Expression of beclin 1 in primary salivary adenoid cystic carcinoma and its relation to Bcl-2 and p53 and prognosis

Beclin 1 plays a critical role in autophagy and functions as a haploinsufficient tumor suppressor. The expression and prognostic significance of beclin 1 in head and neck adenoid cystic carcinoma (ACC) are largely unexplored. Therefore, we investigated the expression of beclin 1, Bcl-2, and p53 in head and neck ACC tissue. Tissue samples from 35 cases (15 females, 20 males) of head and neck ACC were utilized for immunohistochemistry. Beclin 1 expression was observed in 32 cases (91.4%) and considered to be high in 15 cases (42.9%) and low in 20 cases (57.1%). Beclin 1 expression was significantly correlated with a histological growth pattern (P=0.046) and histological grade (P=0.037). Beclin 1 expression was inversely correlated with Bcl-2 expression (P=0.013) and significantly associated with overall survival (P=0.006). Bcl-2 and p53 expression were observed in 21 cases (60.0%) and 16 cases (45.7%). Bcl-2 expression was significantly correlated with perineural invasion (P=0.041) and not associated with overall survival (P=0.053). p53 expression was directly correlated with beclin 1 expression (P=0.044). Our results indicated that beclin 1 may be a novel, promising prognostic factor for clinical outcome in head and neck ACC patients and may play a part in the development of head and neck ACC by interacting with Bcl-2 and p53.     

Expression of Autophagy and Reactive Oxygen Species-Related Proteins in Lacrimal Gland Adenoid Cystic Carcinoma

PurposeTo investigate the difference of expression of autophagy and reactive oxygen species (ROS) related proteins in adenoid cystic carcinoma (ACC) of lacrimal gland in comparison with ACC of salivary gland.ResultsGSTpi in stromal cells was more highly expressed in lacrimal gland ACC (p=0.006), however, MnSOD in epithelial cells was expressed more in salivary gland ACC (p=0.046). LC3B positivity and BNIP3 positivity in epithelial component were associated with shorter disease-free survival (both p=0.002), and LC3A positivity in stromal component was the factor related to shorter OS (p=0.005).ConclusionThis is the first study to demonstrate the expression of autophagy and ROS related proteins in lacrimal gland ACC in comparison with the salivary gland ACC, which would provide a basis for further study of autophagy and ROS mechanism as novel therapeutic targets in lacrimal gland ACC.     

Expression and clinical significances of Beclin1, LC3 and mTOR in colorectal cancer

Autophagy is related to cancer and other diseases, and compromised autophagy could promote chromosome instability associated with carcinogenesis and tumor progression. The role of autophagy in the growth and metastasis of colorectal cancer (CRC) remains poorly understood. Beclin1 mediates autophagic initiation, and LC3 is a specific marker for autophagy. Inactivation of mTOR caused by cellular hypoxia or energy deficiency induces autophagic activity. This study aims to examine the expression and clinical significance of these proteins in CRC. Immunohistochemistry results showed that the positive expression rates of Beclin1, LC3, and mTOR in cancer tissues were 90.50%, 87.19%, and 46.28%, respectively, which were higher than those in adjacent tissues (P < 0.05). Differentiation degree and lymph node metastasis were associated with LC3 overexpression (P < 0.05) but not with Beclin1 (P > 0.05). Lymph node metastasis was also related to mTOR. Spearman analysis results showed that LC3 expression was positively correlated with Beclin1 but negatively correlated with mTOR (r = 0.593 and -0.165, respectively; P < 0.01). Beclin1 expression was also not associated with mTOR (P > 0.05). Survival analysis further indicated that LC3, mTOR, and lymph node metastasis were independent prognostic factors in CRC. Real-time PCR results and Western blot indicated that Beclin1, LC3, and mTOR expression in CRC was significantly higher than that in adjacent tissues (P < 0.01). The aberrant protein expression may be associated with the development and progression of CRC. The LC3 and mTOR genes must be simultaneously detected to evaluate progression and prognosis of CRC.     

