Single-cell analyses reveal evolution mimicry during the specification of breast cancer subtype.

Background: The stem or progenitor antecedents confer developmental plasticity and unique cell identities to cancer cells via genetic and epigenetic programs. A comprehensive characterization and mapping of the cell-of-origin of breast cancer using novel technologies to unveil novel subtype-specific therapeutic targets is still absent. Methods: We integrated 195,144 high-quality cells from normal breast tissues and 406,501 high-quality cells from primary breast cancer samples to create a large-scale single-cell atlas of human normal and cancerous breasts. Potential heterogeneous origin of malignant cells was explored by contrasting cancer cells against reference normal epithelial cells. Multi-omics analyses and both in vitro and in vivo experiments were performed to screen and validate potential subtype-specific treatment targets. Novel biomarkers of identified immune and stromal cell subpopulations were validated by immunohistochemistry in our cohort. Results: Tumor stratification based on cancer cell-of-origin patterns correlated with clinical outcomes, genomic aberrations and diverse microenvironment constitutions. We found that the luminal progenitor (LP) subtype was robustly associated with poor prognosis, genomic instability and dysfunctional immune microenvironment. However, the LP subtype patients were sensitive to neoadjuvant chemotherapy (NAC), PARP inhibitors (PARPi) and immunotherapy. The LP subtype-specific target PLK1 was investigated by both in vitro and in vivo experiments. Besides, large-scale single-cell profiling of breast cancer inspired us to identify a range of clinically relevant immune and stromal cell subpopulations, including subsets of innate lymphoid cells (ILCs), macrophages and endothelial cells. Conclusion: The present single-cell study revealed the cellular repertoire and cell-of-origin patterns of breast cancer. Combining single-cell and bulk transcriptome data, we elucidated the evolution mimicry from normal to malignant subtypes and expounded the LP subtype with vital clinical implications. Novel immune and stromal cell subpopulations of breast cancer identified in our study could be potential therapeutic targets. Taken together, Our findings lay the foundation for the precise prognostic and therapeutic stratification of breast cancer.

A human-specific insertion promotes cell proliferation and migration by enhancing TBC1D8B expression.

Human-specific insertions play important roles in human phenotypes and diseases. Here we reported a 446-bp insertion (Insert-446) in intron 11 of the TBC1D8B gene, located on chromosome X, and traced its origin to a portion of intron 6 of the EBF1 gene on chromosome 5. Interestingly, Insert-446 was present in the human Neanderthal and Denisovans genomes, and was fixed in humans after human-chimpanzee divergence. We have demonstrated that Insert-446 acts as an enhancer through binding transcript factors that promotes a higher expression of human TBC1D8B gene as compared with orthologs in macaques. In addition, over-expression TBC1D8B promoted cell proliferation and migration through "a dual finger" catalytic mechanism (Arg538 and Gln573) in the TBC domain in vitro and knockdown of TBC1D8B attenuated tumorigenesis in vivo. Knockout of Insert-446 prevented cell proliferation and migration in cancer and normal cells. Our results reveal that the human-specific Insert-446 promotes cell proliferation and migration by upregulating the expression of TBC1D8B gene. These findings provide a significant insight into the effects of human-specific insertions on evolution.

Genomic alterations affecting tumor-infiltrating lymphocytes and PD-L1 expression patterns in triple-negative breast cancer.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) and programmed cell death 1 ligand 1 (PD-L1) remain imperfect in predicting clinical outcomes of triple-negative breast cancer because outcomes do not always correlate with the expression of these biomarkers. Genomic and transcriptomic alterations that may contribute to the expression of these biomarkers remain incompletely uncovered. METHODS: We evaluated PD-L1 immunohistochemistry scores (SP142 and 28-8 assays) and TILs in our triple-negative breast cancer multiomics dataset and 2 immunotherapy clinical trial cohorts. Then, we analyzed genomic and transcriptomic alterations correlated with TILs, PD-L1 expression, and patient outcomes. RESULTS: Despite TILs serving as a decent predictor for triple-negative breast cancer clinical outcomes, exceptions remained. Our study revealed that several genomic alterations were correlated with unexpected events. In particular, PD-L1 expression may cause a paradoxical relationship between TILs and prognosis in certain patients. Consequently, we classified triple-negative breast cancers into 4 groups based on PD-L1 and TIL levels. The TIL-negative PD-L1-positive and TIL-positive PD-L1-negative groups were not typical "hot" tumors; both were associated with worse prognoses and lower immunotherapy efficacy than TIL-positive PD-L1-positive tumors. Copy number variation of PD-L1 and oncogenic signaling activation were correlated with PD-L1 expression in the TIL-negative PD-L1-positive group, whereas GSK3B-induced degradation may cause undetectable PD-L1 expression in the TIL-positive PD-L1-negative group. These factors have the potential to affect the predictive function of both PD-L1 and TILs. CONCLUSIONS: Several genomic and transcriptomic alterations may cause paradoxical effects among TILs, PD-L1 expression, and prognosis in triple-negative breast cancer. Investigating and targeting these factors will advance precision immunotherapy for patients with this disease.

