TYMS Knockdown Suppresses Cells Proliferation, Promotes Ferroptosis via Inhibits PI3K/Akt/mTOR Signaling Pathway Activation in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is characterized by a grim prognosis and numerous challenges. The objective of our study was to examine the role of thymidylate synthase (TYMS) in TNBC and its impact on ferroptosis. The expression of TYMS was analyzed in databases, along with its prognostic correlation. TYMS positive expression was identified through immunohistochemistry (IHC), while real-time quantitative PCR (qRTPCR) was employed to measure TYMS mRNA levels in various cell lines. Western blotting was utilized to assess protein expression. Cell proliferation, mobility, apoptosis, and reactive oxygen species (ROS) levels were evaluated using CCK8, wound scratch healing assay, transwell assay, and flow cytometry, respectively. Additionally, a tumor xenograft model was established in BALB/c nude mice for further investigation. Tumor volume and weight were measured, and histopathological analysis using hematoxylin and eosin (H&E) staining was conducted to assess tumor tissue changes. IHC staining was employed to detect the expression of Ki67 in tumor tissues. High expression of TYMS was observed in TNBC and was found to be correlated with poor prognosis in patients. Among various cell lines, TYMS expression was highest in BT549 cells. Knockdown of TYMS resulted in suppression of cell proliferation and mobility, as well as promotion of apoptosis. Furthermore, knockdown of TYMS led to increased accumulation of ROS and Fe2+ levels, along with upregulation of ACLS4 expression and downregulation of glutathione peroxidase 4 (GPX4) expression. In vivo studies showed that knockdown of TYMS inhibited tumor growth. Additionally, knockdown of TYMS was associated with inhibition of mTOR, p-PI3K, and p-Akt expression. Our research showed that the knockdown of TYMS suppressed the TNBC progression by inhibited cells proliferation via ferroptosis. Its underlying mechanism is related to the PI3K /Akt pathway. Our study provides a novel sight for the suppression effect of TYMS on TNBC.

GSH-activable heterotrimeric nano-prodrug for precise synergistic therapy of TNBC.

Combination chemotherapy is an effective approach for triple-negative breast cancer (TNBC) therapy, especially when drugs are administered at specific optimal ratios. However, at present, strategies involving precise and controllable ratios based on effective loading and release of drugs are unavailable. Herein, we designed and synthesized a glutathione (GSH)--responsive heterotrimeric prodrug and formulated it with an amphiphilic polymer to obtain nanoparticles (DSSC2 NPs) for precise synergistic chemotherapy of TNBC. The heterotrimeric prodrug was prepared using docetaxel (DTX) and curcumin (CUR) at the optimal synergistic ratio of 1: 2. DTX and CUR were covalently conjugated by disulfide linkers. Compared with control NPs, DSSC2 NPs had quantitative/ratiometric drug loading, high drug co-loading capacity, better colloidal stability, and less premature drug leakage. After systemic administration, DSSC2 NPs selectively accumulated in tumor tissues and released the encapsulated drugs triggered by high levels of GSH in cancer cells. In vitro and in vivo experiments validated that DSSC2 NPs released DTX and CUR at the predefined ratio and had a highly synergistic therapeutic effect on tumor suppression in TNBC, which can be attributed to ratiometric drug delivery and synchronous drug activation. Altogether, the heterotrimeric prodrug delivery system developed in this study represents an effective and novel approach for combination chemotherapy.

Clinical analysis of a new multifunctional instrument set for gasless endoscopic thyroidectomy with two different approaches.