Taurine induces upregulation of p53 and Beclin1 and has antitumor effect in human nasopharyngeal carcinoma cells in vitro and in vivo

Taurine is an amino acid that has several physiological functions. Previously, we reported the apoptosis-inducing effect of taurine in human nasopharyngeal carcinoma (NPC) cells in vitro. However, the effect of taurine on NPC cell growth in vivo has not been elucidated. Autophagy plays an important role in cell metabolism and exhibits antitumor effects under certain conditions. In this study, we investigated the effects of taurine on apoptosis- and autophagy-related molecules in NPC cells in vitro and in vivo. In our in vitro study, NPC cells (HK1-EBV) were treated with taurine, and Western blot and immunocytochemical analyses revealed that taurine co-upregulated Beclin 1 and p53, with autophagy upregulation. In the in vivo study, we used a nude mouse model with subcutaneous xenografts of HK1-EBV cells. Once the tumors reached 2–3 mm in diameter, the mice were provided with distilled water (control group) or taurine dissolved in distilled water (taurine-treated group) ad libitum (day 1) and sacrificed on day 13. The volume and weight of the tumors were significantly lower in the taurine-treated group. Using immunohistochemistry (IHC), we confirmed that taurine treatment reduced the distinct cancer nest areas. IHC analyses also revealed that taurine promoted apoptosis, as evidenced by an increase in cleaved caspase-3, accompanied by upregulation of p53. Additionally, taurine increased LC3B and Beclin 1 expression, which are typical autophagy markers. The present study demonstrated taurine-mediated tumor growth suppression. Therefore, taurine may be a novel preventive strategy for NPC.     

Expression of autophagy and ER stress-related proteins in primary salivary adenoid cystic carcinoma

Autophagy is the endogenous cellular pathway that facilitates cellular survival by maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation. Endoplasmic reticulum (ER) stress is the process in which disruption of these physiological functions leads to an accumulation of unfolded proteins and induces the unfolded protein response (UPR). ER stress and autophagy are involved in human cancer. We investigated the expression of autophagic proteins (LC3 and beclin 1) and ER stress-related protein (GRP78) in head and neck adenoid cystic carcinoma tissue. Tissue samples from 79 cases of head and neck adenoid cystic carcinoma tissue were utilized for immunohistochemistry. LC3 expression was significantly correlated with lymph node involvement (P = .016) and TNM (P = .021). Beclin 1 expression was significantly correlated with the histological growth pattern (P = .002), the histological grade (P = .000), and longer survival (P = .000). GRP78 expression was significantly correlated with the histological growth pattern (P = .019), the histological grade (P = .019), and longer survival (P = .001). LC3 expression was positively correlated with beclin 1 expression (P = .000); LC3 and beclin 1 expressions were positively correlated with GRP78 expression respectively (P = .035) (P = .008). Our study describes the expression of LC3, beclin 1, and GRP78 in adenoid cystic carcinoma and its relationship with clinicopathologic factors and overall survival. These results suggest that LC3, beclin 1, and GRP78 may play an important role in the tumorigenesis of adenoid cystic carcinoma, and that beclin 1 and GRP78 may serve as new prognostic indicators for the outcome of patients with adenoid cystic carcinoma.     

Inhibiting adenoid cystic carcinoma cells growth and metastasis by blocking the expression of ADAM 10 using RNA interference

Background  

Adenoid cystic carcinoma is one of the most common types of salivary gland cancers. The poor long-term prognosis for patients with adenoid cystic carcinoma is mainly due to local recurrence and distant metastasis. Disintegrin and metalloprotease 10 (ADAM 10) is a transmembrane protein associated with metastasis in a number of diverse of cancers. The aim of this study was to analyze the relationship between ADAM 10 and the invasive and metastatic potentials as well as the proliferation capability of adenoid cystic carcinoma cells in vitro and in vivo.     

