Eugenol-Induced Autophagy and Apoptosis in Breast Cancer Cells via PI3K/AKT/FOXO3a Pathway Inhibition

The use of natural compounds is promising in approaches to prevent and treat cancer. The long-term application of most currently employed chemotherapy techniques has toxic side effects. Eugenol, a phenolic phytochemical extracted from certain essential oils, has an anti-cancer effect. The modulation of autophagy can promote either the survival or apoptosis of cancer cells. Triple-negative (MDA-MB-231) and HER2 positive (SK-BR-3) breast cancer cell lines were treated with different doses of eugenol. Apoptosis was detected by a flow-cytometry technique, while autophagy was detected by acridine orange. Real-time PCR and Western blot assays were applied to investigate the effect of eugenol on the gene and protein expression levels of autophagy and apoptotic genes. Treating cells with different concentrations of eugenol significantly inhibited cell proliferation. The protein levels of AKT serine/threonine kinase 1 (AKT), forkhead box O3 (FOXO3a), cyclin dependent kinase inhibitor 1A (p21), cyclin-dependent kinase inhibitor (p27), and Caspase-3 and -9 increased significantly in Eugenol-treated cells. Eugenol also induced autophagy by upregulating the expression levels of microtubule-associated protein 1 light chain 3 (LC3) and downregulating the expression of nucleoporin 62 (NU p62). Eugenol is a promising natural anti-cancer agent against triple-negative and HER2-positive breast cancer. It appears to work by targeting the caspase pathway and by inducing autophagic cell death.     

Circular Sponge against miR-21 Enhances the Antitumor Activity of Doxorubicin against Breast Cancer Cells

Breast cancer is the most common type of cancer in women, with chemotherapy being the main strategy. However, its effectiveness is reduced by drug resistance mechanisms. miR-21 is upregulated in breast cancer that has been linked to drug resistance and carcinogenic processes. Our aim was to capture miR-21 with a circular sponge (Circ-21) and thus inhibit the carcinogenic processes and drug resistance mechanisms in which it participates. Proliferation, migration, colony formation, cell cycle, and poly [ADP-ribose] polymerase 1 (PARP-1) and vascular endothelial growth factor (VEGF) detection assays were performed with MCF7 breast cancer cells and MCF10A non-tumor cells. In addition, doxorubicin resistance tests and detection of drug resistance gene expression were performed in MCF7 cells. Reduction in proliferation, as well as migration and colony formation, increased PARP-1 expression, inhibition of VEGF expression and cell cycle arrest in G2/M phase were displayed in the Circ-21 MCF7, which were not observed in the MCF10A cells. Furthermore, in the MCF7 cells, the Circ-21 enhanced the antitumor activity of doxorubicin and decreased the expression of resistance genes: ABCA1, ABCC4, and ABCC5. Based on these results, the use of Circ-21 can be considered a first step for the establishment of an effective gene therapy in the treatment of breast cancer.     

The Antitumor Effect of Metformin Is Mediated by miR-26a in Breast Cancer

Metformin, a drug approved for diabetes type II treatment, has been associated with a reduction in the incidence of breast cancer and metastasis and increased survival in diabetic breast cancer patients. High levels of miR-26a expression have been proposed as one of the possible mechanisms for this effect; likewise, this miRNA has also been associated with survival/apoptosis processes in breast cancer. Our aim was to evaluate if miR-26a and some of its targets could mediate the effect of metformin in breast cancer. The viability of MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cell lines was evaluated with an MTT assay after ectopic overexpression and/or downregulation of miR-26a. Similarly, the expression levels of the miR-26a targets CASP3, CCNE2, ABL2, APAF1, XIAP, BCL-2, PTEN, p53, E2F3, CDC25A, BCL2L1, MCL-1, EZH2, and MTDH were assessed by quantitative polymerase chain reaction (PCR). The effect of metformin treatment on breast cancer cell viability and miR-26a, BCL-2, PTEN, MCL-1, EZH2, and MTDH modulation were evaluated. Wound healing experiments were performed to analyze the effect of miR-26a and metformin treatment on cell migration. MiR-26a overexpression resulted in a reduction in cell viability that was partially recovered by inhibiting it. E2F3, MCL-1, EZH2, MTDH, and PTEN were downregulated by miR-26a and the PTEN (phosphatase and tensin homolog) protein was also reduced after miR-26a overexpression. Metformin treatment reduced breast cancer cell viability, increased miR-26a expression, and led to a reduction in BCL-2, EZH2, and PTEN expression. miR-26a inhibition partly prevents the metformin viability effect and the PTEN and EZH2 expression reduction. Our results indicate that metformin effectively reduces breast cancer cell viability and suggests that the effects of the drug are mediated by an increase in miR-26a expression and a reduction of its targets, PTEN and EHZ2 Thus, the use of metformin in breast cancer treatment constitutes a promising potential breast cancer therapy.     

