Combining cell cycle checkpoint kinase inhibitors with the DNA-damaging chemotherapeutic agent gemcitabine offers clinical appeal, with a mechanistic rationale based chiefly on abrogation of gemcitabine-induced G2/M checkpoint activation. However, evidence supporting this mechanistic rationale from chemosensitization studies has not been consistent. Here we report a systematic definition of how pancreatic cancer cells harboring mutant p53 respond to this combination therapy, by combining mathematical models with large-scale quantitative biological analyses of single cells and cell populations. Notably, we uncovered a dynamic range of mechanistic effects at different ratios of gemcitabine and CHK1 inhibitors. Remarkably, effective synergy was attained even where cells exhibited an apparently functional G2/M surveillance mechanism, as exemplified by a lack of both overt premature CDK1 activation and S-phase mitotic entry. Consistent with these findings, S/G2 duration was extended in treated cells, leading to an definable set of lineage-dependent catastrophic fates. At synergistic drug concentrations, global replication stress was a distinct indicator of chemosensitization as characterized molecularly by an accumulation of S-phase cells with high levels of hyperphosphorylated, RPA-loaded single-stranded DNA. In a fraction of these cells, persistent genomic damage was observed, including chromosomal fragmentation with a loss of centromeric regions that prevented proper kinetochore-microtubule attachment. Together, our results suggested a “foot-in-the-door” mechanism for drug synergy where cells were destroyed not by frank G2/M phase abrogation, but rather by initiating a cumulative genotoxicity that deregulated DNA synthesis.

Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies. Cancer Res; 75(16); 1–11. ©2015 AACR.

Cancer stem–like cells (CSC) are a cell subpopulation that can reinitiate tumors, resist chemotherapy, and give rise to metastases. Metadherin (MTDH) contributes widely to tumor growth, drug resistance, relapse, and metastasis, but its molecular mechanisms of action are not well understood. Here, we report that MTDH drives CSC expansion by promoting the expression of TWIST1, a transcription factor critical for cancer cell stemness and metastasis. MTDH activates TWIST1 expression indirectly by facilitating histone H3 acetylation on the TWIST1 promoter, a process mediated by the histone acetyltransferase CBP. Mechanistic investigations showed that MTDH interacts with CBP and prevents its ubiquitin-mediated degradation, licensing its transcriptional activation of TWIST1. In clinical specimens of breast cancer, MTDH expression correlates positively with TWIST1 expression and CSC abundance. Overall, our work revealed that MTDH promotes CSC accumulation and breast tumorigenicity by regulating TWIST1, deepening the understanding of MTDH function in cancer. Cancer Res; 75(17); 1–9. ©2015 AACR.

Cancer stem-like cells (CSC) are a cell subpopulation that can re-initiate tumors, resist chemotherapy and give rise to metastases. Metadherin (MTDH) contributes widely to tumor growth, drug resistance, relapse and metastasis, but its molecular mechanisms of action are not well understood. Here we report that MTDH drives CSC expansion by promoting the expression of TWIST1, a transcription factor critical for cancer cell stemness and metastasis. MTDH activates TWIST1 expression indirectly by facilitating histone H3 acetylation on the TWIST1 promoter, a process mediated by the histone acetyltransferase CBP. Mechanistic investigations showed that MTDH interacts with CBP and prevents its ubiquitin-mediated degradation, licensing its transcriptional activation of TWIST1. In clinical specimens of breast cancer, MTDH expression correlates positively with TWIST1 expression and CSC abundance. Overall, our work revealed that MTDH promotes CSC accumulation and breast tumorigenicity by regulating TWIST1, deepening the understanding of MTDH function in cancer.

Metastasis is the main reason for lung cancer–related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133+/CXCR4+ cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non–small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133+/CXCR4+ cells in residual tumors and their metastatization. Immunophenotypic analysis of lung tumor cells intravenously injected or spontaneously disseminated to murine lungs demonstrated the survival advantage and increased colonization ability of a specific subset of CD133+/CXCR4+ with reduced expression of epithelial cell adhesion molecule (EpCAM−), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing mice enriched for CD133+/CXCR4+/EpCAM− CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133+/CXCR4+/EpCAM− subset. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P = 0.006) and their detection in primary tumors is correlated with poor clinical outcome (disease-free survival: P = 0.03; overall survival: P = 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor–microenvironment interactions. Cancer Res; 75(17); 1–14. ©2015 AACR.