BACKGROUND: Following the rapid development of endoscopic thyroidectomy techniques, various surgical procedures have been developed (e.g., transoral, submandibular, areolar, axillary, retroauricular, and combined procedures), and each of these procedures has its own advantages. In recent years, gasless endoscopic thyroidectomy has emerged as a feasible procedure, and it has replaced traditional CO2 insufflation approaches because of advantages such as stable cavity construction, pollution reduction, resource saving, and risk reduction. However, each gasless procedure requires special instruments for cavity construction, and this results in enormous wastage of medical resources. In the present study, we introduced a set of instruments developed by our team. This set of instruments is designed to be compatible with the current gasless endoscopic thyroidectomy approaches, including transoral, submandibular, transareolar, transaxillary, retroauricular, combined, and lateral cervical lymph node dissection. Here, we introduced this set of instruments for two gasless endoscopic thyroidectomy procedures (transaxillary and transareolar). Following the incorporation of this set of instruments in regular clinical practice, it could be used for more gasless endoscopic thyroidectomy procedures in the future. OBJECTIVE: To investigate the feasibility, safety, and efficacy of the self-developed instruments for gasless endoscopic thyroidectomy in two different approaches. METHODS: A total of 180 patients diagnosed to have papillary thyroid carcinoma (PTC) between January 2020 and April 2022 were retrospectively investigated. The patients were assigned to a gasless transaxillary group (group A) and a gasless transareolar group (group B). The same gasless endoscopic-assisted instruments were used for both groups. The clinical characteristics, treatment results, and complications were compared between the two groups. RESULTS: All 180 patients were successfully operated. The extent of surgical resection in all patients was the same: "unilateral glandular lobectomy + isthmus combined with ipsilateral central zone lymph node dissection." There were 130 and 50 patients in group A and group B, respectively; one patient in the former group was converted to open surgery due to intraoperative bleeding. No significant difference was observed between the two groups in terms of gender, age, body mass index (BMI), education level, and proportion of concomitant Hashimoto's thyroiditis (P > 0.05). The establishment of cavity time was significantly longer in group A than in group B (35.62 ± 5.07 min vs. 17.46 ± 2.55 min, P < 0.01). The number of lymph nodes cleared was slightly less in group A than in group B (4.06 ± 2.93 vs. 4.52 ± 2.38, P = 0.07). Moreover, the two groups showed no significant differences (P > 0.05) in the total operative time (145.54 ± 45.11 min vs. 143.06 ± 46.70 min), tumor size (0.68 ± 0.46 cm vs. 0.71 ± 0.49 cm), postoperative hospital stay (4.08 ± 1.48 days vs. 3.72 ± 1.07 days), vocal cord paralysis [4 (3.1%) vs. 2 (4%)], postoperative swallowing discomfort [24 (18.5%) vs. 5 (10%)], and postoperative recurrence and satisfaction scores (3.27 ± 1.52 vs. 3.28 ± 1.53). CONCLUSION: Although the two approaches of gasless endoscopic surgery have different operative paths and different time periods for cavity construction, both approaches are similar in terms of the principle of cavity construction, safe and reliable postoperative efficacy, and good cosmetic effect. Therefore, the same set of instruments can be used to complete the surgery in both approaches, thus saving medical resources and facilitating the popularization of this technology.

[Development and Clinical Application of Multifunctional Inflation-Free Lumpectomy Aid].

To investigate the value of self-developed air-free laparoscopic auxiliary instruments in the clinical application of thyroid diseases. The clinical data of 70 transaxillary and 45 transareolar air-free laparoscopic surgeries for thyroid cancer and 40 conventional open surgeries were retrospectively compared. The transaxillary and transareolar laparoscopic groups had significantly longer operative times than the open group, while the postoperative satisfaction was higher in the endoscopic group than in the open group. This set of instruments has advantage of novel design, scientific structure, safe application. It can be compatible with a variety of thyroid and breast air-free laparoscopic procedures, which can promote the development and popularization of laparoscopic technology.

LncRNA MIR31HG promotes cell proliferation and invasive properties of the MCF-7 cell line by regulation of receptor-interacting serine-threonine kinase 4.

