Role of p68 in Breast Cancer

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Daojing Wang, Ph.D. -
Award Cycle: 2009 (Cycle 15) Grant #: 15IB-0075 Award: $165,335
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2009)

Two major challenges in diagnosis and therapy of breast cancer lie in variations among tumors and their resistance to drug treatment. The molecular pathways underlying these have not been worked out. In our preliminary studies, we found that high expression of a protein called p68 (also called DDX5) is associated with aggressiveness and drug resistance of breast cancer cells. We and others have found that p68 is involved in a complex interacting network in the cell. Strikingly, p68 was recently shown to be one of the two resistance genes for lung cancer cells during camptothecin (topoisomerase inhibitor) treatment. In this project, we will further define the role of p68 in breast cancer, with particular emphasis on invasion and drug resistance.

We hypothesize that p68 is a key molecular marker contributing to breast cancer heterogeneity, and can be used to stratify tumor aggressiveness and drug resistance. Since p68 has been shown to interact with several key proteins in breast cancer, we will study how p68 regulates the interacting network and therefore influences drug responses. For these studies we will combine detailed biochemical characterization of p68 with a systems biology approach to elucidate its signaling networks in breast cancer. We will modulate the expression of p68 in several breast cancer cell lines using RNAi (inhibition) and gene transfection (increased expression). We will then investigate the effects on invasion and on responses to several commercially available drugs. Finally, we will begin to characterize the reprogramming of p68 signaling network through profiling of interactome (i.e., whole set of molecular interactions in cells), transcriptome (i.e., the set of all messenger RNA molecules), and proteome (all protein constituents).

There is a good chance that p68 may contribute to personalized medicine by serving as a clinically useful molecular marker to characterize breast cancer subtypes, as a predictor for drug resistance, and it may serve as a drug target for a combinational therapy to circumvent the drug resistance commonly seen in aggressive tumors.




Final Report (2011)

Two major challenges in diagnosis and therapy of breast cancer lie in its heterogeneity and drug resistance. RNA helicase p68 (DDX5) has been shown to be involved in all aspects of RNA metabolism and serves as a transcriptional co-regulator. But its functional role in breast cancer remains elusive. This study is designed to mechanistically define the role of p68 in human breast cancer using an integrative biology approach.

We utilized a multifaceted strategy including cellular and molecular biology and various emerging technologies. Specifically, we measured the expression pattern of p68 at both mRNA and protein levels for a panel of ~50 breast cancer cell lines. We performed immunohistochemistry of p68 using human breast tumor microarrays containing a total of 225 cases of normal and malignant (various grades and stages) tissues of the breast. We knocked down p68 in two representative breast cancer cell lines (SKBR3 and MDA-MB-231) and investigated their proliferation and responses to Lapatinib before and after p68 knockdown. Finally, we carried out SILAC-based proteomic profiling of these breast cancer cells after p68 knockdown and identified p68-targeted proteins and networks contributing to the drug responses.

We showed that protein expression of DDX5 increased progressively from the luminal to basal breast cancer cell lines, and correlated positively with that of CD44 in the basal subtypes. Through immunohistochemistry analyses of tissue microarrays containing over 200 invasive human ductal carcinomas, we observed that DDX5 was increased in the majority of malignant tissues, and its expression correlated strongly with those of Ki67 and EGFR in the triple-negative tumors. We demonstrated that DDX5 regulated a subset of MicroRNAs including miR-21 and miR-182 in basal breast cancer cells. Knockdown of DDX5 resulted in reorganization of actin cytoskeleton and reduction of cellular proliferation. The effects were accompanied by an increase in tumor suppressor PDCD4 (a known miR-21 target); as well as an increase in cofilin and profilin, two key proteins involved in actin polymerization and cytoskeleton maintenance, as a consequence of miR-182 decreased amounts. Treatment with miR-182 inhibitors resulted in morphologic phenotypes resembling those induced by DDX5 knockdown. Using bioinformatics tools for pathway and network analyses, we confirmed that the network for regulation of actin cytoskeleton was predominantly enriched for the predicted downstream targets of miR-182.

In conclusion, RNA helicase p68 may contribute to personalized medicine by serving as a new molecular marker to characterize breast cancer subtypes, as a predictor for drug resistance, and as a target for a combinational therapy to circumvent drug resistance of aggressive tumors.




Symposium Abstract (2010)

Background and purpose. Two major challenges in diagnosis and therapy of breast cancer lie in its heterogeneity and drug resistance. RNA helicase p68 (DDX5) has been shown to be involved in all aspects of RNA metabolism and serves as a transcriptional co-regulator. Strikingly, a recent study demonstrated a new role of p53 in modulating miRNA processing, probably through the p53–Drosha/p68 interactions. As a result, the gene expression regulated by p53-Drosha/p68 is under intensive investigation. Previous work has shown that p68 is one of the potential 6-gene predictors of breast cancer resistance to Lapatinib, a dual ErbB2/EGFR tyrosine kinase inhibitor. Furthermore, we and others have shown that p68 is involved in a complex protein-protein interacting network (e.g., Ca2+/CaM binding). This study is designed to mechanistically define the role of p68 in human breast cancer using an integrative biology approach.

Experimental procedures. We utilized a multifaceted strategy including cellular and molecular biology and various emerging technologies. Specifically, we measured the expression pattern of p68 at both mRNA and protein levels for a panel of ~50 breast cancer cell lines. We performed immunohistochemistry of p68 using human breast tumor microarrays containing a total of 225 cases of normal and malignant (various grades and stages) tissues of the breast. We knocked down p68 in two representative breast cancer cell lines (SKBR3 and MDA-MB-231) and investigated their proliferation and responses to Lapatinib before and after p68 knockdown. Finally, we carried out SILAC-based proteomic profiling of these breast cancer cells after p68 knockdown and identified p68-targeted proteins and networks contributing to the drug responses.

Results. P68 expression pattern is distinct among different subtypes of breast cancers. More aggressive basal A and B subtypes have predominantly high p68 protein expression while low p68 expression is predominantly associated with the luminal subtype. Knockdown of p68 inhibits the proliferation of breast cancer cells and sensitize them to cancer drugs such as Lapatinib. Importantly, we identified cofilin-1, a major actin severing and -depolymerization protein, as a key target of p68 through proteomics studies. Knockdown of p68 increases cofilin expression. Cofilin has been previously implicated in platinum-resistance of ovarian cancer cells, and migration and invasion of breast cancer cells. Therefore, our results suggest that high p68 expression may contribute to drug resistance and metastasis of breast cancer cells through cytoskeletal reorganization by promoting actin polymerization.

Conclusions. RNA helicase p68 may contribute to personalized medicine by serving as a new molecular marker to characterize breast cancer subtypes, as a predictor for drug resistance, and as a target for a combinational therapy to circumvent drug resistance of aggressive tumors.



Single cell analysis: the new frontier in 'omics'.
Periodical:Trends in Biotechnology
Index Medicus: Trends Biotechnol
Authors: Wang D, Bodovitz S
Yr: 2010 Vol: 28 Nbr: 6 Abs: Pg:281-90

RNA helicase DDX5 regulates MicroRNA expression and contributes to cytoskeletal reorganization in basal breast cancer cells.
Periodical:Molecular and Cellular Proteomics
Index Medicus: Mol Cell Proteomics
Authors: Wang D, Huang J, Hu Z
Yr: 2011 Vol: 11 Nbr: Abs: Pg:Nov 15 (Epub)