LncRNA PCAT6 mediates UBFD1 expression via sponging miR-545-3p in breast cancer cells.

Background: LncRNA PCAT6 has been shown to involve in carcinogenesis of different tumors. In this study, we investigated underline mechanism by which PCAT6 promoted breast cancer cell progression. Methods: RIP was used to identify lncRNAs associated with IMP1. Bioinformatics assays were used to predict potential miRNAs that interact with PCAT6 and mRNAs that are targeted by miR-545-3p. RNA-seq and RT-qPCR were used to analyze differential expression of lncRNAs and miRNA-targeted genes. Luciferase reporter and RNA pull-down assays were performed to identify the molecular interactions between PCAT6 and individual miRNAs. The role of PCAT6-mediated cell proliferation and invasion were tested by CCK-8 and transwell assays following loss-of-function and gain-of-function effects. Results: We identified that PCAT6 is one of the lncRNAs that associated with IMP1. PCAT6 not only binds to IMP1, but also acts as a ceRNA to interact with multiple miRNAs, including miR-545-3p. Binding of IMP1 destabilized PCAT6, while competitive interaction with miR-545-3p allowed PCAT6 to positively regulate UBFD1 expression. Silencing UBFD1 mRNA could effectively rescue PCAT6-induced cell proliferation and invasive abilities. Conclusions: Our study provided evidence that PCAT6 activates UBFD1 expression via sponging miR-545-3p to increase carcinogenesis of breast cancer cells. Based on the nature of UBFD1 as a polyubiquitin binding protein, our study suggested that ubiquitin pathway might contribute to breast cancer progression.

Mesothelin-targeted CAR-NK Cells Derived From Induced Pluripotent Stem Cells Have a High Efficacy in Killing Triple-negative Breast Cancer Cells as Shown in Several Preclinical Models.

The emergence of immunotherapy has introduced a promising, novel approach to cancer treatment. While multiple chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable clinical efficacy against leukemia, their effect on solid tumors has been limited. One potential option for treating solid tumors is the engineering of natural killer (NK) cells with CARs. Mesothelin (MSLN), a tumor differentiation antigen, is expressed on triple-negative breast cancer (TNBC) cells, making it a potential target for CAR-NK therapy in the treatment of TNBC. We first constructed induced pluripotent stem cells with stable anti-MSLN-CAR expression and subsequently differentiated these cells into mesothelin-targeted CAR-NK (MSLN-NK) cells. We then assessed the effects of MSLN-NK cells on TNBC cells both in vitro (using the MDA-MB-231 cell line), in vivo (in a CDX mouse model), and ex vivo (using patient-specific primary cells and patient-specific organoids), in which MSLN surface expression was confirmed. Our CDX study results indicated that MSLN-NK cells effectively killed MDA-MB-231 (MD231) cells in vitro, reduced tumor growth in the CDX mouse model of TNBC, and lysed patient-specific primary cells and patient-specific organoids derived from the tumor samples of TNBC patients. Our data demonstrated that MSLN-NK cells had high efficacy on killing TNBC cells in in vitro, in vivo, and ex vivo. Therefore, MSLN-NK could be a promising treatment option for TNBC patients.

Safety, immunogenicity, and efficacy of the mRNA vaccine CS-2034 as a heterologous booster versus homologous booster with BBIBP-CorV in adults aged ≥18 years: a randomised, double-blind, phase 2b trial.