LncRNA MIR31HG is involved in many types of cancers, while its roles in breast cancer are still unknown. The current study aimed to explore the function of lncRNA MIR31HG in breast cancer and the underlying mechanisms. Stable expression cell lines were constructed by using lentivirus particles. MTT assay was used to determine cell viability. Wound healing and Transwell assay were used to determine cell migration and invasion, respectively. The changes in biomarkers were determined by using qPR-PCT and Western blotting, respectively. BALB/c nude mice were used to generate a xenograft mouse model. MIR31HG regulated cell proliferation, migration and invasion in MCF7 cells. Besides, MIR31HG regulated N-Cadherin, Vimentin, and E-Cadherin. MIR31HG positively regulated receptor-interacting serine-threonine kinase 4 (RIPK4), as supported by the fact that knockdown of MIR31HG suppressed RIPK4, and the knockdown of RIPK4 did not affect MIR31HG. Additionally, we found that RIPK4 regulated cell proliferation, migration and invasion in MCF7 cells. The changes in RIPK4 regulated N-Cadherin, Vimentin, and E-Cadherin. Consistently, in vivo studies showed that the knockdown of MIR31HG or RIPK4 reduced tumor size in xenograft animal models. The roles of lncRNA MIR31HG in breast cancer were associated with its regulatory effects against RIPK4.

Regulatory T cells are associated with the tumor immune microenvironment and immunotherapy response in triple-negative breast cancer.

Introduction: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high risk of distant metastasis, an extremely poor prognosis, and a high risk of death. Regulatory T cells (Tregs) contribute to the formation of a tumor immunosuppressive microenvironment, which plays an important role in the progression and treatment resistance of TNBC. Methods: A public single-cell sequencing dataset demonstrated increased infiltration of Tregs in TNBC tissues relative to normal breast tissue. Weighted gene co-expression network analysis was used to identify Treg infiltration-related modules for METABRIC TNBC samples. Subsequently, we obtained two Treg infiltration-associated clusters of TNBC by applying consensus clustering and further constructed a prognostic model based on this Treg infiltration-associated gene module. The ability of the selected gene in the prognostic model, thymidine kinase-1 (TK1), to promote the progression of TNBC was evaluated in vitro. Results: We concluded that two Treg infiltration-associated clusters had different prognoses and sensitivities to drugs commonly used in breast cancer treatment, and multi-omics analysis revealed that the two clusters had different copy number variations of key tumor progression genes. The 7-gene risk score based on TNBC Treg infiltration was a reliable prognostic indicator both in the training and validation cohorts. Moreover, patients with TNBC with high Treg infiltration-related scores lacked the activation of immune activation pathways and exhibited resistance to anti-PD1 immunotherapy. Knocking down TK1 led to impaired proliferation, migration, and invasion of TNBC cells in vitro. In addition, specimens from patients with TNBC with high TK1 expression showed significantly higher Treg infiltration in tumors. Results of spatial transcriptome analysis showed that TK1 positive cells mainly localize in tumor area, and Treg cell infiltration in TNBC tissues was associated with high expression of TK1. Pan-cancer analysis also demonstrated that TK1 is associated with poor prognosis and activation of proliferation pathways in multiple cancers. Discussion: We established a prognostic model related to Treg infiltration and this model can be used to establish a clinically relevant classification of TNBC progression. Additionally, our work revealed the underestimable potential of TK1 as a tumor biomarker and immunotherapeutic target.

Assessing the decision quality of artificial intelligence and oncologists of different experience in different regions in breast cancer treatment.

