Cell line authentication: a necessity for reproducible biomedical research.

Immortalized or continuous cell lines are invaluable tools in basic and preclinical research. However, the widespread use of misidentified cell lines is a serious threat to scientific reproducibility. Based on the experiences of mandatory cell line authentication at the International Journal of Cancer (IJC), we provide an overview of the issues pertinent to misidentified cell lines and discuss available solutions. We also summarize the lessons learned, revealing that at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified. About 4% of the considered manuscripts are rejected for severe cell line problems, and most are subsequently published in other journals. In order to diminish such malpractice and its consequences for the scientific record, we postulate that strict multi-layered quality control is essential. Besides journals and publishers, we encourage scientists, research institutions, and funders to take action on the matter and revise their respective policies. Hence, we provide concrete recommendations on introducing regular authentication schemes and staff training, and discuss future steps for enhancing good cell culture practices.

Protein phosphatase 1, regulatory subunit 15B is a survival factor for ERα-positive breast cancer.

Breast cancer is a heterogeneous disease at both the clinical and molecular levels. This heterogeneity may give rise to different therapy responses. Molecular profiling has facilitated identification of signatures for stratifying patients who would potentially benefit from given therapies. Previously, we reported on a subset of genes with the potential for predicting response of primary breast cancer to neoadjuvant chemotherapy. Herein, we report that patients with luminal (estrogen receptor α [ERα]-expressing) breast cancer were enriched for nonresponders. To identify novel factors that contribute to the survival of breast cancer cells, a loss-of-function screen was performed with a subset of genes overexpressed in patients with disease resistant to chemotherapy. This approach led us to identify protein phosphatase 1, regulatory subunit 15B (PPP1R15B) as a factor with a potentially essential role in the survival of ERα-positive breast cancer cells. Functional analyses showed that PPP1R15B depletion results in impaired proliferation due to unsuccessful transition of cells from G1 to S phase of the cell cycle, and apoptosis induction. Moreover, our data revealed a regulatory role for PPP1R15B in activating ERα. Furthermore, a high level of PPP1R15B mRNA expression was associated with poor outcome following tamoxifen-based therapy. Accordingly, knockdown of PPP1R15B expression sensitized tamoxifen-resistant MCF-7 breast cancer cells to tamoxifen while reducing ERα abundance in these cells. Our findings reveal a novel role for PPP1R15B in the survival and therapy response of ERα-positive breast cancer and may open new avenues for tumor subtype-specific therapeutic strategies in the era of personalized medicine.

Antiapoptotic function of charged multivesicular body protein 5: a potentially relevant gene in acute myeloid leukemia.

In recent years, RNA interference (RNAi) has been widely used to uncover gene function or pathway context of novel genes. In our study, we describe a short-hairpin RNA-based RNAi screening of a set of functionally uncharacterized human genes for their possible capability to inhibit apoptosis. We thereby identified a new antiapoptotic function for CHMP5 (charged multivesicular body protein 5), which was confirmed by overexpression and rescue assays. Furthermore, caspase assays showed that CHMP5 silencing induced caspase cascade activation mainly through extrinsic apoptosis pathway. Based on genome-wide expression array profiling, a possible regulatory role of CHMP5 on apoptosis-associated genes and different signaling pathways including nuclear factor kappa B was revealed. In addition, we found significantly higher CHMP5 mRNA levels in acute myeloid leukemia patients. This observation together with the antiapoptotic feature of CHMP5 suggests a possible oncogenic function for this gene in leukemogenesis.

Reprogramming of the ERRα and ERα target gene landscape triggers tamoxifen resistance in breast cancer.

Endocrine treatment regimens for breast cancer that target the estrogen receptor-α (ERα) are effective, but acquired resistance remains a limiting drawback. One mechanism of acquired resistance that has been hypothesized is functional substitution of the orphan receptor estrogen-related receptor-α (ERRα) for ERα. To examine this hypothesis, we analyzed ERRα and ERα in recurrent tamoxifen-resistant breast tumors and conducted a genome-wide target gene profiling analysis of MCF-7 breast cancer cell populations that were sensitive or resistant to tamoxifen treatment. This analysis uncovered a global redirection in the target genes controlled by ERα, ERRα, and their coactivator AIB1, defining a novel set of target genes in tamoxifen-resistant cells. Beyond differences in the ERα and ERRα target gene repertoires, both factors were engaged in similar pathobiologic processes relevant to acquired resistance. Functional analyses confirmed a requirement for ERRα in tamoxifen- and fulvestrant-resistant MCF-7 cells, with pharmacologic inhibition of ERRα sufficient to partly restore sensitivity to antiestrogens. In clinical specimens (n = 1041), increased expression of ERRα was associated with enhanced proliferation and aggressive disease parameters, including increased levels of p53 in ERα-positive cases. In addition, increased ERRα expression was linked to reduced overall survival in independent tamoxifen-treated patient cohorts. Taken together, our results suggest that ERα and ERRα cooperate to promote endocrine resistance, and they provide a rationale for the exploration of ERRα as a candidate drug target to treat endocrine-resistant breast cancer.

Estrogen-related receptor alpha expression and function is associated with the transcriptional coregulator AIB1 in breast carcinoma.

The significance of the estrogen-related receptor alpha (ERRalpha) as prognostic marker for poor clinical outcome in breast carcinoma has recently been reported. Transcriptional activity of nuclear receptors such as ERRalpha depends on coregulatory proteins. Thus, we compared the expression of different receptors, coregulators, and target genes on RNA and protein level in identical primary breast tumor samples (n = 48). We found a positive correlation between the transcripts of ERRalpha and AIB1 (amplified in breast cancer-1), a coactivator overexpressed in breast cancers and associated with resistance to antihormone treatment. These data were confirmed on protein level, studying an independent patient collection (n = 257). Expression of the estrogen-regulated gene pS2 was associated with ERRalpha only in tumors, where estrogen receptor (ERalpha) expression was low or absent. In ERalpha high expressing tumors, no correlation of ERRalpha and pS2 was observed. AIB1 interacts directly with ERRalpha as shown by fluorescence-resonance energy transfer, mammalian two-hybrid, and coimmunoprecipitation assays with endogenous proteins. It enhances ERRalpha transcriptional activity in ERalpha-negative breast cancer cell lines as shown in functional reporter gene assays. Blocking ERRalpha with an inverse agonist abolished interaction and coactivation by AIB1. Recruitment of both proteins to ERRalpha target gene promoters further supports the significance of their interaction. Our findings identify AIB1 as functionally relevant cofactor for ERRalpha in breast carcinoma. ERRalpha/AIB1 complexes may control estradiol-regulated genes in a hormone-independent manner. Accordingly, ERRalpha might be a rewarding target for treatment of endocrine-resistant tumors.