BACKGROUND: Heterologous boosting is suggested to be of use in populations who have received inactivated COVID-19 vaccines. We aimed to assess the safety and immunogenicity of a heterologous vaccination with the mRNA vaccine CS-2034 versus the inactivated BBIBP-CorV as a fourth dose, as well as the efficacy against the SARS-CoV-2 omicron (BA.5) variant. METHODS: This trial contains a randomised, double-blind, parallel-controlled study in healthy participants aged 18 years or older (group A) and an open-label cohort in participants 60 years and older (group B), who had received three doses of inactivated whole-virion vaccines at least 6 months before enrolment. Pregnant women and people with major chronic illnesses or a history of allergies were excluded. Eligible participants in group A were stratified by age (18-59 years and ≥60 years) and then randomised by SAS 9.4 in a ratio of 3:1 to receive a dose of the mRNA vaccine (CS-2034, CanSino, Shanghai, China) or inactivated vaccine (BBIBP-CorV, Sinopharm, Beijing, China). Safety and immunogenicity against omicron variants of the fourth dose were evaluated in group A. Participants 60 years and older were involved in group B for safety observations. The primary outcome was geometric mean titres (GMTs) of the neutralising antibodies against omicron and seroconversion rates against BA.5 variant 28 days after the boosting, and incidence of adverse reactions within 28 days. The intention-to-treat group was involved in the safety analysis, while all patients in group A who had blood samples taken before and after the booster were involved in the immunogenicity analysis. This trial was registered at the Chinese Clinical Trial Registry Centre (ChiCTR2200064575). FINDINGS: Between Oct 13, and Nov 22, 2022, 320 participants were enrolled in group A (240 in the CS-2034 group and 80 in the BBIBP-CorV group) and 113 in group B. Adverse reactions after vaccination were more frequent in CS-2034 recipients (158 [44·8%]) than BBIBP-CorV recipients (17 [21·3%], p<0·0001). However, most adverse reactions were mild or moderate, with grade 3 adverse reactions only reported by eight (2%) of 353 participants receiving CS-2034. Heterologous boosting with CS-2034 elicited 14·4-fold (GMT 229·3, 95% CI 202·7-259·4 vs 15·9, 13·1-19·4) higher concentration of neutralising antibodies to SARS-CoV-2 omicron variant BA.5 than did homologous boosting with BBIBP-CorV. The seroconversion rates of SARS-CoV-2-specific neutralising antibody responses were much higher in the mRNA heterologous booster regimen compared with BBIBP-CorV homologous booster regimen (original strain 47 [100%] of 47 vs three [18·8%] of 16; BA.1 45 [95·8%] of 48 vs two [12·5%] 16; and BA.5 233 [98·3%] of 240 vs 15 [18·8%] of 80 by day 28). INTERPRETATION: Both the administration of mRNA vaccine CS-2034 and inactivated vaccine BBIBP-CorV as a fourth dose were well tolerated. Heterologous boosting with mRNA vaccine CS-2034 induced higher immune responses and protection against symptomatic SARS-CoV-2 omicron infections compared with homologous boosting, which could support the emergency use authorisation of CS-2034 in adults. FUNDING: Science and Technology Commission of Shanghai, National Natural Science Foundation of China, Jiangsu Provincial Science Fund for Distinguished Young Scholars, and Jiangsu Provincial Key Project of Science and Technology Plan. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Two lncRNAs, MACC1-AS1 and UCA1, co-mediate the expression of multiple mRNAs through interaction with individual miRNAs in breast cancer cells.

Background: Increasing studies have shown that lncRNAs often play roles through interaction with miRNAs to control gene expression by inhibiting translation or facilitating degradation of target mRNAs. Here, we report that two lncRNAs, MACC1-AS1 and UCA1 are coordinately expressed in breast cancer cells and share the ability to interact with multiple miRNAs to mediate the expression of different genes. Methods: Targetscan, starBase and miRDB databases were used to predict the relationships of MACC1-AS1/UCA1-miRNA-mRNA network. qRT-PCR, and RNA sequencing were used to study the differential expression of lncRNAs and miRNA-targeted genes in breast cancer cells. RIP, RNA pull-down and luciferase assays were performed to confirm the molecular interactions of MACC1-AS1 or UCA1 with predicted miRNAs. The role of lncRNA-mediated miRNA-mRNA interactions in cell proliferation was examined by MTT assays following loss-of-function and gain-of-function effects. Results: We identified a lncRNA-miRNA-mRNA regulatory network in breast cancer cells, in which a number of mRNAs can be co-regulated by MACC1-AS1 and UCA1 lncRNAs. Each lncRNA possesses the capacity as a ceRNA to compete with various mRNA-targeting miRNAs. Interaction of MACC1-AS1 or UCA1 with individual miRNAs is able to increase the expression of the same target mRNAs, such as TBL1X and MEF2D, thus affecting cancer-cell growth phenotype. Conclusions: Our study suggests that in each cell type, there is a balance of interactions between certain lncRNAs and miRNAs. Disrupting the balance would eventually affect the expression of miRNA-targeted genes and cell proliferation.

