scRNA-seq characterizing the heterogeneity of fibroblasts in breast cancer reveals a novel subtype SFRP4+ CAF that inhibits migration and predicts prognosis.

Introduction: Cancer-associated fibroblasts (CAFs) are a diverse group of cells that significantly impact the tumor microenvironment and therapeutic responses in breast cancer (BC). Despite their importance, the comprehensive profile of CAFs in BC remains to be fully elucidated. Methods: To address this gap, we utilized single-cell RNA sequencing (scRNA-seq) to delineate the CAF landscape within 14 BC normal-tumor paired samples. We further corroborated our findings by analyzing several public datasets, thereby validating the newly identified CAF subtype. Additionally, we conducted coculture experiments with BC cells to assess the functional implications of this CAF subtype. Results: Our scRNA-seq analysis unveiled eight distinct CAF subtypes across five tumor and six adjacent normal tissue samples. Notably, we discovered a novel subtype, designated as SFRP4+ CAFs, which was predominantly observed in normal tissues. The presence of SFRP4+ CAFs was substantiated by two independent scRNA-seq datasets and a spatial transcriptomics dataset. Functionally, SFRP4+ CAFs were found to impede BC cell migration and the epithelial-mesenchymal transition (EMT) process by secreting SFRP4, thereby modulating the WNT signaling pathway. Furthermore, we established that elevated expression levels of SFRP4+ CAF markers correlate with improved survival outcomes in BC patients, yet paradoxically, they predict a diminished response to neoadjuvant chemotherapy in cases of triple-negative breast cancer. Conclusion: This investigation sheds light on the heterogeneity of CAFs in BC and introduces a novel SFRP4+ CAF subtype that hinders BC cell migration. This discovery holds promise as a potential biomarker for refined prognostic assessment and therapeutic intervention in BC.

SIRT3 Expression Predicts Overall Survival and Neoadjuvant Chemosensitivity in Triple-Negative Breast Cancer.

Background: The Sirtuin (SIRT) family consists of seven evolutionary conserved NAD-dependent deacetylases that play important roles in various cancers, including breast cancer (BC). SIRTs expression has been reported to have prognostic value in BC, but these studies used limited sample size and yielded inconsistent conclusions. This study evaluated the association of SIRT3 and other SIRT family members with survival and neoadjuvant chemotherapy outcomes. Methods: BC patients' data was obtained from the TCGA-BRCA, METABRIC and GEO databases, comprising 4336 samples. SIRTs expression and overall survival (OS) were analyzed using Kaplan-Meier analysis and Cox proportional hazards regression. SIRT3 expression levels were compared between pathologic complete response (pCR) and non-pCR groups after neoadjuvant chemotherapy in triple-negative breast cancer (TNBC). Protein-protein interaction networks were constructed using the STRING database. Gene set enrichment analysis (GSEA) was performed to explore potential functions of SIRT3. Results: Through systematic analysis of SIRTs expression and OS of BC using three independent cohorts: TCGA-BRCA, METABRIC and GSE16446, we found that high SIRT3 expression was significantly associated with worse OS in TNBC in the TCGA-BRCA cohort, which was validated in the METABRIC and GSE16446 cohorts. SIRT3 expression was correlated with BC subtypes and American Joint Committee on Cancer (AJCC) T stage, but not with age-at-diagnosis, race, or tumor stage. Moreover, TNBC patients with higher SIRT3 expression had lower pCR rates after neoadjuvant chemotherapy (p = 6.40e-03) and SIRT3 expression was significantly lower in the pCR group than in the non-pCR group in TNBC (p = 4.2e-03). GSEA indicated that SIRT3 was involved in drug-related pathways such as oxidative phosphorylation, metabolism of xenobiotics by cytochrome P450, and drug metabolism. Conclusion: Our study suggests that SIRT3 is a potential biomarker for both OS and neoadjuvant chemosensitivity in TNBC. It may also assist in selecting suitable candidates and treatment options for TNBC patients.