Diagnostic role of DOG1 and p63 immunohistochemistry in salivary gland carcinomas

Background: The differential diagnosis of salivary carcinomas is always difficult and challenging. Salivary neoplasms often shows more than one growth pattern and significant morphologic variability may exist within a single tumor and between different tumors. The aim of this study was to examine the role of DOG1 (discovered on gastrointestinal tumor-1) and p63 immunohistochemistry in the diagnosis and differential diagnosis of salivary carcinomas. Methods: we examined the expression of DOG1 and p63 immunohistochemistry in 33 mucoepidermoid carcinomas (MEC), 9 acinic cell carcinomas (ACC), 10 adenoid cystic carcinomas (AdCC) and 4 myoepithelial carcinomas. Results: All ACC showed strong to moderate positivity for DOG1 (P=0.001) and all were totally negative for p63. All MEC expressed strong to moderate positivity for p63 (P=0.001) while only (9.1%) were weak to moderately positive for DOG1. (80%) AdCC were moderately positive for DOG1 in ductal and myoepithelial components and (100%) showed moderate positivity for p63 in myoepithelial cells only (P=0.001). All myoepithelial carcinomas were DOG1 negative, 2 (50%) were weakly positive for p63 while the other 2 were moderately positive (P=0.5). Conclusion: DOG1 is a sensitive marker in the diagnosis of acinic cell carcinoma, p63 is sensitive in the diagnosis of mucoepidermoid carcinoma, the combined use of both markers is helpful and statistically significant in the differential diagnosis of acinic cell carcinoma versus mucoepidermoid carcinoma, both markers can help in the diagnosis of adenoid cystic carcinoma but they have no role in the diagnosis of myoepithelial carcinoma.     

Cell-type-specific tumour sensitivity identified with a bromodomain targeting PROTAC in adenoid cystic carcinoma

Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy with limited treatment options. The development of novel therapies is hindered by a lack of preclinical models. We have generated ACC patient-derived xenograft (PDX) lines that retain the physical and genetic properties of the original tumours, including the presence of the common MYB::NFIB or MYBL1::NFIB translocations. We have developed the conditions for the generation of both 2D and 3D tumour organoid patient-derived ACC models that retain MYB expression and can be used for drug studies. Using these models, we show in vitro and in vivo sensitivity of ACC cells to the bromodomain degrader, dBET6. Molecular studies show a decrease in BRD4 and MYB protein levels and target gene expression with treatment. The most prominent effect of dBET6 on tumours in vivo was a change in the relative composition of ACC cell types expressing either myoepithelial or ductal markers. We show that dBET6 inhibits the progenitor function of ACC cells, particularly in the myoepithelial marker-expressing population, revealing a cell-type-specific sensitivity. These studies uncover a novel mechanistic effect of bromodomain inhibitors on tumours and highlight the need to impact both cell-type populations for more effective treatments in ACC patients. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.     

Potential role for inhibition of protein phosphatase 2A tumor suppressor in salivary gland malignancies

The aetiology and pathogenesis of salivary gland malignancies remain unknown. To reveal novel molecular factors behind the development of salivary gland cancer, we performed gene expression analyses from Smgb‐Tag mouse salivary gland samples. The overall purpose was to apply these results for clinical use to find new approaches for both possible therapeutic targets and more accurate diagnostic tools. Smgb‐Tag mouse strain, in which salivary neoplasms arise through a dysplastic phase in submandibular glands, was investigated using genome‐wide microarray expression analysis, ingenuity pathway analysis, RT‐PCR, and immunohistochemistry. Thirty‐eight human salivary gland adenoid cystic carcinoma samples were investigated using immunohistochemistry for validation purposes. Our genome‐wide study showed that Ppp2r1b, a PP2A subunit encoding tumor suppressor gene, is underexpressed in submandibular gland tumors of Smgb‐Tag mice. mTOR signaling pathway was significantly enriched and mTOR linked PP2A subunit gene B55 gamma was significantly underexpressed in the analyses. Furthermore, parallel immunohistochemical analysis of three PP2A inhibitors demonstrated that two PP2A inhibitors, CIP2A and SET, are highly expressed in both dysplastic and adenocarcinomatous tumors of the Smgb‐Tag mice. In addition, all 38 investigated human salivary adenoid cystic carcinoma samples stained positively for CIP2A and most for SET. Finally, p‐S6 staining showed activation of mTOR pathway in human adenoid cystic carcinoma samples. Our results suggest that PP2A inhibition either via PP2A subunit underexpression or PP2A inhibitor overexpression play an important role in the formation of salivary gland malignancy, potentially due to mTOR signaling activation. © 2015 Wiley Periodicals, Inc.     