Mutant p53 and Twist1 Co-Expression Predicts Poor Prognosis and Is an Independent Prognostic Factor in Breast Cancer.

PURPOSE: The basic helix-loop-helix transcription factor (bHLH) transcription factor Twist1 plays a key role in embryonic development and tumorigenesis. p53 is a frequently mutated tumor suppressor in cancer. Both proteins play a key and significant role in breast cancer tumorigenesis. However, the regulatory mechanism and clinical significance of their co-expression in this disease remain unclear. The purpose of this study was to analyze the expression patterns of p53 and Twist1 and determine their association with patient prognosis in breast cancer. We also investigated whether their co-expression could be a potential marker for predicting patient prognosis in this disease. METHODS: Twist1 and mutant p53 expression in 408 breast cancer patient samples were evaluated by immunohistochemistry. Kaplan-Meier Plotter was used to analyze the correlation between co-expression of Twist1 and wild-type or mutant p53 and prognosis for recurrence-free survival (RFS) and overall survival (OS). Univariate analysis, multivariate analysis, and nomograms were used to explore the independent prognostic factors in disease-free survival (DFS) and OS in this cohort. RESULTS: Of the 408 patients enrolled, 237 (58%) had high mutant p53 expression. Two-hundred twenty patients (53.9%) stained positive for Twist1, and 188 cases were Twist1-negative. Furthermore, patients that co-expressed Twist1 and mutant p53 (T+P+) had significantly advanced-stage breast cancer [stage III, 61/89 T+P+ (68.5%) vs. 28/89 T-P- (31.5%); stage II, 63/104 T+P+ (60.6%)vs. 41/104 T-P- (39.4%)]. Co-expression was negatively related to early clinical stage (i.e., stages 0 and I; P = 0.039). T+P+ breast cancer patients also had worse DFS (95% CI = 1.217-7.499, P = 0.017) and OS (95% CI = 1.009-9.272, P = 0.048). Elevated Twist1 and mutant p53 expression predicted shorter RFS in basal-like patients. Univariate and multivariate analysis identified three variables (i.e., lymph node involvement, larger tumor, and T+P+) as independent prognostic factors for DFS. Lymph node involvement and T+P+ were also independent factors for OS in this cohort. The total risk scores and nomograms were reliable for predicting DFS and OS in breast cancer patients. CONCLUSIONS: Our results revealed that co-expression of mutant p53 and Twist1 was associated with advanced clinical stage, triple negative breast cancer (TNBC) subtype, distant metastasis, and shorter DFS and OS in breast cancer patients. Furthermore, lymph nodes status and co-expression of Twist1 and mutant p53 were classified as independent factors for DFS and OS in this cohort. Co-evaluation of mutant p53 and Twist1 might be an appropriate tool for predicting breast cancer patient outcome.

Notch3 inhibits cell proliferation and tumorigenesis and predicts better prognosis in breast cancer through transactivating PTEN.

