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Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with unfavorable outcomes. Developing therapeutic targets for TNBC remains a challenge. Here, we identified that acetyl-CoA acyltransferase 1 (ACAA1) is highly expressed in the luminal androgen receptor (LAR) subtype of TNBC compared with adjacent normal tissues in our TNBC proteomics dataset. Inhibition of ACAA1 restrained TNBC proliferation and potentiated the response to the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor abemaciclib. Mechanistically, ACAA1 interacted with CDK4, and the inhibition of ACAA1 blocked RB transcriptional corepressor 1 (RB1) phosphorylation, resulting in G1–S cell-cycle arrest. Importantly, trimetazidine, a traditional drug for ischemic heart disease, caused a decrease in ACAA1 protein levels and enhanced the efficacy of abemaciclib in preclinical TNBC models. In conclusion, this study identifies that ACAA1 is a therapeutic target in TNBC and suggests the combination of trimetazidine and abemaciclib could be beneficial for ACAA1-high TNBCs. Significance: ACAA1 is highly expressed in TNBC, serving as a potential therapeutic target in ACAA1-high tumors and a predictive biomarker of resistance to CDK4/6 inhibitors for RB1-proficient patients.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with unfavorable outcomes. Developing therapeutic targets for TNBC remains a challenge. Here, we identified that acetyl-CoA acyltransferase 1 (ACAA1) is highly expressed in the luminal androgen receptor (LAR) subtype of TNBC compared with adjacent normal tissues in our TNBC proteomics dataset. Inhibition of ACAA1 restrained TNBC proliferation and potentiated the response to the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor abemaciclib. Mechanistically, ACAA1 interacted with CDK4, and the inhibition of ACAA1 blocked RB transcriptional corepressor 1 (RB1) phosphorylation, resulting in G1–S cell-cycle arrest. Importantly, trimetazidine, a traditional drug for ischemic heart disease, caused a decrease in ACAA1 protein levels and enhanced the efficacy of abemaciclib in preclinical TNBC models. In conclusion, this study identifies that ACAA1 is a therapeutic target in TNBC and suggests the combination of trimetazidine and abemaciclib could be beneficial for ACAA1-high TNBCs.Significance:ACAA1 is highly expressed in TNBC, serving as a potential therapeutic target in ACAA1-high tumors and a predictive biomarker of resistance to CDK4/6 inhibitors for RB1-proficient patients.

Supplementary methods, figures, figure legends, and tables

Supplementary Figure S1. Flowchart of study design. Supplementary Figure S2. Protein structure visualizations of the HER2 somatic mutations. Supplementary Figure S3. HER2 mutations L768S and V773L induce HER2 internalization. Supplementary Figure S4. Kaplan-Meier estimates of relapse-free survival (RFS) according to HER2 mutation in different regions.

Supplementary Methods: Immunofluorescence

Purpose: By integrating expression profiles of mRNAs and long noncoding RNAs (lncRNA), we tried to develop and validate novel multigene signatures to facilitate individualized treatment of triple-negative breast cancer (TNBC) patients.Experimental Design: We analyzed 165 TNBC samples and 33 paired normal breast tissues using transcriptome microarrays. Tumor-specific mRNAs and lncRNAs were identified and correlated with patients' recurrence-free survival (RFS). Using Cox regression model, we built two multigene signatures incorporating mRNAs and lncRNAs. The prognostic and predictive accuracy of the signatures were tested in a training set of 165 TNBC patients and validated in other 101 TNBC patients.Results: We successfully developed an mRNA and an integrated mRNA–lncRNA signature based on eight mRNAs and two lncRNAs. In the training set, patients in the high-risk group were more likely to suffer from recurrent disease than patients in the low-risk group in both signatures [HR, 10.00; 95% confidence interval (CI), 2.53–39.47, P = 0.001; HR = 4.46, 95% CI, 1.34–14.91, P = 0.015 for integrated signature and mRNA signature, respectively). Results were validated in the validation set (P = 0.019 and 0.030, respectively). In addition, time-dependent receiver operating curve showed that the integrated mRNA–lncRNA signature had a better prognostic value than both the eight-mRNA-only signature and the clinicopathologic risk factors in both sets. We also found through interaction analysis that patients classified into the low-risk group by the integrated mRNA–lncRNA signature had a more favorable response to adjuvant taxane chemotherapy.Conclusions: The multigene signature we developed can accurately predict clinical outcome and benefit of taxane chemotherapy in TNBC patients. Clin Cancer Res; 22(7); 1653–62. ©2016 AACR.

