Ambra1 in cancer: implications for clinical oncology.

Activating molecule in Beclin-1-regulated autophagy protein 1 (Ambra1) is well known to mediate the autophagy process and promote the formation of autophagosomes. In addition, Ambra1 is involved in the execution of apoptosis. A growing number of studies have revealed that this protein modifies the sensitivity of cancer cells to anticancer drugs by controlling the balance between autophagy and apoptosis. In addition, Ambra1 is a key factor in regulating the cell cycle, proliferation, invasion and migration. Therefore, it plays a key role in tumorigenesis and progression. Moreover, Ambra1 is highly expressed in a variety of cancers and is closely related to the prognosis of patients. Thus, it appears that Ambra1 has multiple roles in tumorigenesis and progression, which may have implications for clinical oncology. The present review focuses on recent advances in the study of Ambra1, especially the role of the protein in tumorigenesis, progression and effects on anticancer drug sensitivity.

Ambra1 regulates apoptosis and chemosensitivity in breast cancer cells through the Akt-FoxO1-Bim pathway.

The sensitivity of cells to chemotherapeutic agents has a major effect on disease outcome in breast cancer patients. Unfortunately, there are numerous factors involved in the regulation of chemosensitivity, and the mechanisms need to be further investigated. Autophagy/Beclin 1 regulator 1 (Ambra1) is a key protein in the crosstalk between autophagy and apoptosis. It controls the switch between these two processes, which determines whether cells survive or die. Induction of apoptosis is the primary mechanism by which most chemotherapeutic drugs eliminate cancer cells. Recently, Ambra1 has been shown to modulate paclitaxel-induced apoptosis in breast cancer cells via the Bim/mitochondrial pathway, thereby modifying the sensitivity of cells to paclitaxel. However, how Ambra1 regulates Bim expression remains unclear. Here, we further confirmed that Bim plays an indispensable role in Ambra1's regulation of apoptosis and chemosensitivity in breast cancer cells. Furthermore, Ambra1 was found to regulate Bim expression at the transcriptional level through the Akt-FoxO1 pathway. Therefore, we propose a novel pathway, Ambra1-Akt-FoxO1-Bim, which regulates apoptosis and chemosensitivity in breast cancer cells. Thus, Ambra1 may represent a potential target for breast cancer treatment.

Ambra1 modulates the sensitivity of breast cancer cells to epirubicin by regulating autophagy via ATG12.

The sensitivity of breast cancer cells to epirubicin (EPI) is closely related to the efficacy of the drug and the prognosis of patients. A growing body of research has suggested that autophagy is involved in the treatment of a variety of cancers, including breast cancer, and modifies the sensitivity of anticancer drugs. However, the mechanism by which autophagy participates in cancer therapy and modulates drug sensitivity has not been fully elucidated. In this study, we showed that the expression of Autophagy/Beclin 1 regulator 1 (Ambra1), a key protein of autophagy, was negatively correlated with EPI sensitivity in breast cancer cells. In addition, it altered the sensitivity of breast cancer cells to EPI by regulating EPI-induced autophagy. As a potential mechanism, we demonstrated that autophagy-related protein 12 (ATG12) was a downstream protein that Ambra1-regulated EPI-induced autophagy. Therefore, Ambra1 plays an important role in regulating the sensitivity of breast cancer cells to EPI. And the regulatory effect of Ambra1 on EPI sensitivity is achieved through the regulation of autophagy by targeting ATG12. Overall, we propose a novel mechanism by which autophagy modulates the sensitivity of breast cancer cells to EPI. ATG12 is a novel targeting protein of Ambra1 in regulating EPI-induced autophagy. In addition, the important role of Ambra1 in modulating the sensitivity of breast cancer cells to EPI is confirmed in vivo. This finding indicates that Ambra1 might be a target for developing breast cancer treatments.

The Clinicopathological Significance and Correlative Signaling Pathways of an Autophagy-Related Gene, Ambra1, in Breast Cancer: a Study of 25 Microarray RNA-Seq Datasets and in-House Gene Silencing.