Upregulated PARP1 confers breast cancer resistance to CDK4/6 inhibitors via YB-1 phosphorylation.

BACKGROUND: Cyclic-dependent kinase (CDK) 4/6 kinases, as the critical drivers of the cell cycle, are involved in the tumor progression of various malignancies. Pharmacologic inhibitors of CDK4/6 have shown significant clinical prospects in treating hormone receptor-positive and human epidermal growth factor receptor-negative (HR + /HER2-) breast cancer (BC) patients. However, acquired resistance to CDK4/6 inhibitors (CDK4/6i), as a common issue, has developed rapidly. It is of great significance that the identification of novel therapeutic targets facilitates overcoming the CDK4/6i resistance. PARP1, an amplified gene for CDK4/6i-resistant patients, was found to be significantly upregulated during the construction of CDK4/6i-resistant strains. Whether PARP1 drives CDK4/6i resistance in breast cancer is worth further study. METHOD: PARP1 and p-YB-1 protein levels in breast cancer cells and tissues were quantified using Western blot (WB) analysis, immunohistochemical staining (IHC) and immunofluorescence (IF) assays. Bioinformatics analyses of Gene Expression Profiling Interactive Analysis (GEPIA), Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets were applied to explore the relationship between YB-1/PARP1 protein levels and CDK4/6i IC50. Cell Counting Kit-8 (CCK-8) and crystal violet staining assays were performed to evaluate cell proliferation rates and drug killing effects. Flow cytometry assays were conducted to assess apoptosis rates and the G1/S ratio in the cell cycle. An EdU proliferation assay was used to detect the DNA replication ratio after treatment with PARP1 and YB-1 inhibitors. A ChIP assay was performed to assess the interaction of the transcription factor YB-1 and associated DNA regions. A double fluorescein reporter gene assay was designed to assess the influence of WT/S102A/S102E YB-1 on the promoter region of PARP1. Subcutaneous implantation models were applied for in vivo tumor growth evaluations. RESULTS: Here, we reported that PARP1 was amplified in breast cancer cells and CDK4/6i-resistant patients, and knockdown or inhibition of PARP1 reversed drug resistance in cell experiments and animal models. In addition, upregulation of transcription factor YB-1 also occurred in CDK4/6i-resistant breast cancer, and YB-1 inhibition can regulate PARP1 expression. p-YB-1 and PARP1 were upregulated when treated with CDK4/6i based on the WB and IF results, and elevated PARP1 and p-YB-1 were almost simultaneously observed during the construction of MCF7AR-resistant strains. Inhibition of YB-1 or PAPR1 can cause decreased DNA replication, G1/S cycle arrest, and increased apoptosis. We initially confirmed that YB-1 can bind to the promoter region of PARP1 through a ChIP assay. Furthermore, we found that YB-1 phosphorylated at S102 was crucial for PARP1 transcription according to the double fluorescein reporter gene assay. The combination therapy of YB-1 inhibitors and CDK4/6i exerted a synergistic antitumor effect in vitro and in vivo. The clinical data suggested that HR + /HER2- patients with low expression of p-YB-1/PARP1 may be sensitive to CDK4/6i in breast cancer. CONCLUSION: These findings indicated that a ''YB-1/PARP1'' loop conferred resistance to CDK4/6 inhibitors. Furthermore, interrupting the loop can enhance tumor killing in the xenograft tumor model, which provides a promising strategy against drug resistance in breast cancer.

Identification of GZMA as a Potential Therapeutic Target Involved in Immune Infiltration in Breast Cancer by Integrated Bioinformatical Analysis.