Background: AI-based clinical decision support system (CDSS) has important prospects in overcoming the current informational challenges that cancer diseases faced, promoting the homogeneous development of standardized treatment among different geographical regions, and reforming the medical model. However, there are still a lack of relevant indicators to comprehensively assess its decision-making quality and clinical impact, which greatly limits the development of its clinical research and clinical application. This study aims to develop and application an assessment system that can comprehensively assess the decision-making quality and clinical impacts of physicians and CDSS. Methods: Enrolled adjuvant treatment decision stage early breast cancer cases were randomly assigned to different decision-making physician panels (each panel consisted of three different seniority physicians in different grades hospitals), each physician made an independent "Initial Decision" and then reviewed the CDSS report online and made a "Final Decision". In addition, the CDSS and guideline expert groups independently review all cases and generate "CDSS Recommendations" and "Guideline Recommendations" respectively. Based on the design framework, a multi-level multi-indicator system including "Decision Concordance", "Calibrated Concordance", " Decision Concordance with High-level Physician", "Consensus Rate", "Decision Stability", "Guideline Conformity", and "Calibrated Conformity" were constructed. Results: 531 cases containing 2124 decision points were enrolled; 27 different seniority physicians from 10 different grades hospitals have generated 6372 decision opinions before and after referring to the "CDSS Recommendations" report respectively. Overall, the calibrated decision concordance was significantly higher for CDSS and provincial-senior physicians (80.9%) than other physicians. At the same time, CDSS has a higher " decision concordance with high-level physician" (76.3%-91.5%) than all physicians. The CDSS had significantly higher guideline conformity than all decision-making physicians and less internal variation, with an overall guideline conformity variance of 17.5% (97.5% vs. 80.0%), a standard deviation variance of 6.6% (1.3% vs. 7.9%), and a mean difference variance of 7.8% (1.5% vs. 9.3%). In addition, provincial-middle seniority physicians had the highest decision stability (54.5%). The overall consensus rate among physicians was 64.2%. Conclusions: There are significant internal variation in the standardization treatment level of different seniority physicians in different geographical regions in the adjuvant treatment of early breast cancer. CDSS has a higher standardization treatment level than all physicians and has the potential to provide immediate decision support to physicians and have a positive impact on standardizing physicians' treatment behaviors.

Comparison of surgical smoke between two approaches for endoscopic thyroidectomy and open thyroidectomy.

BACKGROUND: Surgical smoke has been recognized as a potential health risk by an increasing number of researchers. Moreover, the counts of surgical smoke produced during different surgical approaches are different. This study aimed to measure and compare the particulate matter (PM) of surgical smoke generated during open thyroidectomy and two endoscopic approaches for thyroidectomy to provide guidance for safe clinical practices. METHODS: Forty-eight patients with thyroid cancer admitted to our hospital from June 2020 to December 2021 and treated with different surgical approaches were enrolled in this study. The total and peak counts of PM, dynamic changes, and other characteristics of surgical smoke produced during surgery were recorded. PM was classified as PM2.5 (size ≤ 2.5 µm) and PM10 (size ≤ 10 µm). RESULTS: In a single cut, both the peak and total counts of PM2.5 and PM10 of surgical smoke in the open thyroidectomy group (n = 15) were significantly higher than those in the breast approach endoscopic thyroidectomy with CO2 insufflation group (n = 15) and the gasless transaxillary endoscopic thyroidectomy group (n = 18) (p < 0.001). Moreover, the latter two groups showed no significant differences in the peak and total counts of PM2.5 and PM10 (p > 0.05). CONCLUSION: In thyroid surgery, more surgical smoke is produced during open thyroidectomy than during endoscopic thyroidectomy, while different endoscopic approaches showed no significant difference in surgical smoke production. Thus, endoscopic approaches outperform the open thyroidectomy approach with regard to surgical smoke production.

Lobetyolin inhibits the proliferation of breast cancer cells via ASCT2 down-regulation-induced apoptosis.

This study aimed to investigate the anti-cancer effect of lobetyolin on breast cancer cells. Lobetyolin was incubated with MDA-MB-231 and MDA-MB-468 breast cancer cells for 24 h. Glucose uptake and the mRNA expression of GLUT4 (SLC2A4), HK2 and PKM2 were detected to assess the effect of lobetyolin on glucose metabolism. Glutamine uptake and the mRNA expression of ASCT2 (SLC1A5), GLS1, GDH and GLUL were measured to assess the effect of lobetyolin on glutamine metabolism. Annexin V/PI double staining and Hoechst 33342 staining were used to investigate the effect of lobetyolin on cell apoptosis. Immunoblot was employed to estimate the effect of lobetyolin on the expression of proliferation-related markers and apoptosis-related markers. SLC1A5 knockdown with specific siRNA was performed to study the role of ASCT2 played in the anti-cancer effect of lobetyolin on MDA-MB-231 and MDA-MB-468 breast cancer cells. C-MYC knockdown with specific siRNA was performed to study the role of c-Myc played in lobetyolin-induced ASCT2 down-regulation. Myr-AKT overexpression was performed to investigate the role of AKT/GSK3ß signaling played in lobetyolin-induced down-regulation of c-Myc and ASCT2. The results showed that lobetyolin inhibited the proliferation of both MDA-MB-231 and MDA-MB-468 breast cancer cells. Lobetyolin disrupted glutamine uptake via down-regulating ASCT2. SLC1A5 knockdown attenuated the anti-cancer effect of lobetyolin. C-MYC knockdown attenuated lobetyolin-caused down-regulation of ASCT2 and Myr-AKT overexpression reversed lobetyolin-caused down-regulation of both c-Myc and ASCT2. In conclusion, the present work suggested that lobetyolin exerted anti-cancer effect via ASCT2 down-regulation-induced apoptosis in breast cancer cells.