Supplementary Figure 1. Kaplan-Meier estimates of recurrence-free survival (RFS) according to the expression of every ten mRNA/lncRNA incorporated in the integrated signature in the training set (n=165). RNA expression level was coded as high or low according to the median expression level. Supplementary Figure 2. Validation of the expression of each mRNA/lncRNA incorporated in the integrated signature in the 33 paired tumor and normal breast tissues from the training set using quantitative real-time polymerase chain reaction (qRT-PCR). Supplementary Figure 3. Flowchart of the study design, patient selection and analytical strategy. Supplementary Figure 4. Details of the filtration procedures. For lncRNAs, either lncRNAs with fold change >1.5 and P2 and P

While recognized as a generally aggressive disease, triple-negative breast cancer (TNBC) is highly diverse in different patients with variable outcomes. In this prospective observational study, we aimed to develop an RNA signature of TNBC patients to improve risk stratification and optimize the choice of adjuvant therapy. Transcriptome microarrays for 33 paired TNBC and adjacent normal breast tissue revealed tumor-specific mRNAs and long noncoding RNAs (lncRNA) that were associated with recurrence-free survival. Using the Cox regression model, we developed an integrated mRNA-lncRNA signature based on the mRNA species for FCGR1A, RSAD2, CHRDL1, and the lncRNA species for HIF1A-AS2 and AK124454. The prognostic and predictive accuracy of this signature was evaluated in a training set of 137 TNBC patients and then validated in a second independent set of 138 TNBC patients. In addition, we enrolled 82 TNBC patients who underwent taxane-based neoadjuvant chemotherapy (NCT) to further verify the predictive value of the signature. In both the training and validation sets, the integrated signature had better prognostic value than clinicopathologic parameters. We also confirmed the interaction between the administration of taxane-based NCT and different risk groups. In the NCT cohort, patients in the low-risk group were more likely to achieve pathologic complete remission after taxane-based NCT (P = 0.014). Functionally, we showed that HIF1A-AS2 and AK124454 promoted cell proliferation and invasion in TNBC cells and contributed there to paclitaxel resistance. Overall, our results established an integrated mRNA-lncRNA signature as a reliable tool to predict tumor recurrence and the benefit of taxane chemotherapy in TNBC, warranting further investigation in larger populations to help frame individualized treatments for TNBC patients. Cancer Res; 76(8); 2105–14. ©2016 AACR.

Supplementary Methods. Supplementary Table S1. Selected mRNAs and lncRNAs by comparing the expression profiles in 33 paired tumor and adjacent normal tissues of triple-negative breast cancer. Supplementary Table S2. Primers for the real-time RT-PCR analysis of mRNAs and lncRNAs included in the signature. Supplementary Table S3. Cutoff value for each RNA. Supplementary Table S4. Univariate Cox proportional hazards regression analysis of the integrated RNA signature and clinicopathological characteristics with recurrence-free survival. Supplementary Table S5. Clinicopathologic characteristics of patients with triple-negative breast cancer who received neoadjuvant chemotherapy. Supplementary Table S6. Multivariate logistic regression analysis of the integrated RNA signature and clinicopathological characteristics with pathological complete remission. Supplementary Table S7. Top ten mRNAs co-expressed with lncRNAs AK124454 and HIF1A-AS2. Supplementary Table S8. Top ten gene-ontology terms and pathways in which the co-expressed mRNAs involved. Supplementary Figure S1. Flowchart of RNA filtration. Supplementary Figure S2. Flowchart of study design, patient selection and analytical strategy. Supplementary Figure S3. Hierarchical clustering of 33 paired tumor and adjacent normal breast tissues with the 5 differentially expressed RNAs using Euclidean distance and average linkage clustering. Supplementary Figure S4. Validation of the expression of each mRNA/lncRNA incorporated in the integrated signature in the 33 paired tumor and adjacent normal tissues from the training set using quantitative real-time polymerase chain reaction (qRT-PCR). Supplementary Figure S5. Predictive values of taxane benefit using the integrated signature.

