmacological inhibition of S100A4. The direct transcriptional and functional relationship of MACC1 and S100A4 was probed by combined targeting with repositioned drugs. In fact, the MACC1-β-catenin-S100A4 axis by statins (MACC1) and niclosamide (S100A4) synergized in inhibiting cancer cell motility in vitro and metastasis in vivo. The MACC1-β-catenin-S100A4 signaling axis is causal for CRC metastasis. Selectively repositioned drugs synergize in restricting MACC1/S100A4-driven metastasis with cross-entity potential.

Deregulated Wnt-signaling is a key mechanism driving metastasis in adenocarcinoma of the gastroesophageal junction and stomach (AGE/S). The oncogene S100A4 was identified as a Wnt-signaling target gene and is known to promote metastasis. In this project, we illuminate the role of S100A4 for metastases development and disease prognosis of AGE/S. Five gastric cancer cell lines were assessed for S100A4 expression. Two cell lines with endogenous high S100A4 expression were used for functional phenotyping including analysis of proliferation and migration after stable S100A4 knock-down. The prognostic value of S100A4 was evaluated by analyzing the S100A4 expression of tissue microarrays with samples of 277 patients with AGE/S. S100A4 knock-down induced lower migration in FLO1 and NCI-N87 cells. Treatment with niclosamide in these cells led to partial inhibition of S100A4 and to reduced migration. Patients with high S100A4 expression showed lower 5-year overall and disease-specific survival. In addition, a larger share of patients in the S100A4 high expressing group suffered from metachronous metastasis. This study identifies S100A4 as a negative prognostic marker for patients with AGE/S. The strong correlation between S100A4 expression, metastases development and patient survival might open opportunities to use S100A4 to improve the prognosis of these patients and as a therapeutic target for intervention in this tumor entity.

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease, which is thought to be initiated by colon cancer stem cells, according to the cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcomes. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our MACC1 transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and the CSC-associated invasive phenotype based on patient-derived tumor organoids (PDOs), patient-derived xenografts (PDXs) and human CRC cell lines. We showed that CD44-enriched CSCs from PDO models express significantly higher levels of MACC1 and LGR5 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PDO and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with the CSC markers CD44, NANOG and LGR5 in PDO models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter, uncovering the long-known CSC marker LGR5 as a novel essential signaling mediator employed by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by a significant positive correlation of MACC1 with LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis inducer MACC1 acts as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs and prevent cancer recurrence and distant metastasis formation.

The coxsackievirus B3 strain PD-0 has been proposed as a new oncolytic virus for the treatment of colorectal carcinoma. Here, we generated a cDNA clone of PD-0 and analyzed the virus PD-H, newly generated from this cDNA, in xenografted and syngenic models of colorectal cancer. Replication and cytotoxic assays revealed that PD-H replicated and lysed colorectal carcinoma cell lines in vitro as well as PD-0. Intratumoral injection of PD-H into subcutaneous DLD-1 tumors in nude mice resulted in strong inhibition of tumor growth and significantly prolonged the survival of the animals, but virus-induced systemic infection was observed in one of the six animals. In a syngenic mouse model of subcutaneously growing Colon-26 tumors, intratumoral administration of PD-H led to a significant reduction of tumor growth, the prolongation of animal survival, the prevention of tumor-induced cachexia, and the elevation of CD3⁺ and dendritic cells in the tumor microenvironment. No virus-induced side effects were observed. After intraperitoneal application, PD-H induced weak pancreatitis and myocarditis in immunocompetent mice. By equipping the virus with target sites of miR-375, which is specifically expressed in the pancreas, organ infections were prevented. Moreover, employment of this virus in a syngenic mouse model of CT-26 peritoneal carcinomatosis resulted in a significant reduction in tumor growth and an increase in animal survival. The results demonstrate that the immune status of the host, the route of virus application, and the engineering of the virus with target sites of suitable microRNAs are crucial for the use of PD-H as an oncolytic virus.