Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells. Cancer Res; 1–15. ©2015 AACR.

Gastric cancer remains the third leading cause of cancer-related mortality worldwide, and invasion and metastasis of gastric cancer represent the major reason for its poor prognosis. In this study, we found that loss of the receptor for activated C-kinase 1 (RACK1) promoted the metastasis of gastric cancer by enhancing the autocrine of interleukin (IL)-8 in vitro and in vivo. MicroRNA array identified that RACK1 modulated the expression of a series of microRNAs including microRNA-302 cluster, and RACK1 modulated the IL-8 expression and tumor invasion through microRNA-302c. Moreover, up-regulation of IL-8 in turn decreased the level of microRNA-302c and induced IL-8 expression in feedback manner. Tissue microarray also indicated that RACK1 was correlated with invasion/metastasis phenotype, IL-8 expression as well as five-year survival in clinical cases of gastric cancer. Together, our results imply that loss of RACK1 in gastric cancer links epigenetics to inflammatory cytokines to promote tumor metastasis.

While a small number of plasma membrane ABC transporters can export chemotherapeutic drugs and confer drug resistance, it is unknown if these transporters are expressed or functional in less therapeutically tractable cancers such as Group3 medulloblastoma (G3 MB). Herein we show that among this class of drug transporters only ABCG2 was expressed at highly increased levels in human G3 MB and a mouse model of this disease. In the mouse model, Abcg2 protein was expressed at the plasma membrane where it functioned as expected based on export of prototypical substrates. By screening ABC substrates against mouse G3 MB tumorspheres in vitro, we found that Abcg2 inhibition could potentiate responses to the clinically employed drug topotecan, producing a >9-fold suppression cell proliferation. Extended studies in vivo in this model confirmed that Abcg2 inhibition was sufficient to enhance antiproliferative responses to topotecan, producing a significant survival advantage compared to subjects treated with topotecan alone. Our findings offer a preclinical proof of concept for blockade of ABCG2 transporter activity as a strategy to empower chemotherapeutic responses in Group3 medulloblastoma.

Tristetraprolin (TTP) is an inducible zinc finger AU-rich RNA-binding protein essential for enforcing degradation of mRNAs encoding inflammatory chemokines and cytokines. Most studies on TTP center on the connection between mRNA half-life and inflammatory output, because loss of TTP amplifies inflammation by increasing the stability of AU-rich mRNAs. Here, we focused on how TTP controls cytokine and chemokine production in the nonresolving inflammation of cancer using tissue-specific approaches. In contrast with model in vitro macrophage systems, we found constitutive TTP expression in late-stage tumor-associated macrophages (TAM). However, TTP’s effects on AU-rich mRNA stability were negligible and limited by constitutive p38α MAPK activity, which was the main driver of proinflammatory cytokine production in TAMs at the posttranscriptional level. Instead, elimination of TTP caused excessive protein production of inflammatory mediators, suggesting TTP-dependent translational suppression of AU-rich mRNAs. Manipulation of the p38α–TTP axis in macrophages has significant effects on the growth of tumors and therefore represents a means to manipulate inflammation in the tumor microenvironment. Cancer Res; 75(15); 1–11. ©2015 AACR.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells from carcinogenic effects of alkylating agents; however, MGMT is silenced by promoter hypermethylation during carcinogenesis. A single-nucleotide polymorphism (SNP) in an enhancer in the MGMT promoter was previously identified to be highly significantly associated with risk for MGMT methylation in lung cancer and sputum from smokers. To further genetic investigations, a genome-wide association and replication study was conducted in two smoker cohorts to identify novel loci for MGMT methylation in sputum that were independent of the MGMT enhancer polymorphism. Two novel trans-acting loci (15q15.2 and 17q24.3) that were identified acted together with the enhancer SNP to empower risk prediction for MGMT methylation. We found that the predisposition to MGMT methylation arising from the 15q15.2 locus involved regulation of the ubiquitin protein ligase E3 component UBR1. UBR1 attenuation reduced turnover of MGMT protein and increased repair of O6-methylguanine in nitrosomethylurea-treated human bronchial epithelial cells, while also reducing MGMT promoter activity and abolishing MGMT induction. Overall, our results substantiate reduced gene transcription as a major mechanism for predisposition to MGMT methylation in the lungs of smokers, and support the importance of UBR1 in regulating MGMT homeostasis and DNA repair of alkylated DNA adducts in cells. Cancer Res; 75(15); 1–10. ©2015 AACR.