Notch receptors (Notch1-4) play critical roles in tumorigenesis and metastasis of malignant tumors, including breast cancer. Although abnormal Notch activation is related to various tumors, the importance of single receptors and their mechanism of activation in distinct breast cancer subtypes are still unclear. Previous studies by our group demonstrated that Notch3 may inhibit the emergence and progression of breast cancer. PTEN is a potent tumor suppressor, and its loss of function is sufficient to promote the occurrence and progression of tumors. Intriguingly, numerous studies have revealed that Notch1 is involved in the regulation of PTEN through its binding to CBF-1, a Notch transcription factor, and the PTEN promoter. In this study, we found that Notch3 and PTEN levels correlated with the luminal phenotype in breast cancer cell lines. Furthermore, we demonstrated that Notch3 transactivated PTEN by binding CSL-binding elements in the PTEN promoter and, at least in part, inhibiting the PTEN downstream AKT-mTOR pathway. Notably, Notch3 knockdown downregulated PTEN and promoted cell proliferation and tumorigenesis. In contrast, overexpression of the Notch3 intracellular domain upregulated PTEN and inhibited cell proliferation and tumorigenesis in vitro and in vivo. Moreover, inhibition or overexpression of PTEN partially reversed the promotion or inhibition of cell proliferation induced by Notch3 alterations. In general, Notch3 expression positively correlated with elevated expression of PTEN, ER, lower Ki-67 index, and incidence of involved node status and predicted better recurrence-free survival in breast cancer patients. Therefore, our findings demonstrate that Notch3 inhibits breast cancer proliferation and suppresses tumorigenesis by transactivating PTEN expression.

A negative binomial regression model for risk estimation of 0-2 axillary lymph node metastases in breast cancer patients.

Extensive clinical trials indicate that patients with negative sentinel lymph node biopsy do not need axillary lymph node dissection (ALND). However, the ACOSOG Z0011 trial indicates that patients with clinically negative axillary lymph nodes (ALNs) and 1-2 positive sentinel lymph nodes having breast conserving surgery with whole breast radiotherapy do not benefit from ALND. The aim of this study is therefore to identify those patients with 0-2 positive nodes who might avoid ALND. A total of 486 patients were eligible for the study with 212 patients in the modeling group and 274 patients in the validation group, respectively. Clinical lymph node status, histologic grade, estrogen receptor status, and human epidermal growth factor receptor 2 status were found to be significantly associated with ALN metastasis. A negative binomial regression (NBR) model was developed to predict the probability of having 0-2 ALN metastases with the area under the curve of 0.881 (95% confidence interval 0.829-0.921, P < 0.001) in the modeling group and 0.758 (95% confidence interval 0.702-0.807, P < 0.001) in the validation group. Decision curve analysis demonstrated that the model was clinically useful. The NBR model demonstrated adequate discriminative ability and clinical utility for predicting 0-2 ALN metastases.

Comparison of indocyanine green and methylene blue use for axillary reverse mapping during axillary lymph node dissection.

Axillary reverse mapping (ARM) is a technique to identify arm lymphatic drainage during axillary lymph node dissection (ALND). This study compared the feasibility of ARM using indocyanine green (ICG) or methylene blue (MB), and accessed the oncologic safety of the procedure. Overall, 158 patients qualified for ALND were enrolled. The characteristics of ARM-identified nodes were recorded with ICG (n = 78) or MB (n = 80) visualization. Fine-needle aspiration cytology (FNAC) of the nodes were performed and validated by histologic analysis. The nodal identification rate in the ICG group significantly surpassed that of the MB group (87.2% vs 52.5%, P < .05) with fewer complications. Note that 10.9% of the patients had metastatic involvement of the ARM-identified nodes. Also 80% of the positive nodes were found in areas B and D, while the ARM-identified nodes mainly located in area A. All the 51 nodes diagnosed as negative of malignancy by FNAC were free of metastasis. Nodal metastasis was significantly correlated with extensive nodel involvement, advanced disease, and the characteristics of identified nodes. In conclusion, ICG appears superior to MB for ARM nodes identification. FNAC, together with the features of primary tumors and ARM nodes, can delineate which nodes could be preserved during ALND.

L1 Cell Adhesion Molecule and Its Soluble Form sL1 Exhibit Poor Prognosis in Primary Breast Cancer Patients.

