Platinum-based chemotherapeutic drugs are front-line therapies for the treatment of non-small cell lung cancer. However, intrinsic drug resistance limits the clinical efficacy of these agents. Recent evidence suggests that loss of the translesion polymerase, Polζ, can sensitize tumor cell lines to cisplatin, although the relevance of these findings to the treatment of chemoresistant tumors in vivo has remained unclear. Here, we describe a tumor transplantation approach that enables the rapid introduction of defined genetic lesions into a preclinical model of lung adenocarcinoma. Using this approach, we examined the effect of impaired translesion DNA synthesis on cisplatin response in aggressive late-stage lung cancers. In the presence of reduced levels of Rev3, an essential component of Polζ, tumors exhibited pronounced sensitivity to cisplatin, leading to a significant extension in overall survival of treated recipient mice. Additionally, treated Rev3-deficient cells exhibited reduced cisplatin-induced mutation, a process that has been implicated in the induction of secondary malignancies following chemotherapy. Taken together, our data illustrate the potential of Rev3 inhibition as an adjuvant therapy for the treatment of chemoresistant malignancies, and highlight the utility of rapid transplantation methodologies for evaluating mechanisms of chemotherapeutic resistance in preclinical settings.
Summary
In normal cells p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21cip1 and Gadd45α. In p53 mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the “successor” to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53 this is through the post-transcriptional stabilization of p27Kip1 and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer demonstrating the importance of post-transcriptional RNA control to chemotherapy response.
There are several hundreds of genes overexpressed in a typical breast cancer cell, and their overexpression is often assumed to have a pro-cancer function, but what percentage of these overexpressed genes have a role in promoting and maintaining tumorigenicity isn’t known. To address this question, we screened an unbiased set of seventy-five genes overexpressed in breast cancer for their ability to induce malignant transformation and for breast cancer cell expression dependency. Barcoded open-reading frames under the control of a constitutive promoter were tested by pooled screening for their ability to promote tumor formation of premalignant mammary epithelial cells and growth in 2D and 3D culture. We found that growth in 3D cell culture was more strongly correlated with tumorigenic growth than was 2D growth, validating a long-standing hypothesis. Genes with high activity in promoting both 3D growth and in vivo tumor formation included CEACAM5, CXCL8, NKX2-5, PYGO2 and WWTR1/TAZ. To screen for corresponding tumor dependencies on these six genes, we performed shRNA screening of breast cancer cell lines and found uniform dependency on expression of the Hippo pathway transcriptional activator WWTR1, selective dependencies on CEACAM5 expression, but no consistent effects for the other three genes. Since breast cancer dependency upon WWTR1 is well established, we focused our follow-up studies on CEACAM5. We confirmed that CEACAM5 overexpression conferred tumorigenicity to pre-malignant mammary epithelial cells and that reduced expression selectively decreased the clonogenicity of luminal breast cancer cell lines. Protein analysis indicated that CEACAM5 activated Cdk4/6 and mTOR signaling. We also determined that CEACAM5 overexpression strongly promoted cell number increase in 3D spheroid culture by a novel mechanism that increased the number of epithelial cell layers from a single layer to multiple layers. These results outline a strategy for functional genomic identification of cancer targets, starting with a set of genes overexpressed in cancer, then using pooled approaches to identify those that promote tumorigenicity and cancer cell dependency, followed by validation experiments and exploration of oncogenic mechanisms. We propose that larger screens would substantially increase the number of potential targets for treating breast cancer.
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