Cytologic diagnosis of adenoid cystic carcinoma of salivary glands.

The cytomorphologic features in fine-needle aspiration (FNA) biopsies from 31 primary and 33 recurrent adenoid cystic carcinomas (ACC) were investigated. The correct FNA diagnosis was established in 24 of 31 primary ACC (77%). The diagnostic clue in aspirates from ACC are large globules of extracellular matrix, partially surrounded by basaloid tumor cells. In FNAs with predominance of basaloid tumor cells, but lacking characteristic globules, all other benign and malignant salivary gland tumors of epithelial-myoepithelial differentiation should be considered in the cytologic diagnosis. Pleomorphic adenoma is most frequently confused with ACC, and therefore, the cytologic findings in FNAs from 50 pleomorphic adenomas were compared with those diagnosed as ACC. Furthermore, rare neoplasms of salivary glands with epithelial-myoepithelial cell differentiation, including basal-cell adenoma and carcinoma, epithelial-myoepithelial carcinoma, and polymorphous low-grade adenocarcinoma, as well as some nonsalivary gland neoplasms presenting an adenoid cystic pattern, must be considered. The cytologic features of these entities are discussed in detail with respect to the cytologic diagnostic criteria of ACC.     

Clinicopathologic significance of CXCR4 expressions in patients with esophageal squamous cell carcinoma

AimsThis study was designed to investigate the biological function of CXCR4 in esophageal squamous cell carcinoma and to explore the underlying mechanism to provide potential targets for esophageal squamous cell carcinoma.MethodsA total of 101 patients with esophageal squamous cell carcinoma were included, and the relationship between CXCR4 and clinicopathological factors was analyzed. Laser scanning confocal microscopy was used to observe numbers of autophagosomes in TE-1 cell line and the ability of proliferation and invasion were evaluated meanwhile.ResultsCXCR4 is overexpressed in ESCC specimens and is associated with poor differentiation and lymphocyte metastasis. In the survival analysis, CXCR4 predicted a poor overall survival prognosis. The number of autophagosomes in the siR-CXCR4 group was decreased compared with negative group (P < 0.05), while was increased in the pcDNA3.1-CXCR4 group (P < 0.05).Western blot result show upregulation of LC3II, the ratio of LC3II/LC3I and Beclin1 in pcDNA3.1-CXCR4 group and decreased expression of LC3II, the ratio of LC3II/LC3I and Beclin1 in siR-CXCR4 group. Transwell assay show CXCR4 overexpression promote the invasion of TE-1 cells and was attenuated by autophagy inhibitor 3-Methyladenine.On the contrary, invasion cell numbers decreased in siR-CXCR4 group and was rescued by autophagy inducer Rapamycin.ConclusionCXCR4 is an indicator of poor prognosis for ESCC. CXCR4 promote autophagy and regulate cell invasion through autophagy in ESCC. Our study provides new insights for the treatment of esophageal squamous cell carcinoma and CXCR4 may serve as a therapeutic target for ESCC.     

Additive cytotoxic effects of radiation and mTOR inhibitors in a cervical cancer cell line