Purpose: Somatic mutations in the tyrosine kinase domain of human epidermal growth factor receptor 2 (HER2) may be an alternative mechanism to HER2 activation and can affect the sensitivity toward HER2-targeted therapies. We aimed to investigate the prevalence, clinicopathologic characteristics, and functional relevance of novel HER2 mutations in breast cancer.Experimental Design: We performed Sanger sequencing of all exons of the HER2 gene in 1,248 primary tumors and 18 paired metastatic samples. Novel HER2 mutations were functionally characterized.Results: The total HER2 somatic mutation rate was 2.24% (28/1,248). Of the seven novel HER2 mutations, L768S and V773L were only detected in HER2-negative tumors, whereas K753E was found in HER2-positive disease. L768S and V773L mutations exhibited a significant increase in tyrosine kinase–specific activity and strongly increased the phosphorylation of signaling proteins in various cell lines. Xenograft experiments showed that NIH3T3 cells bearing the L768S and V773L mutations displayed more rapid growth. MCF10A, BT474, and MDA-MB-231 cells bearing the K753E mutation were resistant to lapatinib, but could be inhibited by neratinib. Finally, comparison of HER2 mutations in 18 pairs of primary and metastatic lesions revealed that the drug-resistant HER2 mutations (K753E and L755S) were enriched in metastatic lesions.Conclusions: HER2-negative breast cancer with activating mutations can benefit from HER2-targeted therapies. Meanwhile, mutations in the HER2 kinase domain might be a key mechanism of resistance to HER2-targeted therapy, and irreversible tyrosine kinase inhibitors such as neratinib may offer alternative treatment options. Clin Cancer Res; 22(19); 4859–69. ©2016 AACR.

Supplementary Table S1. Sanger sequencing primers for all 27 exons of HER2 gene. Supplementary Table S2. Clinicopathologic characteristics of breast cancer patients enrolled in our study. Supplementary Table S3. Clinicopathologic characteristics of HER2-mutated breast cancer. Supplementary Table S4. Clinical information of patients with HER2-mutated breast cancer

Purpose: Triple-negative breast cancer (TNBC) is a highly heterogeneous disease. Patients with early-stage TNBCs have distinct likelihood of distant recurrence. Current therapeutic guidance is still limited.Methods: We extracted transcriptome data for 189 pathologically confirmed pT1-2N0M0 TNBC patients at Fudan University Shanghai Cancer Center. Candidate mRNAs were filtered, which was followed by differential expressed mRNAs analysis, survival analysis, and LASSO Cox regression model. All-subsets regression program was used for constructing a multi-mRNA signature in the training set (n=159); the accuracy and prognostic value were then validated using an independent validation set (n=158). Results: Here, we profiled the transcriptome data from 189 early-stage TNBC patients along with 50 paired normal tissues. Early-stage TNBCs are featured of basal-like and immune-suppressed subtype and homologous recombination ability deficiency. We developed a prognostic signature contained seven mRNAs from transcriptome data (ACAN, KRT5, TMEM101, LCA5, RPP40, LAGE3, CDKL2). In both the training (n=159) and validation cohorts (n=158), the signature could identify patients with relatively high recurrence risks and serve as an independent prognostic factor. The signature had better prognostic value than traditional clinicopathological features in both sets. Among the seven mRNAs, TMEM101 is highly expressed in TNBC and represents a potential therapeutic target. Inhibition of TMEM101 impaired tumor progression.Conclusions: Our 7-mRNA signature could accurately predict recurrence risks of early-stage TNBCs. Clinical and genomic low risk TNBC patients may have the opportunity to avoid adjuvant chemotherapy