BACKGROUND/AIMS: The activating molecule in Beclin1-regulated autophagy (Ambra1) has been observed to be over-expressed in several cancers, but the clinical contribution of Ambra1 in breast cancer (BC) remains unknown. Hence, in this study, we conducted a comprehensive investigation into the expression, biological role, and underlying functional mechanism of Ambra1 in BC. METHODS: Microarray and RNA-seq datasets providing Ambra1 expression data were obtained from Gene Expression Omnibus (GEO), ArrayExpress, Oncomine, and The Cancer Genome Atlas (TCGA). Both standard mean deviation (SMD) and summary receiver operating characteristic methods were employed to assess Ambra1 expression in BC. We then silenced Ambra1 in MDA-MB-231 cells and performed in vitro experiments to explore the biological effects of Ambra1 on BC cells. Furthermore, differentially expressed genes (DEGs) after Ambra1 knock-down were profiled with a microarray and overlapped with the genes correlated with Ambra1 from Multi Experiment Matrix (MEM) and genes similar to Ambra1 from Gene Expression Profiling Interactive Analysis. These overlapping genes were collected for further bioinformatics analyses to investigate the underlying molecular mechanism of Ambra1 in BC. RESULTS: A total of 25 microarray and RNA-seq datasets involving 2460 breast cancer samples were included. The pooled results demonstrated that Ambra1 was markedly up-regulated in BC tissues (SMD=0.39, 95% CI=0.15-0.63; P=0.002), and the Ambra1 level was also significantly related to the progression of BC, especially metastasis status (P=0.004). In vitro experiments suggested that the proliferation of MDA-MB-231 cells transfected with Ambra1 short hairpin RNA (sh-RNA 2450) showed a decreasing trend at 48 h compared with the control (CK) group. However, apoptosis was similar in cells transfected with Ambra1 sh-RNAs and in the CK cells. Furthermore, we performed a microarray-based comparison of genes after Ambra1 knock-down. The 828 DEGs from microarray analysis were intersected with 4266 Ambra1 co-expressed genes from MEM. Eventually, the overlapped 183 genes were found to be enriched in several well-known cancer-related pathways, including the MAPK signaling pathway, chronic myeloid leukemia pathway, and VEGF signaling pathway. CONCLUSION: These results indicate that the level of Ambra1 up-regulation is clearly related to tumorigenesis and progression of BC, probably via influencing several vital pathways. However, this hypothesis needs to be validated with more in-depth experiments in the future.

Mammalian target of rapamycin signaling inhibition ameliorates vascular calcification via Klotho upregulation.

Vascular calcification (VC) is a major risk factor for cardiovascular mortality in chronic renal failure (CRF) patients, but the pathogenesis remains partially unknown and effective therapeutic targets should be urgently explored. Here we pursued the therapeutic role of rapamycin in CRF-related VC. Mammalian target of rapamycin (mTOR) signal was activated in the aortic wall of CRF rats. As expected, oral rapamycin administration significantly reduced VC by inhibiting mTOR in rats with CRF. Further in vitro results showed that activation of mTOR by both pharmacological agent and genetic method promoted, while inhibition of mTOR reduced, inorganic phosphate-induced vascular smooth muscle cell (VSMC) calcification and chondrogenic/osteogenic gene expression, which were independent of autophagy and apoptosis. Interestingly, the expression of Klotho, an antiaging gene that suppresses VC, was reduced in calcified vasculature, whereas rapamycin reversed membrane and secreted Klotho decline through mTOR inhibition. When mTOR signaling was enhanced by either mTOR overexpression or deletion of tuberous sclerosis 1, Klotho mRNA was further decreased in phosphate-treated VSMCs, suggesting a vital association between mTOR signaling and Klotho expression. More importantly, rapamycin failed to reduce VC in the absence of Klotho by using either siRNA knockdown of Klotho or Klotho knockout mice. Thus, Klotho has a critical role in mediating the observed decrease in calcification by rapamycin in vitro and in vivo.

Cucurbitacin B induces apoptosis in colorectal cells through reactive oxygen species generation and endoplasmic reticulum stress pathways.

Cucurbitacin B (CuB) is a member of the cucurbitacin family, which has shown potent anticancer pharmacological activity. Prolonged or severe endoplasmic reticulum stress (ERS) induces apoptosis; therefore, the present study investigated whether CuB may activate the ERS pathway to induce apoptosis. HT-29 and SW620 colorectal cancer (CRC) cells were treated with a range of concentrations of CuB for 48 h, and the viability and proliferation of cells were determined using Cell Counting Kit 8 (CCK8) and colony formation assays. Subsequently, the appropriate CuB concentration (5 µM) was selected for treatment of CRC cells for 48 h. Western blot analysis was used to measure the expression levels of ERS-related proteins, flow cytometry was used to evaluate apoptosis, the dichlorodihydrofluorescein diacetate fluorescent probe was used to detect reactive oxygen species (ROS) production, and the relationship between ROS and ERS was determined by western blot analysis. Furthermore, flow cytometry was used to evaluate apoptosis after treatment with the ERS inhibitor 4-phenylbutyric acid, the ROS inhibitor N-acetylcysteine and following knockdown of CHOP expression. In addition, western blot analysis was performed to measure Bax and Bcl2 protein expression levels, and a CCK8 assay was performed to evaluate the viability of cells following knockdown of CHOP. Notably, CuB treatment increased apoptosis and inhibited cell proliferation in CRC cell lines, and these effects were mediated by ROS and ROS-regulated activation of the PERK and XBP1 ERS pathways. In conclusion, CuB may induce apoptosis in HT-29 and SW620 CRC cells via ROS and ERS.

c-Jun N-terminal kinase mediates microtubule-depolymerizing agent-induced microtubule depolymerization and G2/M arrest in MCF-7 breast cancer cells.