The knockdown of the sepiapterin reductase gene suppresses the proliferation of breast cancer by inducing ROS-mediated apoptosis.

BACKGROUND: Sepiapterin reductase (SPR) is an important regulator of the biosynthesis of tetrahydrobiopterin (BH4), which has been shown to be a promoter of different kinds of tumors. This study aims to investigate the role of SPR in breast cancer and to explore its molecular mechanism. METHODS: SPR expressions in breast cancer tissues with different pathological stages were compared with the corresponding pericarcinomatous tissues and were analyzed using immunohistochemical staining and western blot. SPR knockdown was performed in MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cells using specific siRNAs. Quantitative real-time PCR and western blot were used to determine the efficiency of the SPR knockdown. The intracellular BH4 levels were measured using HPLC, and the intracellular ROS levels were measured using an ROS detection kit. Clone formation and cell proliferation assays were used to study the effect of the SPR knockdown on cell proliferation. Annexin V/PI double staining, cell mitochondria isolation, and western blot were performed to study the effect of the SPR knockdown on cell apoptosis. ROS scavenger NAC was used to inhibit increased ROS caused by the SPR knockdown. RESULTS: SPR is highly expressed in breast cancer tissues compared with the pericarcinomatous tissues and positively correlated with the pathological stages. The knockdown of SPR causes decreased intracellular BH4 and increased intracellular ROS and inhibits the proliferation of MDA-MB-231 and MDA-MB-468 cells. The knockdown of SPR also induces mitochondrial pathway-mediated apoptosis. NAC suppresses the SPR knockdown-caused cell apoptosis and cell death. CONCLUSIONS: SPR promotes the proliferation of breast cancer cells. The knockdown of SPR suppresses the proliferation of breast cancer cells by inducing ROS-mediated apoptosis.

Molecular phenotype discordance between primary and synchronous metastatic lesions of breast cancer and its determinant role in guiding treatment decisions: a case report.

Molecular phenotype discordance between primary and metastatic tumors exists in a small proportion of breast cancer (BC) patients with accessible synchronous metastases. Reduced therapeutic effect and delays in treatment can occur when decisions on systemic therapy are determined by ignoring the differences in tumor type. Here we report a 54-year-old post-menopausal locally advanced BC patient, who showed no tumor response following routine treatment which included targeting anti-HER2, based on the phenotype of primary tumor (Luminal B, HER2-positive), during neoadjuvant therapy. However, following a secondary biopsy of the metastatic subclavian lymph node, a distinct pathological feature (Triple-negative) was revealed; chemotherapy was adjusted accordingly and resulted in a positive tumor response. Various subclones within primary and metastatic lesions were identified which might be attributed to tumor heterogeneity and in turn resulting in the phenotypic discordance in the receptor status. The patient died due to tumor progression related to triple-negative-featured lung metastasis, with overall survival time of 26.4 months. This study strengthens the value of concurrent biopsies of both primary and synchronous metastatic lesions in BC patients, and provides a reference for treating this kind of tumor when discordance in the molecular phenotype is observed.

Induction of Necroptosis in Human Breast Cancer Drug-Resistant Cells by SMAC Analog LCL161 After Caspase Inhibition Requires RIP3.