INTRODUCTION: The L1 cell adhesion molecule (L1-CAM) and its soluble form sL1 play a prominent role in invasion and metastasis in several cancers. However, its association with breast cancer is still unclear. PATIENTS AND METHODS: We analyzed L1-CAM expression and serum sL1 levels in cancer and para-carcinoma tissues from 162 consecutive patients with primary invasive breast cancer (PBC) using immunohistochemistry and an enzyme-linked immunosorbent assay, respectively. The serum sL1 levels were also examined in 38 patients with benign breast disease and 36 healthy controls. RESULTS: L1-CAM was expressed more frequently in cancer tissues than in para-carcinoma tissues (24.1% vs. 5.6%; P < .001), and the mean sL1 levels were significantly greater in PBC than in those with benign breast disease and healthy controls (P = .027). Both L1-CAM+ expression and higher mean sL1 levels correlated significantly with larger tumor size, lymph node involvement, higher histologic grade, advanced TNM stage, and shorter disease-free survival for PBC patients. Moreover, higher mean sL1 levels were also significantly associated with estrogen receptor-α-negative expression, human epidermal growth factor receptor 2-positive (HER2+) expression, HER2-enriched and triple-negative molecular subtypes, and L1-CAM+ expression (P < .05). On multivariate analysis, larger tumor size, nodal involvement, HER2+, and higher sL1 levels (≥ 0.7 ng/mL) were independent factors associated with L1-CAM+ expression (P < .05). No association was found between L1-CAM expression or sL1 level with age, gender, histologic type, or expression of progesterone receptor, Ki-67, p53, or vascular endothelial growth factor C (P > .05). CONCLUSION: These results indicate that L1-CAM and sL1 are elevated in PBC and both might affect the prognosis of PBC patients. In addition, sL1 might be a useful marker for screening and diagnosis.

Activated-PAK4 predicts worse prognosis in breast cancer and promotes tumorigenesis through activation of PI3K/AKT signaling.

The p21-activated kinase 4 (PAK4) is sufficient to transform noncancerous mammary epithelial cells and to form tumors in the mammary glands of mice. The accumulated information suggests that PAK4 might be an oncogenic protein in breast cancer. In this study, we sought to identify the role for PAK4 in breast cancer progression. Immunohistochemical study revealed that high PAK4 expression is associated with larger tumor size, lymph node metastasis, and advanced stage cancer in 93 invasive breast carcinoma patients. Moreover, high PAK4 expression was significantly associated with poor overall and disease-free survival. PAK4 remained an independent adverse prognosticator after univariate and multivariate analysis. Ectopic expression of wild-type PAK4 in MDA-MB-231 cells activated PI3K/AKT signaling and resulted in the enhancement of the cell proliferation, migration, and invasion, whereas PAK4-induced effects were blocked by the PAK4 kinase inhibitor PF- 3758309, PAK4 siRNAs or the PI3K inhibitor LY294002. Furthermore, a kinase-active PAK4 (S474E) strongly induced PI3K/AKT activation, and promoted proliferation, migration and invasion in breast cancer cells. A kinase-inactive PAK4 KD (K350A/K351A) did partially upregulate PI3K/AKT, and promoted invasive phenotype. Taken together, these findings suggest that PAK4-activated PI3K/AKT signaling is both kinase-dependent and -independent, which contributes to breast cancer progression. Thus, our results imply that dual inhibition of PAK4 and PI3K/AKT signaling might be a potential therapeutic approach for breast cancer therapy.

EGFR expression is associated with cytoplasmic staining of CXCR4 and predicts poor prognosis in triple-negative breast carcinomas.