Microtubule-binding agents (MBAs) form one of the most important anticancer-drug families, but their molecular mechanisms are poorly understood. MBAs such as paclitaxel (PTX) stabilize microtubules, whereas XRP44X (a novel pyrazole) and combretastatins A4 (CA4) destabilize microtubules. These two different types of MBAs have potent antitumor activity. Comparisons of their effects on signal transduction and cellular responses will help uncover the molecular mechanism by which MBAs affect tumor cells. We used MCF-7 cells to compare the effects of the three MBAs on the cytoskeleton, cell cycle distribution, and activation of the three major mitogen-activated protein kinase (MAPK) signaling cascades [extracellular signal-related kinases, c-Jun N-terminal kinase (JNK), and p38 MAPK] using pharmacological inhibitors. The G2/M phase arrest was induced following polymerization of microtubules by PTX and depolymerization by XRP44X and CA4. The three major MAPKs were rapidly activated by XRP44X, and extracellular signal-related kinases and p38 by PTX, whereas JNK did not quickly respond to PTX. Pharmacological inhibitors indicated that activation of JNK is principally required for XRP44X- and CA4-induced microtubule depolymerization and G2/M phase arrest. Our results suggest that early phosphorylation of JNK is a specific mechanism involved in microtubule depolymerization by certain MBAs.

Mixed neuroendocrine carcinoma of the gastric stump: A case report.

BACKGROUND: Gastric stump cancer, also known as gastric remnant cancer (GRC), is one of the main complications of postgastrectomy syndrome, which usually occurs following Billroth II reconstruction. The predominant histological subtype of GRC is adenocarcinoma, whereas neuroendocrine carcinoma is relatively rare. In particular, there are few recently reported cases of mixed neuroendocrine carcinoma (MNEC) in the English literature. Here, we present an extremely rare case of MNEC of the gastric stump. CASE SUMMARY: A 59-year-old patient presented to our department owing to chronic constipation. He had undergone subtotal gastric resection 35 years prior to admission because of benign peptic ulcer. After admission, the patient underwent several tests, and gastroendoscopy showed evidence of Billroth II gastrectomy and local thickening of the gastric stump mucosa at the gastrojejunostomy site, with bile reflux; pathological biopsy revealed adenocarcinoma. He was then diagnosed with GRC and underwent total gastrectomy, D2 Lymphadenectomy, and esophagojejunal Roux-en-Y reconstruction. Histopathological examination of the specimen identified MNEC comprising MNEC (60%), adenocarcinoma (30%), and squamous cell carcinoma (10%). Postoperative adjuvant chemotherapy was initiated on September 17, 2020. Taxol plus cisplatin was administered for only one cycle because of severe liver function damage, and the regimen was changed to etoposide plus cisplatin on October 10, 2020 for five cycles. The patient recovered, with no recurrence after 6 mo of follow-up. CONCLUSION: Gastric MNECs (GMNECs) is a rare type of GRC. This study presented the unusual occurrence of GMNEC in the gastric stump. This case will contribute to improvements in our understanding of the carcinogenesis, biology, pathology, and behavior of GMNEC and GRC.

[Establishment and identification of human lung adenocarcinoma cell line stably expressing the alpha1, 3-galaetosyltransferase gene from pig].

OBJECTIVES: To establish a human lung adenocarcinoma cell subline A549 that can stably express the Chinese Banna minipig inbred-line (BMI) alpha1 ,3-galactosyltransferase (alpha1 ,3GT) gene and alpha-galactosyl (Gala1-3Galb1-4GlcNAc-R, alpha-gal) epitopic, providing a cell model which expressed xenotransplantation antigens for the further research on the effect of complement dependent cytotoxic lysis of the tumor cells triggered by human natural serum. METHODS: The pEGFP-CMV-GT plasmid containing Banna minipig alpha1 ,3-GT gene was ransfected into A549 cells with lipofectin in vitro. After screened with G418,the single clones were got out and then amplified, the stable transfected cells was named A549-GT. The transcription of alpha1, 3-GT gene in A549-GT cells was detected by RT-PCR. Direct immunofluonrescence methods and flow cytometer were performed to observe the expression of alpha-gal and the binding conditions of IgM and complement C3 in human serum on A549-GT cells. The biological characters of A549-GT cells including morphology, proliferation, and tumorigenesis in nude mice were also examined. RESULTS: After G418 screening, A549-GT that stablely transfected with alpha1, 3-GT gene was obtained and has been passaged for 2 years. The expression of alpha1,3-GT mRNA and alpha-gal was detected continuously and stably in A549-GT. The expression rate of alpha-gal positive cells reached 80.1% +/- 3.2%. The binding of human serum IgM and C3 in human serum on A549-GT cells were founded. Compared with parental A549 cells, its biological characteristics did not change. CONCLUSION: A549-GT cell line stably and continuously expressing alpha1, 3-GT and alpha-gal was established successfully. It provided a useful cell model for the further study of pig alpha1,3-GT gene in tumor immunotherapy.