Purpose: Granzyme A (GZMA) is a potential prognostic target for various cancer types. However, its therapeutic significance in breast cancer with immune infiltration remains controversial. We analyzed GZMA expression and its prognostic value in breast cancer with immune cell infiltration. Patients and methods: Data was obtained from patients with breast cancer registered at The Cancer Genome Atlas. A correlation was performed between GZMA expression and patient's clinicopathological features such as age, pathologic stage, metastasis stage, overall survival (OS), disease-specific survival (DSS), and progress free interval (PFI). Kaplan-Meier analyses and Cox proportional hazard regression model were used to examine the predictive significance of GZMA expression for breast cancer. The co-expression pattern of GZMA was assessed by the LinkedOmics web portal. The relationship between GZMA expression and immune cells was analyzed using the TIMER database. The correlation between GZMA and lymphocytes and immunomodulators was established with the TISIDB database. Results: There was a lower GZMA expression in breast cancer tissue than in normal tissue. Interestingly, GZMA expression was associated with age, pathologic stage, and the Tumour, Node, and Metastasis stage. Overexpression of GZMA was also associated with better OS, DSS, and PFI. Based on the Cox regression analysis, GZMA was identified as an independent favorable prognostic factor for breast cancer. Our findings demonstrated a strong association between GZMA and T-cell checkpoints (PD-1, PD-L1, and cytotoxic T lymphocyte-associated antigen (CTLA-4)) in breast cancer. Moreover, we evaluated the interactions between GZMA expression and markers of dendritic and CD8+ T cells using quantitative immunofluorescence. We discovered that increased infiltration of dendritic and CD8+ T cells was associated with GZMA expression in breast cancer. Conclusion: GZMA expression is associated with a favorable prognosis in breast cancer and is significantly correlated with immune cell infiltration. GZMA may be considered a promising therapeutic target for patients with breast cancer.

Predictive and Prognostic Value of TRIM58 Protein Expression in Patients with Breast Cancer Receiving Neoadjuvant Chemotherapy.

Introduction: Tripartite motif-containing protein (TRIM) family members play crucial roles in carcinogenesis and chemotherapy resistance. In this study, we aimed to determine whether TRIM58 protein expression is related to patient responses to neoadjuvant therapy (NAT) and their survival outcome. Methods: Immunohistochemistry was performed on female breast cancer samples from biopsies before NAT in Shenzhen Second People's Hospital. Univariate and multivariate logistic regression tests were used to analyze the association between TRIM58 protein expression and pathological complete response (pCR). The Cox proportional hazards model was used to calculate the adjusted hazard ratio (HR) with a 95% confidence interval (95% CI). The Kaplan-Meier plotter database was used to analyze the prognostic value of TRIM58. Results: High TRIM58 expression was associated with small tumor size in all the patients (n = 58). Multivariate analysis suggested that low TRIM58 expression was an independent predictive factor for higher pCR (odds ratio = 0.06, 95% CI 0.005-0.741, P = 0.028). The Kaplan-Meier Plotter dataset suggested that the TRIM58 high-expression group showed a worse 5-year overall survival than the low-expression group (HR = 1.34, 95% CI 1.07-1.67, P = 0.01). Pathway analysis revealed the potential mechanisms of TRIM58 in chemoresistance. Discussion: Our study suggests that TRIM58 is a promising biomarker for both neoadjuvant chemosensitivity and long-term clinical outcomes in breast cancer. It may also help to identify candidate responders and determine treatment strategies.

Clonal relationship and alcohol consumption-associated mutational signature in synchronous hypopharyngeal tumours and oesophageal squamous cell carcinoma.