S-Adenosylmethionine Inhibits Cell Growth and Migration of Triple Negative Breast Cancer Cells through Upregulating MiRNA-34c and MiRNA-449a

Triple-negative breast cancer (TNBC) is one of the most common malignancies worldwide and shows maximum invasiveness and a high risk of metastasis. Recently, many natural compounds have been highlighted as a valuable source of new and less toxic drugs to enhance breast cancer therapy. Among them, S-adenosyl-L-methionine (AdoMet) has emerged as a promising anti-cancer agent. MicroRNA (miRNA or miR)-based gene therapy provides an interesting antitumor approach to integrated cancer therapy. In this study, we evaluated AdoMet-induced modulation of miRNA-34c and miRNA-449a expression in MDA-MB-231 and MDA-MB-468 TNBC cells. We demonstrated that AdoMet upregulates miR-34c and miR-449a expression in both cell lines. We found that the combination of AdoMet with miR-34c or miR-449a mimic strongly potentiated the pro-apoptotic effect of the sulfonium compound by a caspase-dependent mechanism. For the first time, by video time-lapse microscopy, we showed that AdoMet inhibited the in vitro migration of MDA-MB-231 and MDA-MB-468 cells and that the combination with miR-34c or miR-449a mimic strengthened the effect of the sulfonium compound through the modulation of β-catenin and Small Mother Against Decapentaplegic (SMAD) signaling pathways. Our results furnished the first evidence that AdoMet exerts its antitumor effects in TNBC cells through upregulating the expression of miR-34c and miR-449a.     

Platelet-Derived miR-126-3p Directly Targets AKT2 and Exerts Anti-Tumor Effects in Breast Cancer Cells: Further Insights in Platelet-Cancer Interplay

Among the surrounding cells influencing tumor biology, platelets are recognized as novel players as they release microvesicles (MVs) that, once delivered to cancer cells, modulate signaling pathways related to cell growth and dissemination. We have previously shown that physiological delivery of platelet MVs enriched in miR-126 exerted anti-tumor effects in different breast cancer (BC) cell lines. Here, we seek further insight by identifying AKT2 kinase as a novel miR-126-3p direct target, as assessed by bioinformatic analysis and validated by luciferase assay. Both ectopic expression and platelet MV-mediated delivery of miR-126-3p downregulated AKT2 expression, thus suppressing proliferating and invading properties, in either triple negative (BT549 cells) or less aggressive Luminal A (MCF-7 cells) BC subtypes. Accordingly, as shown by bioinformatic analysis, both high miR-126 and low AKT2 levels were associated with favorable long-term prognosis in BC patients. Our results, together with the literature data, indicate that miR-126-3p exerts suppressor activity by specifically targeting components of the PIK3/AKT signaling cascade. Therefore, management of platelet-derived MV production and selective delivery of miR-126-3p to tumor cells may represent a useful tool in multimodal therapeutic approaches in BC patients.     

PI3K/AKT/mTOR Signaling Pathway in Breast Cancer: From Molecular Landscape to Clinical Aspects

Breast cancer is a serious health problem worldwide, representing the second cause of death through malignancies among women in developed countries. Population, endogenous and exogenous hormones, and physiological, genetic and breast-related factors are involved in breast cancer pathogenesis. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) is a signaling pathway involved in cell proliferation, survival, invasion, migration, apoptosis, glucose metabolism and DNA repair. In breast tumors, PIK3CA somatic mutations have been reported, located in exon 9 and exon 20. Up to 40% of PIK3CA mutations are estrogen receptor (ER) positive and human epidermal growth factor receptor 2 (HER2) -negative in primary and metastatic breast cancer. HER2 is overexpressed in 20–30% of breast cancers. HER1, HER2, HER3 and HER4 are membrane receptor tyrosine kinases involved in HER signaling to which various ligands can be attached, leading to PI3K/AKT activation. Currently, clinical studies evaluate inhibitors of the PI3K/AKT/mTOR axis. The main purpose of this review is to present general aspects of breast cancer, the components of the AKT signaling pathway, the factors that activate this protein kinase B, PI3K/AKT-breast cancer mutations, PI3K/AKT/mTOR-inhibitors, and the relationship between everolimus, temsirolimus and endocrine therapy.     