Evaluation of Local Injection of Bevacizumab against Triple-Negative Breast Cancer Xenograft Tumors.

BACKGROUND AND OBJECTIVE: Bevacizumab (BVZ) is a recombinant humanized antibody that inhibits the vascular endothelial growth factor A (VEGFA) and is used for the treatment of various types of cancer. BVZ is primarily given by the intravenous drip (I.V.), which often leads to low efficacy and various side effects. Therefore, the present study was to evaluate the effect of local delivery of BVZ against triple-negative breast cancer (TNBC) xenograft tumors. METHODS: Mice 4T1 TNBC cells were engrafted in female BALB/c mice. After the tumors reached about 5 mm (diameter), animals were treated with BVZ through the local injection from four directions around the tumors. The tumor growth, survival and potential mechanisms of action were evaluated. RESULTS: The growth and microvessel density of engrafted tumors were dramatically reduced with the tumor inhibition rate of 32.8 ± 3%. No obvious side effects were observed. The expression of VEGFA, VEGF receptor (VEGFR), matrix metalloproteinase (MMP)-2, MMP-9, Delta-like ligand 4 (DLL4) and Integrin-5 was significantly reduced in TNBC tumor tissues. In contrast, tissue inhibitor of matrix metalloproteinase (TIMP)-2 was significantly upregulated in xenograft tumors. Additionally, local delivery of BVZ led to the reduction of VEGFA and tumor necrosis factor (TNF)-alpha in the serum. Protein-protein interaction (PPI) analysis revealed that the proteins altered by the local delivery of BVZ were associated with angiogenesis and regulation of cell migration. CONCLUSION: This study provided evidence associated with local delivery of BVZ against TNBC tumors supporting the use of BVZ local injections to overcome some of the disadvantages associated with I.V. therapy with BVZ.

Ambra1 in autophagy and apoptosis: Implications for cell survival and chemotherapy resistance.

Increasing studies suggest that autophagy has a protective role in cancer treatment and may even be involved in chemotherapy resistance. Nevertheless, the mechanism of autophagy in cancer treatment and drug resistance has not yet been established. There is a complex association between autophagy and apoptosis. Accordingly, these two processes can mutually regulate and transform to determine the fate of a cell, depending on the context. Activating molecule in Beclin 1-regulated autophagy protein 1 (Ambra1) is an important factor at the crossroad between autophagy and apoptosis. The expression level and intracellular distributions of Ambra1 may control the balance and conversion between autophagy and apoptosis, and modify the effectiveness of chemotherapy. Therefore, Ambra1 may provide a novel target for cancer treatment, particularly for overcoming anticancer drug resistance. The present review focuses on the role of Ambra1 in autophagy and apoptosis and assesses the implications for cell survival and chemotherapy resistance.

Autophagy protects breast cancer cells from epirubicin-induced apoptosis and facilitates epirubicin-resistance development.

Epirubicin (EPI) is one of the most effective drugs against cancer. But the acquired resistance of cancer cells to EPI is becoming a major obstacle for successful cancer therapy. Recently, some studies have revealed that macroautophagy (here referred to as autophagy) may protect the cancer cell from anticancer drug-induced death, so autophagy might be related to the development of drug resistance to these reagents. However, the relationship between autophagy and drug resistance has yet to be defined. Our study showed that EPI induced autophagy in human breast cancer MCF-7 cells. And the EPI-induced autophagy protected MCF-7 cells from EPI-induced apoptosis. Furthermore, autophagy was elevated in EPI-resistant MCF-7 cells (MCF-7er cells), and inhibition of autophagy restored the sensitivity of MCF-7er cells to EPI. Therefore, autophagy is a prosurvival factor and has a role in the development of EPI-acquired resistance in EPI-treated MCF-7 cells. Also, this finding indicates that the use of clinically applicable autophagy inhibitors might be one of the important strategies for breast cancer therapy.