LCL161 is a second mitochondrial activator of caspases (SMAC) mimetic and inhibitor of apoptosis protein (IAP) antagonist that has oral bioavailability, exhibits anti-tumor effects and improves the chemical sensitivity of many cancers. The aim of this study was to ascertain the effects and mechanisms of the SMAC analog LCL161 on breast cancer drug-resistant cells after undergoing caspase inhibition. This was achieved through use of colony formation and CCK-8 assays to detect cell proliferation. Flow cytometry, Western blot analysis, ATP assay, immunofluorescence and siRNA transfection were used to study the molecular mechanisms of LCL161-induced death of cisplatin-resistant MCF-7 cells after caspase inhibition. LCL161 exhibited an inhibitory effect on MCF-7/DDP cells including after inhibition of caspase. However, LCL161 could not on its own induce a necroptosis effect on MCF-7/DDP cells (P < 0.01 or P < 0.001). When used jointly with the caspase inhibitor z-VAD-fmk, it significantly decreased intracellular ATP levels (P < 0.01 or P < 0.05). This induction of necroptosis occurred through the activation of the RIP1-RIP3-MLKL programmed cell necrosis cascade. Knockdown of RIP3 using siRNA protected against the combined LCL161 / z-VAD-fmk-induced cell death (P < 0.01 or P < 0.001). These findings support the hypothesis that LCL161 combined with caspase inhibition can induce a necroptosis effect on MCF-7/DDP cells, suggesting that it has potential to be an effective treatment for breast cancer.

Immature colon carcinoma transcript-1 promotes proliferation of gastric cancer cells.

Gastric cancer is the fourth most common malignant tumor and has been considered as one of the leading causes of cancer-related death worldwide. The identification of the molecular mechanism during gastric cancer progression is urgently needed, which will help to develop more effective treatment strategies. As a component of the human mitoribosome, immature colon carcinoma transcript-1 (ICT1) might be involved in tumor formation and progression. However, its biological function and the corresponding mechanism in gastric cancer have been poorly characterized. To study the mechanism of ICT1 in gastric cancer, we first investigated the mRNA levels of ICT1 in human normal and gastric cancer tissues using datasets from the publicly available Oncomine database. The results showed that ICT1 is overexpressed in gastric cancer tissues. Then in order to study the role of ICT1 in gastric cancer, two shRNAs were used to silence ICT1 in MGC80-3 and AGS cells. Functional analysis showed ICT1 knockdown significantly inhibited the proliferation of gastric cancer cells and induced apoptosis. Further, mechanistic study demonstrated that ICT1 silencing induced cell-cycle arrest at G2/M phase via the suppression of cyclin A2 and cyclin B1. In addition, ICT1 silencing also increased cleaved caspase-3 and activated PARP in gastric cancer cells. These findings suggest that ICT1 may play a crucial role in promoting gastric cancer proliferation in vitro.

Smac mimetic­induced caspase­independent necroptosis requires RIP1 in breast cancer.

There is an urgent requirement for the development of novel targeted therapies to treat breast cancer, which is the most comment type of malignancy among women. The evasion of apoptosis is a hallmark of cancer, and is often due to the upregulation of inhibitor of apoptosis proteins (IAPs) in tumor cells. Second mitochondrial­derived activator of caspase/direct IAP­binding protein with low PI is a natural IAP antagonist, which is found in the mitochondrion; this protein has a motif, which binds to a surface groove on the baculovirus IAP repeat domains of the IAPs. In the present study, the effects of the LCL161 Smac mimetic, a small molecule IAP antagonist, on breast cell lines was examined. The results from MTT and colony formation assays demonstrated that LCL161 markedly inhibited the proliferation and induced the apoptosis of MDA­MB­231 and MCF­7 cell lines. As determined by western blotting, cIAP1 was degraded in the breast cancer cells, which occurred in an LCL161­dependent manner. Upon caspase activation, LCL161 treatment induced necroptosis, another form of programmed cell death. The downregulation of receptor­interacting protein kinase­1 via small interfering RNA protected the cells from LCL161­induced necroptosis. Taken together, the results of the present study showed that LCL161 can induce multiple forms of programmed cell death in breast cancer cells, and may thus offer promise as an anticancer agent in diverse genotypic backgrounds.