The PI3 K/AKT/mTOR signaling pathway is frequently activated in HPV-positive cervical squamous cell cancer (CC). This study investigated the biological effects of mTOR inhibitors associated with radiotherapy in a CC cell line (HeLa). A human keratinocyte cell line (HaCaT) was used as control. Temsirolimus, everolimus, resveratrol, curcumin and epigallocatechin gallate (EGCG) were the mTOR inhibitors assessed. The 50% cell cytotoxicity rate (CC₅₀) for each treatment was determined by MTT cell viability assay. Cells were pre-treated with mTOR inhibitors at CC₅₀ followed by radiotherapy (RT) at 2 Gy. Cell death profile after treatment with temsirolimus, resveratrol and curcumin was assessed with flow cytometry. Everolimus, temsirolimus, EGCG, resveratrol and curcumin were cytotoxic to HeLa. Radiation induced a statistically significant (p < 0.01) supra-additive cytotoxic effect in the cervical cancer cell line when combined with mTOR inhibitors. After a 24-h treatment, EGCG and resveratrol were more cytotoxic to HeLa cells than to HaCaT cells. After 48 h of treatment, resveratrol, curcumin and everolimus were more cytotoxic to HeLa cells when compared to HaCaT cells. After 24 h, temsirolimus induced late apoptosis or necrosis in HeLa cells. Based on these data, new studies with mTOR inhibitors as treatment options for cervical cancer are recommended, mainly combined to radiotherapy.     

Observations by chromosome banding, FISH and immunohistochemistry in an adenoid cystic carcinoma with del(17)(p13) as the sole deviation

We report on an adenoid cystic carcinoma (ACC) with a clonal deletion of 17p as the only karyotypic abnormality. Using different chromosome 17-derived probes we showed by FISH that the deletion encompassed thep53 tumour suppressor gene. Immunohistochemical analysis revealed overexpression ofp53 protein in a subpopulation of cells, suggesting a mutation in the remainingp53 allele in these cells. Our findings provide novel information about possible progressional pathways in ACC, and demonstrate the value of combining conventional cytogenetic analysis with molecular cytogenetic and immunohistochemical methods. This approach is particularly useful in cases with minor cytogenetic abnormalities at the border of visibility.     

Basal cell adenoma: a diagnostic dilemma on fine needle aspiration cytology

Basal cell adenoma (BCA) is a rare neoplasm which is one of the basaloid tumors of salivary gland. Basaloid tumors are the most difficult problem in salivary gland fine needle aspiration cytology (FNAC). There are various benign and malignant tumors such as; cellular pleomorphic adenoma, basal cell adenocarcinoma, adenoid cystic carcinoma, metastatic basal cell carcinoma, metastatic basaloid squamous carcinoma and small cell carcinoma in differential diagnosis. We present a case of BCA, membranous type in a 39-year-old female with right submandibular swelling misinterpreted as adenoid cystic carcinoma (ACC) on FNAC.     

Expression of KIT (CD117) in neoplasms of the head and neck: an ancillary marker for adenoid cystic carcinoma.

Adenoid cystic carcinoma is an indolent salivary gland malignancy that is associated with a poor long-term prognosis. The distinction of adenoid cystic carcinoma from other head and neck neoplasms can occasionally be problematic, particularly in small biopsies. Recent studies suggest that KIT (CD117) might be useful as an ancillary marker for adenoid cystic carcinoma; however, the expression of KIT in other benign and malignant head and neck neoplasms, including those that might mimic adenoid cystic carcinoma, has not been well studied. Here we use two different antibodies against KIT to evaluate its expression in a series of 66 adenoid cystic carcinomas compared with its expression in 98 other neoplasms of the head and neck. Overall, 94% (n = 62) of adenoid cystic carcinomas from various anatomic sites and of various histologic subtypes were positive for at least one of the KIT antibodies, and 77% (n = 50) of adenoid cystic carcinoma cases were positive for both antibodies. This contrasted with only 8% (n = 8) of other head and neck neoplasms that were positive for both KIT antibodies (P <.001). It was of note that certain neoplasms, including pleomorphic adenoma, basal cell adenoma, polymorphous low-grade adenocarcinoma, and basal cell carcinoma, that can show histologic overlap with adenoid cystic carcinoma had significantly less KIT immunoreactivity than did adenoid cystic carcinoma (P <.001). In contrast, KIT expression did not reliably distinguish adenoid cystic carcinoma from basal cell adenocarcinoma and basaloid squamous carcinoma (P >.05). The overall sensitivity of the two KIT antibodies for adenoid cystic carcinoma was 82-89%, and the specificity was 87-88%. The findings in this study support the potential use of KIT immunoexpression for distinguishing adenoid cystic carcinoma from many other benign and malignant head and neck neoplasms.     