Breast cancer animal models and applications.

Breast cancer is the most common malignancy in women. Basic and translational breast cancer research relies heavily on experimental animal models. Ideally, such models for breast cancer should have commonality with human breast cancer in terms of tumor etiology, biological behavior, pathology, and response to therapeutics. This review introduces current progress in different breast cancer experimental animal models and analyzes their characteristics, advantages, disadvantages, and potential applications. Finally, we propose future research directions for breast cancer animal models.

A new Schiff base copper(II) complex induces cancer cell growth inhibition and apoptosis by multiple mechanisms.

A new Schiff base copper(II) complex [N,N'-bis(2'-hydroxyphenylacetone)-o-ethanediamine] copper (II) (M1) has been synthesized and characterized by single X-ray crystallography. The cytotoxicity of complex M1 was evaluated against HeLa, LoVo, A549, A549/cis cancer cell lines, and the normal cell lines LO2 and HUVEC, by MTT (3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazoliumbromide) assays. The IC50 (50% inhibition concentrations) is in the range of 5.13-11.68 µM, which is somewhat lower than cisplatin on the basis of platinum molar concentration. Furthermore, anticancer mechanistic studies showed that the complex M1 inhibited cell proliferation by blocking DNA synthesis and then acted on nuclear division of HeLa cells over time. Moreover, M1 increased intracellular ROS (Reactive oxygen species) levels in a dose-dependent manner. Western blot analysis indicated M1 dramatically decrease c-Myc transcription factor and KLF5 (Krüppel-like factor 5) protein expression levels in HeLa. M1 did not inhibit proteasomal activity. Finally, M1 induced DNA damages and activated the DNA damage repair pathways.

UCH-L1-mediated Down-regulation of Estrogen Receptor α Contributes to Insensitivity to Endocrine Therapy for Breast Cancer.

Purpose: To determine the role of UCH-L1 in regulating ERα expression, and to evaluate whether therapeutic targeting of UCH-L1 can enhance the efficacy of anti-estrogen therapy against breast cancer with loss or reduction of ERα. Methods: Expressions of UCH-L1 and ERα were examined in breast cancer cells and patient specimens. The associations between UCH-L1 and ERα, therapeutic response and prognosis in breast cancer patients were analyzed using multiple databases. The molecular pathways by which UCH-L1 regulates ERα were analyzed using immunoblotting, qRT-PCR, immunoprecipitation, ubiquitination, luciferase and ChIP assays. The effects of UCH-L1 inhibition on the efficacy of tamoxifen in ERα (-) breast cancer cells were tested both in vivo and in vitro. Results: UCH-L1 expression was conversely correlated with ERα status in breast cancer, and the negative regulatory effect of UCH-L1 on ERα was mediated by the deubiquitinase-mediated stability of EGFR, which suppresses ERα transcription. High expression of UCH-L1 was associated with poor therapeutic response and prognosis in patients with breast cancer. Up-regulation of ERα caused by UCH-L1 inhibition could significantly enhance the efficacy of tamoxifen and fulvestrant in ERα (-) breast cancer both in vivo and in vitro. Conclusions: Our results reveal an important role of UCH-L1 in modulating ERα status and demonstrate the involvement of UCH-L1-EGFR signaling pathway, suggesting that UCH-L1 may serve as a novel adjuvant target for treatment of hormone therapy-insensitive breast cancers. Targeting UCH-L1 to sensitize ER negative breast cancer to anti-estrogen therapy might represent a new therapeutic strategy that warrants further exploration.