The purpose of the present study was to investigate the significance of C-X-C motif chemokine receptor type 4 (CXCR4) and epidermal growth factor receptors (EGFRs) in triple-negative breast cancer (TNBC). CXCR4 and EGFR expression levels were immunohistochemically determined in 207 primary breast cancer specimens. The associations between receptor expression and clinicopathological characteristics were analyzed, and receptor expression was also assessed as a prognostic factor. In the human MDA-MB-231 TNBC cell line, CXCR4 or EGFR was stably knocked down by short hairpin RNA, and the biological behavior of the cells, including migration, invasion and tumorigenesis, was investigated. The results revealed that TNBC was associated with younger age, higher histological grade and an aggressive phenotype. CXCR4 and EGFR were highly expressed in patients with TNBC, and those with high CXCR4 or EGFR expression exhibited an unfavorable prognosis in terms of disease-free survival and overall survival. In MDA-MB-231 cells, the expression of CXCR4 protein was decreased following EGFR silencing, while CXCR4 knockdown also caused a decrease in EGFR protein levels. The migratory and invasive capabilities of MDA-MB-231 cells were decreased following the knockdown of CXCR4 or EGFR expression. A strong correlation between CXCR4 and EGFR expression was identified in patients with TNBC. The results suggest that elevated expression levels of these two receptors may serve as predictive factors for poor prognosis in patients with TNBC. In addition, tumor proliferation, migration, invasion and tumorigenesis are weakened in MDA-MB-231 cells following suppression of CXCR4 or EGFR expression. Therefore, EGFR and CXCR4 may be potential therapeutic targets for TNBC.

Breast reconstruction with single-pedicle TRAM flap in breast cancer patients with low midline abdominal scar.

Breast reconstruction with transverse rectus abdominis myocutaneous (TRAM) flap is challenging in patients with low midline abdominal scar. In this study, we aimed to investigate the clinical feasibility of immediate breast reconstruction using single-pedicle TRAM (SP-TRAM) flaps in patients with low midline abdominal scar. There were 4 strict selection criteria: 1) presence at least 3 perforators on the pedicle side; 2) perforators with regional average flow velocity of >20 cm/s; 3) upper edge of the abdominal scar at least 4 cm from the umbilicus; and 4) scar age >1 year. Eight breast cancer patients with low midline abdominal scar (scar group) and 20 without (control group) underwent immediate breast reconstruction with SP-TRAM flaps consisting of zone I and III and zone II tissues. Flap complications, donor-site complications, and cosmetic results were compared between the two groups. All flaps survived and both groups presented similar flap and donor site complications, including fat necrosis, seroma, hematoma, infection, delayed wound healing, and abdominal hernia, and patients in both groups had similar aesthetic results (p > 0.05). Thus, the study demonstrated that breast reconstruction using SP-TRAM flap was a safe approach in carefully selected patients with low midline abdominal scar.

A nomogram to predict the probability of axillary lymph node metastasis in early breast cancer patients with positive axillary ultrasound.

Among patients with a preoperative positive axillary ultrasound, around 40% of them are pathologically proved to be free from axillary lymph node (ALN) metastasis. We aimed to develop and validate a model to predict the probability of ALN metastasis as a preoperative tool to support clinical decision-making. Clinicopathological features of 322 early breast cancer patients with positive axillary ultrasound findings were analyzed. Multivariate logistic regression analysis was performed to identify independent predictors of ALN metastasis. A model was created from the logistic regression analysis, comprising lymph node transverse diameter, cortex thickness, hilum status, clinical tumour size, histological grade and estrogen receptor, and it was subsequently validated in another 234 patients. Coefficient of determination (R(2)) and the area under the ROC curve (AUC) were calculated to be 0.9375 and 0.864, showing good calibration and discrimination of the model, respectively. The false-negative rates of the model were 0% and 5.3% for the predicted probability cut-off points of 7.1% and 13.8%, respectively. This means that omission of axillary surgery may be safe for patients with a predictive probability of less than 13.8%. After further validation in clinical practice, this model may support increasingly limited surgical approaches to the axilla in breast cancer.

Brachial plexus palsy after a left-side modified radical mastectomy with immediate latissimusdorsi flap reconstruction: report of a case.

Brachial plexus injury is a rare complication during operation and anesthesia; it can occur as a result of various mechanisms such as inappropriate positioning, over-abduction and stretching the upper limbs. Brachial plexus injury can cause the poor function of the upper limb before recovery, and sometimes serious injury is unable to completely recovered the function permanently. Here, we report a female breast cancer patient who sustained a left brachial plexus palsy after modified radical mastectomy with immediate breast reconstruction with latissimusdorsi flap (LDF). The patient had fully recovered with normal function of her left upper limb six months postoperation after conservative treatment.