BACKGROUND: The patients with dual oesophageal squamous cell carcinoma (ESCC) and hypopharyngeal cancer (HPC) have poor prognosis; their underlying genetic pathogenesis is unclear. We hypothesise that development of synchronous ESCC/HPC depends on multicentricity or independent origin, rather than multifocality due to local or lateral spreading. METHOD: Multiple region whole-exome sequencing (M-WES) and clonality analysis were used to assess clonal relationship and spatial inter- or intra-tumour heterogeneity (ITH) in 62 tumour regions from eight dual ESCC/HPC and ten ESCC patients. RESULTS: All synchronous ESCC/HPC patients had COSMIC 16 mutation signatures, compared to only 40% ESCC in the current study (p = 0.013) and public data set (n = 165, p = 0.003). This alcohol consumption-related mutation signature 16, commonly involved in multiple alcohol-related cancers, was significantly associated with drinking and alcohol metabolism-related ADH1B rs1229984. The mutational landscape and copy number profiles were completely distinct between the two primary tumours; clonality analysis further suggested the two primary tumours shared no or only one clone accompanying independent subclone evolution. M-WES strategy demonstrated higher sensitivity and accuracy for detection of mutational prevalence and the late branch mutations among different regions in the ESCC tumours, compared to traditional sequencing analysis based on single biopsy strategy. Patients with high ITH assessed by cancer cell fraction analysis after M-WES were significantly associated with both relapse and survival. CONCLUSIONS: Our hypothesis-generating M-WES ITH assessment data have implications for prognostication. Collectively, our findings support multicentric independent clonal evolution, the field cancerisation theory, and suggest novel insights implicating an aetiologic role of alcohol metabolism in dual ESCC/HPC carcinogenesis.

Comprehensive Analysis of the Expression and Prognosis for Tripartite Motif-Containing Genes in Breast Cancer.

Tripartite motif-containing genes (TRIMs), with a ubiquitin ligase's function, play critical roles in antitumor immunity by activating tumor-specific immune responses and stimulating tumor proliferation, thus affecting patient outcomes. However, the expression pattern and prognostic values of TRIMs in breast cancer (BC) are not well clarified. In this study, several datasets and software were integrated to perform a comprehensive analysis of the expression pattern in TRIMs and investigate their prognosis values in BC. We found that TRIM59/46 were significantly upregulated and TRIM66/52-AS1/68/7/2/9/29 were decreased in BC and validated them using an independent cohort. The expression of numerous TRIMs are significantly correlated with BC molecular subtypes, but not with tumor stages or patient age at diagnosis. Higher expression of TRIM3/14/69/45 and lower expressions of TRIM68/2 were associated with better overall survival in BC using the Kaplan-Meier analysis. The multivariate Cox proportional hazards model identified TRIM45 as an independent prognostic marker. Further analysis of single-cell RNA-seq data revealed that most TRIMs are also expressed in nontumor cells. Higher expression of some TRIMs in the immune or stromal cells suggests an important role of TRIMs in the BC microenvironment. Functional enrichment of the co-expression genes indicates that they may be involved in muscle contraction and interferon-gamma signaling pathways. In brief, through the analysis, we provided several TRIMs that may contribute to the tumor progression and TRIM45 as a potential new prognostic biomarker for BC.

RAD50 Loss of Function Variants in the Zinc Hook Domain Associated with Higher Risk of Familial Esophageal Squamous Cell Carcinoma.

Unbiased whole-exome sequencing approaches in familial esophageal squamous cell carcinoma (ESCC) initially prioritized RAD50 as a candidate cancer predisposition gene. The combined study with 3289 Henan individuals from Northern China identified two pathogenic RAD50 protein truncation variants, p.Q672X and a recurrent p.K722fs variant at the zinc hook domain significantly conferring increased familial ESCC risk. Effects of ~10-fold higher familial ESCC risk were observed, when compared to East Asians from the gnomAD database. Functional characterization suggested that the RAD50Q672X mutation contributes a dominant-negative effect in DNA repair of double-stranded breaks. Overexpression of the RAD50Q672X and RAD50L1264F missense mutation also sensitized cell death upon replication stress stimuli induced by formaldehyde treatment and the CHK1 inhibitor, AZD7762. Our study suggested the novel insight of the potential for synthetic lethal therapeutic options for RAD50Q672X and the East-Asian-specific RAD50L1264F variants and CHK1 inhibitors. Our study also suggested the association of RAD50 LOF variants in the zinc hook domain with a higher risk of familial ESCC in Chinese.