NVP-BEZ235诱导多囊肾大鼠胆管上皮细胞自噬的机制研究

 目的:探讨PI3K/Akt/mTOR双靶点抑制剂NVP-BEZ235诱导多囊肾(polycystic kidney,PCK)大鼠胆管上皮细胞自噬的作用。方法:免疫组化法检测p-mTOR和p-Akt在PCK大鼠胆管上皮细胞中的水平。WST-1比色法检测NVP-BEZ235对胆管细胞活力的抑制作用以及LC3、Beclin 1基因沉默和自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)对细胞活力的影响。蛋白免疫印迹法检测NVP-BEZ235对PI3K/Akt/mTOR信号通路相关蛋白及自噬标志蛋白LC3和Beclin 1的变化。结果:p-mTOR和p-Akt在PCK大鼠胆管上皮细胞中显著升高,NVP-BEZ235可明显抑制胆管上皮细胞的活力,且呈浓度和时间依赖性改变(P<0.05);NVP-BEZ235明显抑制PI3K/Akt/mTOR信号通路相关蛋白的水平;并上调自噬标志蛋白LC3 II/LC3 I比值和Beclin 1蛋白的表达水平。LC3、Beclin 1基因沉默和3-MA均可明显减弱NVP-BEZ235对细胞活力的抑制作用(P<0.01)。结论:NVP-BEZ235可抑制PCK大鼠胆管上皮细胞的活力,其机制与自噬密切相关。     

Primary adenoid cystic carcinoma of the esophagus

Immunohistochemical and electron microscopic findings of a primary adenoid cystic carcinoma of the esophagus from a 71-year-old male patient are described. An oval tumor in the middle intrathoracic esophagus was resected. The tumor appeared similar to a submucosal tumor and was histologically diagnosed as primary adenoid cystic carcinoma (ACC) of the esophagus. The patient has survived for 10 years postoperatively with no evidence of recurrence. Histologically, the tumor showed a cribriform, tubular or solid pattern. A small focus of squamous cell carcinoma (SCC) was found independently. Immunohistochemical results for keratin groups, S-100 protein and smooth muscle actin (αSMA) revealed similar profiles in the ACC tumor tissues and small ducts of the normal esphageal glands. By electron microscopy, tumor cells were found markedly similar to the small duct cells of the normal esophageal gland. These findings. indicate that ACC may originate from the esophageal glands, especially from cells of the small duct including intercalated duct.     

Cedrus atlantica Extract Suppress Glioblastoma Growth through Promotion of Genotoxicity and Apoptosis: In Vitro and In Vivo Studies

Glioblastoma (GBM) is the most common malignant primary brain tumor in humans, exhibiting highly infiltrative growth and drug resistance to conventional chemotherapy. Cedrus atlantica (CAt) extract has been shown to decrease postoperative pain and inhibit the growth of K562 leukemia cells. The aim of this study was to assess the anti-GBM activity and molecular mechanism of CAt extract in vitro and in vivo. The results showed that CAt extract greatly suppressed GBM cells both in vitro and in vivo and enhanced the survival rate in subcutaneous and orthotopic animal models. Moreover, CAt extract increased the level of ROS and induced DNA damage, resulting in cell cycle arrest at the G₀/G₁ phase and cell apoptosis. Western blotting results indicated that CAt extract regulates p53/p21 and CDK4/cyclin D1 protein expression and activates extrinsic and intrinsic apoptosis. Furthermore, CAt extract enhanced the cytotoxicity of Temozolomide and decreased AKT/mTOR signaling by combination treatment. In toxicity assays, CAt extract exhibited low cytotoxicity toward normal cells or organs in vitro and in vivo. CAt extract suppresses the growth of GBM by induction of genotoxicity and activation of apoptosis. The results of this study suggest that CAt extract can be developed as a therapeutic agent or adjuvant for GBM treatment in the future.