The regulatory mechanisms and functional roles of the Hippo signaling pathway in breast cancer.

Hippo signaling pathway has attracted broad attention due to its essential roles in controlling organ size and tumorigenesis. TAZ/YAP, two core downstream molecules of the Hippo signaling pathway in mammals, are tightly regulated by a wide range of extracellular and intrinsic signals in both Hippo signaling pathway-dependent and -independent manners. Besides their roles in the development and function of normal mammary glands, TAZ/YAP display remarkable potency and relevance to multiple aspects of human breast carcinogenesis, including cellular proliferation, differentiation, apoptosis, migration, invasion, epithelial-mesenchymal transition and stemness. In this review, we summarize the regulators of TAZ/YAP, discuss the significant contribution of the Hippo signaling pathway to human breast cancers and highlight their potential therapeutic roles.

Tree shrew (Tupaia belangeri) as a novel laboratory disease animal model.

The tree shrew (Tupaia belangeri) is a promising laboratory animal that possesses a closer genetic relationship to primates than to rodents. In addition, advantages such as small size, easy breeding, and rapid reproduction make the tree shrew an ideal subject for the study of human disease. Numerous tree shrew disease models have been generated in biological and medical studies in recent years. Here we summarize current tree shrew disease models, including models of infectious diseases, cancers, depressive disorders, drug addiction, myopia, metabolic diseases, and immune-related diseases. With the success of tree shrew transgenic technology, this species will be increasingly used in biological and medical studies in the future.

The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models.

Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio-medicine.

Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

[Tree shrews under the spot light: emerging model of human diseases].

Animal models are indispensible in biomedical research and have made tremendous contributions to answer fundamental questions on human biology, disease mechanisms, and to the development of new drugs and diagnostic tools. Due to the limitations of rodent models in translational medicine, tree shrews (Tupaia belangeri chinensis), the closest relative of primates, have attracted increasing attention in modeling human diseases and therapeutic responses. Here we discuss the recent progress in tree shrew biology and the development of tree shrews as human disease models including infectious diseases, metabolic diseases, neurological and psychiatric diseases, and cancers. Meanwhile, the current problems and future perspectives of the tree shrew model are explored.

Genome of the Chinese tree shrew.

Chinese tree shrews (Tupaia belangeri chinensis) possess many features valuable in animals used as experimental models in biomedical research. Currently, there are numerous attempts to employ tree shrews as models for a variety of human disorders: depression, myopia, hepatitis B and C virus infections, and hepatocellular carcinoma, to name a few. Here we present a publicly available annotated genome sequence for the Chinese tree shrew. Phylogenomic analysis of the tree shrew and other mammalians highly support its close affinity to primates. By characterizing key factors and signalling pathways in nervous and immune systems, we demonstrate that tree shrews possess both shared common and unique features, and provide a genetic basis for the use of this animal as a potential model for biomedical research.

Characterization of spontaneous breast tumor in tree shrews (Tupaia belangeri chinenesis).

Breast cancer is a common malignant tumor. It is essential to develop suitable animal models for discovering novel preventive and therapeutic approaches. Tree shrews (Tupaia belangeri chinensis) have a closer evolutionary relationship with humans than do rodents, which have been widely used in laboratory research. Spontaneous breast tumors were identified in tree shrews in 1960s; however, no detailed studies about tree shrew breast tumors have been conducted to date. Here, we characterized a spontaneous breast tumor from tree shrews by Haematoxylin Eosin (H&E) staining. This tumor was identified as a papillary tumor. Immunohistochemical staining (IHC) for progesterone receptor (PR), Ki-67 and cleaved caspase-3 showed that tumor cells were positive for PR, highly proliferative, and less apoptotic compared to normal breast epithelial cells. Thus, the spontaneous tumor of tree shrew is very close to human papillary tumors in terms of morphology and pathology and we concluded that tree shrew may be a suitable animal model for breast cancer research.