Functional Proteomics Approach to Investigate the Biological Activities of cDNAs Implicated in Breast Cancer

Witt, Abigail E.; Hines, Lisa M.; Collins, Nicole L.; Hu, Yanhui; Gunawardane, Ruwanthi N.; Moreira, Donna; Raphael, Jacob; Jepson, Daniel A.; Koundinya, Malvika; Rolfs, Andreas; Taron, Barbara W.; Isakoff, Steven J.; Brugge, Joan S.; LaBaer, Joshua

doi:10.1021/pr050395r

Cited by 62 publications

(62 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For the in vivo screen, we employed the Cancer 1000 shRNA library consisting of 2,204 shRNAs, targeting a panel of about 1,000 cancer-associated mouse genes (8,23), with a subpool complexity of 96 shRNAs (24 subpools). The screen strategy is outlined in Fig.…”

Section: Resultsmentioning

confidence: 99%

An In Vivo Functional Screen Identifies JNK Signaling As a Modulator of Chemotherapeutic Response in Breast Cancer

Ashenden

Weverwijk

Murugaesu

et al. 2017

Molecular Cancer Therapeutics

View full text Add to dashboard Cite

Chemotherapy remains the mainstay of treatment for advanced breast cancer; however, resistance is an inevitable event for the majority of patients with metastatic disease. Moreover, there is little information available to guide stratification of firstline chemotherapy, crucial given the common development of multidrug resistance. Here, we describe an in vivo screen to interrogate the response to anthracycline-based chemotherapy in a syngeneic metastatic breast cancer model and identify JNK signaling as a key modulator of chemotherapy response. Combining in vitro and in vivo functional analyses, we demonstrate that JNK inhibition both promotes tumor cell cytostasis and blocks activation of the proapoptotic protein Bax, thereby antagonizing chemotherapy-mediated cytotoxicity. To investigate the clinical relevance of this dual role of JNK signaling, we developed a proliferation-independent JNK activity signature and demonstrate high JNK activity to be enriched in triple-negative and basal-like breast cancer subtypes. Consistent with the dual role of JNK signaling in vitro, high-level JNK pathway activation in triplenegative breast cancers is associated both with poor patient outcome in the absence of chemotherapy treatment and, in neoadjuvant clinical studies, is predictive of enhanced chemotherapy response. These data highlight the potential of monitoring JNK activity as early biomarker of response to chemotherapy and emphasize the importance of rational treatment regimes, particularly when combining cytostatic and chemotherapeutic agents.

show abstract

Section: Resultsmentioning

confidence: 99%

An In Vivo Functional Screen Identifies JNK Signaling As a Modulator of Chemotherapeutic Response in Breast Cancer

Ashenden

Weverwijk

Murugaesu

et al. 2017

Molecular Cancer Therapeutics

View full text Add to dashboard Cite

show abstract

“…We next tested the in vitro translation products of ;1000 cDNAs previously linked to breast cancer (Witt et al 2006). Each cDNA was tested in quadruplicate in separate plates (one cDNA per well per plate 3 four plates).…”

Section: Screen For Novel Egln2 Substratesmentioning

confidence: 99%

“…We adapted a previously reported 96-well decarboxylation assay to screen for proteins that can be hydroxylated by EglN2 in vitro (Zhang et al 1999). We focused on 1000 proteins previously linked to breast cancer because EglN2 À/À mice exhibit mammary gland hypoproliferation and because loss of EglN2 inhibits breast cancer growth (Witt et al 2006;Zhang et al 2009). We identified FOXO3a as an EglN2 prolyl hydroxylase substrate.…”

mentioning

confidence: 99%

Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase

et al. 2014

View full text Add to dashboard Cite

The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity.

show abstract

“…Importantly, these shRNAs can stably and efficiently knockdown target genes when expressed at single copy in the genome (13). We chose to survey shRNAs targeting the ''cancer 1000,'' a set of known or putative cancer-relevant genes compiled by manual curation, microarray expression data, and literature mining (17). To improve gene knockdown and facilitate in vivo experiments (13), all of the existing murine shRNAs targeting the cancer 1000 set (Ϸ2,300 shRNAs, two to three shRNAs per gene) were cloned into a murine stem cell virus (MSCV)-based vector that coexpressed green f luorescent protein.…”

Section: Rnai Screens Identify Shrnas Mediating Doxorubicin Resistancementioning

confidence: 99%

Topoisomerase levels determine chemotherapy response in vitro and in vivo

Burgess

Doles²,

Zender

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

280

236

View full text Add to dashboard Cite

Topoisomerase poisons are chemotherapeutic agents that are used extensively for treating human malignancies. These drugs can be highly effective, yet tumors are frequently refractory to treatment or become resistant upon tumor relapse. Using a pool-based RNAi screening approach and a well characterized mouse model of lymphoma, we explored the genetic basis for heterogeneous responses to topoisomerase poisons in vitro and in vivo. These experiments identified Top2A expression levels as major determinants of response to the topoisomerase 2 poison doxorubicin and showed that suppression of Top2A produces resistance to doxorubicin in vitro and in vivo. Analogously, using a targeted RNAi approach, we demonstrated that suppression of Top1 produces resistance to the topoisomerase 1 poison camptothecin yet hypersensitizes cancer cells to doxorubicin. Importantly, lymphomas relapsing after treatment display spontaneous changes in topoisomerase levels as predicted by in vitro gene knockdown studies. These results highlight the utility of pooled shRNA screens for identifying genetic determinants of chemotherapy response and suggest strategies for improving the effectiveness of topoisomerase poisons in the clinic.A myriad of genetic factors influence the efficacy of cancer chemotherapy, including both somatic changes in the tumor itself as well as genetic polymorphisms present in the patient. These factors include increased expression of detoxification pumps that prevent access of the drug to its target (1), point mutations that disrupt the drug-target interaction (2, 3), and mutations in stress response pathways [e.g., p53 loss (4)]. To tailor treatment successfully to the individual patient, a more complete understanding of the genetic determinants of therapy response is necessary.RNA interference (RNAi) exploits a mechanism of gene regulation whereby double-stranded RNAs are processed by a conserved cellular machinery to suppress the expression of genes containing homologous sequences (5). Importantly, libraries of DNA-based vectors encoding short hairpin RNAs (shRNAs) capable of targeting most genes in the human and mouse genomes have been produced and enable forward genetic screens to be performed in mammalian cells. Indeed, by using human tumor-derived cell lines treated in vitro, RNAi has been used to evaluate potential drug targets (6) or to investigate mechanisms of drug action and drug resistance by screening for new molecules that modulate the response of tumor-derived cell lines to a given chemotherapeutic agent (7-10).Here, we evaluate the suitability of combining mouse models and RNAi to identify genetic modifiers of drug action in tumors in their natural site. Initially, we chose to investigate resistance to doxorubicin in the E -Myc mouse lymphoma system. Doxorubicin (Adriamycin) is an anthracycline DNA-damaging agent that exerts its effects primarily by targeting of the topoisomerase 2 activity and DNA intercalation (11). Along with etoposide and the camptothecin derivatives, doxorubicin is one of several t...

show abstract

Functional Proteomics Approach to Investigate the Biological Activities of cDNAs Implicated in Breast Cancer

Cited by 62 publications

References 71 publications

An In Vivo Functional Screen Identifies JNK Signaling As a Modulator of Chemotherapeutic Response in Breast Cancer

An In Vivo Functional Screen Identifies JNK Signaling As a Modulator of Chemotherapeutic Response in Breast Cancer

Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase

Topoisomerase levels determine chemotherapy response in vitro and in vivo

Contact Info

Product

Resources

About