miR-34a慢病毒表达质粒的构建及其对人结肠癌SW480细胞增殖的作用

目的构建miR-34a慢病毒表达质粒,并检测其对人结肠癌SW480细胞增殖的影响。方法以hsa-miR-34a前体序列pre-miR-34a为模板,设计末端部分互补的引物,进行引物退火反应,形成引物二聚体,进行PCR扩增,再以此DNA双链为模板进行PCR扩增,PCR产物酶切后,插入线性化的慢病毒载体pGIPZ-GFP中,构建重组慢病毒表达质粒pGIPZ-miR-34a,与包装质粒Helper 1.0、Helper 2.0共转染293T细胞,进行慢病毒的包装,梯度稀释法检测重组慢病毒的滴度。将包装好的重组慢病毒感染SW480细胞,qPCR法检测感染细胞中miR-34a基因mRNA的转录水平;MTT和平板克隆法检测感染细胞的增殖活力;流式细胞术检测感染细胞的细胞周期分布。结果 PCR及测序结果证实重组慢病毒表达质粒构建正确;重组慢病毒的滴度为6.52×108TU/ml;重组慢病毒感染SW480细胞后,显著上调了细胞中miR-34a基因mRNA的转录水平,明显抑制了细胞的增殖活力(P<0.01),G0-G1期细胞比例明显增加(P<0.01)。结论成功构建了miR-34a慢病毒表达质粒,慢病毒感染SW480细胞后,能显著提高miR-34a的内源性表达,并抑制细胞的增殖,为进一步研究miR-34a的功能及作用机制奠定了基础。     

A Novel Mechanism of Immunoproteasome Regulation via miR-369-3p in Intestinal Inflammatory Response

The immunoproteasome is a multi-catalytic protein complex expressed in hematopoietic cells. Increased expression of immuno-subunits followed by increased proteasome activities is associated with the pathogenesis of IBD. Therefore, the identification of molecules that could inhibit the activities of this complex has been widely studied. microRNAs are small molecules of non-coding RNA that regulate the expression of target genes. Our purpose was to demonstrate that miR-369-3p is able to reduce the expression of the PSMB9 subunit and consequently modulate the catalytic activities of immunoproteasome. After bioinformatics prediction of the gene target of miR-369-3p, we validated its modulation on PSMB9 expression in the RAW264.7 cell line in vitro. We also found that miR-369-3p indirectly reduced the expression of other immunoproteasome subunits and that this regulation reduced the catalytic functions of the immunoproteasome. Increased levels of PSMB9 were observed in colon samples of acute IBD patients compared to the remission IBD group and control group. Our data suggest that miR-369-3p may be a future alternative therapeutic approach to several compounds currently used for the treatment of inflammatory disorders including IBD.     

A Regulatory Loop Involving miR-200c and NF-κB Modulates Mortalin Expression and Increases Cisplatin Sensitivity in an Ovarian Cancer Cell Line Model

Ovarian cancer is currently the most lethal gynecological cancer. At present, primary debulking surgery combined with platinum-based chemotherapy is the standard treatment strategy for ovarian cancer. Although cisplatin-based chemotherapy has greatly improved the prognosis of patients, the subsequent primary or acquired drug resistance of cancer cells has become an obstacle to a favorable prognosis. Mortalin is a chaperone that plays an important role in multiple cellular and biological processes. Our previous studies have found that mortalin is associated with the proliferation and migration of ovarian cancer cells and their resistance to cisplatin-based chemotherapy. In this study, microRNA (miR)-200b/c downregulated mortalin expression and inhibited the proliferation and migration of the paired cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) epithelial ovarian cancer cell lines. Moreover, miR-200c increased the sensitivity of ovarian cancer cells to cisplatin treatment by regulating mortalin levels. Nuclear factor (NF)-κB directly regulated mortalin and miR-200b/c expression levels, while NF-κB and miR-200b/c jointly regulated the expression of mortalin. The combination of cisplatin and miR-200c significantly enhanced the therapeutic effects on ovarian cancer in vivo, suggesting that miR-200c may serve as a potential therapeutic agent for ovarian cancer.     

Targeting the Highly Expressed microRNA miR-146b with CRISPR/Cas9n Gene Editing System in Thyroid Cancer

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.     