Differentiation-related zinc finger protein 750 suppresses cell growth in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a deadly squamous cell carcinoma (SCC) of the esophagus. Development of SCCs is associated with the deregulation of the squamous cell lineage program and/or keratinocyte terminal differentiation by genomic and genetic aberrations; thus, these processes must be tightly controlled to maintain normal squamous cell development. Zinc finger protein 750 (ZNF750) is a gene involved in keratinocyte terminal differentiation and is frequently mutated and putatively silenced in ESCC, which implicates its function as a potential differentiation-related suppressor of ESCC. The present study aimed to elucidate the relationship between ZNF750 function to induce keratinocyte differentiation and tumor suppression in ESCC. The results demonstrated that chemical manipulation of esophageal keratinocyte differentiation in mouse normal esophageal epithelial organoids (mNEEO) implicated the involvement of the mouse homologue of ZNF750, Zfp750, in keratinocyte differentiation in premalignant cells. Bioinformatics analyses of data from high ZNF750-expressing ESCC tumors obtained from public databases and ZNF750-overexpressing ESCC cells compared with low ZNF750-expressing ESCC tumors and GFP-expressing ESCC cells, respectively, revealed enrichment of keratinocyte differentiation-related gene sets in these samples. Finally, the induction through to terminal differentiation of the keratinocyte by all-trans retinoic acid on parental ESCC cell lines led to the upregulation of the terminal differentiation marker Involucrin and a decrease in cell viability similar to that observed in ZNF750-overexpressing ESCC cells. The results of the present study demonstrated a functional link between the ability of ZNF750 to induce cell differentiation through to terminal differentiation and its function as a growth suppressor in ESCC. This study provides improved understanding of the role of ZNF750, a frequently mutated differentiation-related gene in ESCC, and its effects in ESCC pathogenesis.

Nasopharyngeal carcinoma MHC region deep sequencing identifies HLA and novel non-HLA TRIM31 and TRIM39 loci.

Despite pronounced associations of major histocompatibility complex (MHC) regions with nasopharyngeal carcinoma (NPC), causal variants underlying NPC pathogenesis remain elusive. Our large-scale comprehensive MHC region deep sequencing study of 5689 Hong Kong Chinese identifies eight independent NPC-associated signals and provides mechanistic insight for disrupted transcription factor binding, altering target gene transcription. Two novel protective variants, rs2517664 (Trs2517664 = 4.6%, P = 6.38 × 10-21) and rs117495548 (Grs117495548 = 3.0%, P = 4.53 × 10-13), map near TRIM31 and TRIM39/TRIM39-RPP21; multiple independent protective signals map near HLA-B including a previously unreported variant, rs2523589 (P = 1.77 × 10-36). The rare HLA-B*07:05 allele (OR < 0.015, P = 5.83 × 10-21) is absent in NPC, but present in controls. The most prevalent haplotype lacks seven independent protective alleles (OR = 1.56) and the one with additional Asian-specific susceptibility rs9391681 allele (OR = 2.66) significantly increased NPC risk. Importantly, this study provides new evidence implicating two non-human leukocyte antigen (HLA) genes, E3 ubiquitin ligases, TRIM31 and TRIM39, impacting innate immune responses, with NPC risk reduction, independent of classical HLA class I/II alleles.

FANCD2 Confers a Malignant Phenotype in Esophageal Squamous Cell Carcinoma by Regulating Cell Cycle Progression.