BACKGROUND Bile duct ligation (BDL) in animals is a classical method for mimicking cholestatic fibrosis. Although different surgical techniques have been described in rats and rabbits, mouse models can be more cost-effective and reproducible for investigating cholestatic fibrosis. Magnetic resonance imaging (MRI) has made great advances for noninvasive assessment of liver fibrosis. More comprehensive liver fibrotic features of BDL on MRI are important. However, the utility of multiparameter MRI to detect liver fibrosis in a BDL mouse model has not been assessed. AIM To evaluate the correlation between the pathological changes and multiparameter MRI characteristics of liver fibrosis in a BDL mouse model. METHODS Twenty-eight healthy adult male balb/c mice were randomly divided into four groups: sham, week 2 BDL, week 4 BDL, and week 6 BDL. Multiparameter MRI sequences, included magnetic resonance cholangiopancreatography, T1-weighted, T2-weighted, T2 mapping, and pre- and post-enhanced T1 mapping, were performed after sham and BDL surgery. Peripheral blood and liver tissue were collected after MRI. For statistical analysis, Student’s t-test and Pearson’s correlation coefficient were used. RESULTS Four mice died after BDL surgery; seven, six, five and six mice were included separately from the four groups. Signal intensities of liver parenchyma showed no difference on TI- and T2-weighted images. Bile duct volume, ΔT1 value, T2 value, and the rate of liver fibrosis increased steadily in week 2 BDL, week 4 BDL and week 6 BDL groups compared with those in the sham group (P < 0.01). Alanine aminotransferase and aspartate transaminase levels initially surged after surgery, followed by a gradual decline over time. Strong correlations were found between bile duct volume (r = 0.84), T2 value (r = 0.78), ΔT1 value (r = 0.62), and hepatic fibrosis rate (all P < 0.01) in the BDL groups. CONCLUSION The BDL mouse model induces changes that can be observed on MRI. The MRI parameters correlate with the hepatic fibrosis rate and allow for detection of cholestatic fibrosis.

Background: Aberrant splicing is a hallmark of cancer. For MYC-driven cancers, such as triple-negative breast cancer, hypersensitivity to spliceosome-targeted therapy is a novel anticancer strategy since it also triggers antitumorimmunity. Of the approximately 200 known spliceosomal genes, core spliceosomes are essential for cancer cell survival, yet the underlying mechanisms remain elusive. Methods: First, SNPRE representing the core spliceosomal Sm gene was screened using a multiomics database analysis approach. Then, we investigated the effect of SNRPE on the growth of TNBC cells in vitro and in vivo. We identified DDX20 from the interacting partners of SNPRE as a potential molecule involved in spliceosome vulnerability using coimmunoprecipitationtechniques. The mechanism of the SNRPE-DDX20 axis in tumor spliceosome therapeutic vulnerability was determined by various techniques,including RNA-seq, chromatin immunoprecipitation and other technologies. Results: In our research, SNRPE was upregulated in TNBC and predicted poor prognosis. Downregulation of SNRPE attenuated the vitality of TNBC in vivo and in vitro. We hypothesized and confirmed DDX20 as an important factor in core spliceosome therapeutic vulnerability. In DDX20-knockout TNBC cells, cell survival is no longer dependent onintracellular splicing activity orSNPRE expression. In DDX20/SNRPE double-downregulated cells, growth ability was restored due to MTOR reactivation via a splicing-independent pathway. In SNRPE-knockdown TNBC cells, ETV3 and its corepressor DDX20 function together to block MTOR expression to induce growth suppression. Once SNRPE expression was high, ETV3 bound to the C-terminus of DDX20 was replaced by SNRPE-containing components, whilethe binding affinity of released ETV3 to MTOR was reduced, and eventually, MTOR was expressed sufficiently to promote proliferation. Conclusions: Our results reveal that DDX20 is an adaptor in the crosstalk between RNA splicing (SnRNP components such as SNRPE) and the MTOR pathway in MYC-driven TNBC. Patients with high DDX20 expression are likely suitable for treatment with spliceosome modulators, leading to double killing effects by cell arrest and antitumorimmunity. Targeting DDX20 may serve as a therapeutic target for MYC-driven cancers.