Inhibition of the PI3K/mTOR Pathway in Breast Cancer to Enhance Response to Immune Checkpoint Inhibitors in Breast Cancer

Objectives: Inhibition of the PI3K/mTOR pathway suppresses breast cancer (BC) growth, enhances anti-tumor immune responses, and works synergistically with immune checkpoint inhibitors (ICI). The objective here was to identify a subclass of PI3K inhibitors that, when combined with paclitaxel, is effective in enhancing response to ICI. Methods: C57BL/6 mice were orthotopically implanted with syngeneic luminal/triple-negative-like PyMT cells exhibiting high endogenous PI3K activity. Tumor growth in response to treatment with anti-PD-1 + anti-CTLA-4 (ICI), paclitaxel (PTX), and either the PI3Kα-specific inhibitor alpelisib, the pan-PI3K inhibitor copanlisib, or the broad spectrum PI3K/mTOR inhibitor gedatolisib was evaluated in reference to monotherapy or combinations of these therapies. Effects of these therapeutics on intratumoral immune populations were determined by multicolor FACS. Results: Treatment with alpelisib + PTX inhibited PyMT tumor growth and increased tumor-infiltrating granulocytes but did not significantly affect the number of tumor-infiltrating CD8⁺ T cells and did not synergize with ICI. Copanlisib + PTX + ICI significantly inhibited PyMT growth and increased activation of intratumoral CD8⁺ T cells as compared to ICI alone, yet did not inhibit tumor growth more than ICI alone. In contrast, gedatolisib + ICI resulted in significantly greater inhibition of tumor growth compared to ICI alone and induced durable dendritic-cell, CD8⁺ T-cell, and NK-cell responses. Adding PTX to this regimen yielded complete regression in 60% of tumors. Conclusion: PI3K/mTOR inhibition plus PTX heightens response to ICI and may provide a viable therapeutic approach for treatment of metastatic BC.     

The Role of MicroRNAs in Breast Cancer Migration, Invasion and Metastasis

MicroRNAs (miRNAs) are a major class of small, noncoding RNA molecules that regulate gene expression by targeting mRNAs to trigger either translational repression or mRNA degradation. They have recently been more widely investigated due to their potential role as targets for cancer therapy. Many miRNAs have been implicated in several human cancers, including breast cancer. miRNAs are known to regulate cell cycle and development, and thus may serve as useful targets for exploration in anticancer therapeutics. The link between altered miRNA signatures and breast cancer development and metastasis can be observed either through the loss of tumor suppressor miRNAs, such as let-7s, miR-30a/31/34a/125s/200s/203/205/206/342 or the overexpression of oncogenic miRNAs, such as miR-10b/21/135a/155/221/222/224/373/520c in breast cancer cells. Some of these miRNAs have also been validated in tumor specimens of breast cancer patients, underscoring their potential roles in diagnostics, as well as targets for novel therapeutics for breast cancer. In this review article, we will provide an overview and update of our current understanding of the mode of action of several of these well characterized miRNAs in breast cancer models. Therefore, better understanding of the gene networks orchestrated by these miRNAs may help exploit the full potential of miRNAs in regards to cancer diagnosis, treatment, and therapeutics.     

SIRT1/FOXO Signaling Pathway in Breast Cancer Progression and Metastasis

Breast cancer is the second most common cancer in women. The roles of the SIRT and FoxO proteins in tumor progression are known, but their roles in metastasis have not yet been clearly elucidated. In our study, we investigated the roles of SIRT and FoxO proteins their downstream pathways, proteins p21 and p53, in tumor progression and metastasis. We evaluated these proteins in vitro using metastatic 4TLM and 67NR cell lines, as well as their expression levels in tumor-bearing mice. In addition, the regulatory role of SIRT and FoxO proteins in different transduction cascades was examined by IPA core analysis, and clinicopathological evidence was investigated in the TCGA database. In primary tumors, the expression levels of SIRT1, p21, p53, E2F1 and FoxO proteins were higher in 67NR groups. In metastatic tissues, the expression levels of SIRT1, E2F1 and FoxO proteins were found to be enhanced, whereas the levels of p53 and p21 expression were noted to be reduced. IPA analysis also provided empirical evidence of the mechanistic involvement of SIRT and FoxO proteins in tumor progression and metastasis. In conclusion, SIRT1 was found to co-operate with FoxO proteins and to play a critical role in metastasis. Additional research is required to determine why overexpression of SIRT1 in metastatic tissues has oncogenic effects.     

Function and Application of Flavonoids in the Breast Cancer

Breast cancer is one of the top causes of death, particularly among women, and it affects many women. Cancer can also be caused by various factors, including acquiring genetic alteration. Doctors use radiation to detect and treat breast cancer. As a result, breast cancer becomes radiation-resistant, necessitating a new strategy for its treatment. The approach discovered by the researchers is a flavonoid, which is being researched to see if it might help treat radiation-resistant breast cancer more safely than an approved medicine already being used in the field. As a result, this study focuses on the role of flavonoids in breast cancer suppression, breast cancer gene anomalies, and the resulting apoptotic mechanism.