Fanconi anemia patients with germline genetic defects in FANCD2 are highly susceptible to cancers. Esophageal squamous cell carcinoma (ESCC) is a deadly cancer. Little is known about the function of FANCD2 in ESCC. For detailed molecular and mechanistic insights on the functional role of FANCD2 in ESCC, in vivo and in vitro assays and RNA sequencing approaches were used. Utilizing Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology, FANCD2 knockout models were established to examine the functional impact in mouse models for tumor growth and metastasis and in vitro assays for cell growth, cell cycle, and cellular localization. Our RNA sequence analyses were integrated with public datasets. FANCD2 confers a malignant phenotype in ESCC. FANCD2 is significantly upregulated in ESCC tumors, as compared to normal counterparts. Depletion of FANCD2 protein expression significantly suppresses the cancer cell proliferation and tumor colony formation and metastasis potential, as well as cell cycle progression, by involving cyclin-CDK and ATR/ATM signaling. FANCD2 translocates from the nucleus to the cytoplasm during cell cycle progression. We provide evidence of a novel role of FANCD2 in ESCC tumor progression and its potential usefulness as a biomarker for ESCC disease management.

BRCA2 loss-of-function germline mutations are associated with esophageal squamous cell carcinoma risk in Chinese.

Esophageal squamous cell carcinoma (ESCC) occurs with highest frequency in China with over 90% mortality, highlighting the need for early detection and improved treatment strategies. We aimed to identify ESCC cancer predisposition gene(s). Our study included 4,517 individuals. The discovery phase using whole-exome sequencing (WES) included 186 familial ESCC patients from high-risk China. Targeted gene sequencing validation of 598 genes included 3,289 Henan and 1,228 moderate-risk Hong Kong Chinese. A WES approach identified BRCA2 loss-of-function (LOF) mutations in 3.23% (6/186) familial ESCC patients compared to 0.21% (9/4300) in the ExAC East Asians (odds ratio [OR] = 15.89, p = 2.48 × 10-10 ). BRCA2 LOF mutation frequency in the combined Henan cohort has significantly higher prevalence (OR = 10.55, p = 0.0035). Results were independently validated in an ESCC Hong Kong cohort (OR = 10.64, p = 0.022). One Hong Kong pedigree was identified to carry a BRCA2 LOF mutation. BRCA2 inactivation in ESCC was via germline LOF mutations and wild-type somatic allelic loss via loss of heterozygosity. Gene-based association analysis, including LOF mutations and rare deleterious missense variants defined with combined annotation dependent depletion score ≥30, confirmed the genetic predisposition role of BRCA2 (OR = 9.50, p = 3.44 × 10-5 ), and provided new evidence for potential association of ESCC risk with DNA repair genes (POLQ and MSH2), inflammation (TTC39B) and angiogenesis (KDR). Our findings are the first to provide compelling evidence of the role of BRCA2 in ESCC genetic susceptibility in Chinese, suggesting defective homologous recombination is an underlying cause in ESCC pathogenesis, which is amenable to therapeutic options based on synthetic lethality approaches such as targeting BRCA2 with PARP1 inhibitors in ESCC.

Endoplasmic reticulum-localized ECM1b suppresses tumor growth and regulates MYC and MTORC1 through modulating MTORC2 activation in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a deadly disease with dismal 5-year survival. Extracellular matrix protein 1 (ECM1) was identified as one of the most downregulated genes by transcriptomic analysis of normal esophageal/ESCC paired tissue samples. ECM1 plays oncogenic roles in cancer development in various cancer types. However, little is known about its role in ESCC. In vivo and in vitro functional assays coupled with analyses on public datasets and detailed molecular and mechanistic analyses were used to study the gene. We demonstrate that as opposed to the previously identified oncogenic role of ECM1a, ECM1b is a novel tumor suppressor in ESCC. ECM1 is significantly downregulated in ESCC and several other squamous cell carcinomas. ECM1b encodes a cellular protein that suppresses MYC protein expression and MTORC1 signaling activity. MTORC2 inactivation leads to suppressed MYC expression and MTORC1 signaling. ECM1b localizes to the endoplasmic reticulum and suppresses MTORC2 activation by inhibiting MTORC2/ribosome association. By regulating MTORC2/MYC/MTORC1 signaling, ECM1b suppresses general protein translation and enhances chemosensitivity. We provide evidence establishing a novel role of ECM1 in cancer that suggests ECM1b as a biomarker for ESCC disease management.