Loss Of Beclin Research Articles

Abstract 349: Beclin1 as a Critical Regulator of the Endosomal Degradation Pathway

Effective cellular quality control is crucial for cellular homeostasis and cardiac health. Defects in the pathways that regulate the removal of damaged proteins and organelles contribute to heart failure. Autophagy is well known for its role in removing protein aggregates and organelles; however, we recently found that the small GTPase Rab5 and early endosomes also participate in Parkin-mediated mitochondrial clearance. Beclin1 is a scaffolding protein which can form distinct PI3K protein complexes to regulate different processes such as autophagy. Here, we have investigated the role of Beclin1 in regulating the endosomal degradation pathway in mice. We have generated inducible cardiac-specific Beclin1 KO mice to investigate the in vivo role of Beclin1 in myocytes. We found significant contractile dysfunction as early as one week after deletion of Beclin1, which led to increased mortality. Loss of Beclin1 led to rapid development of cardiac hypertrophy as characterized by increased Myh7, Nppa, Nppb , myocyte size, and heart weight/body weight. Furthermore, we found that loss of Beclin1 led to increased inflammatory markers Il6 and Tgfb1 , as well as increased DNA damage/apoptosis in the heart. Ultrastructural analysis revealed Beclin1 deficiency led to significant myocardial disarray, demonstrating the importance of Beclin1 for proper myocyte alignment. These mice also had reduced autophagic flux but increased IGFR-beta receptor protein levels, indicating defects in both the autophagy and endosomal degradation pathways. Additionally, immunofluorescence analysis revealed that Beclin1 colocalizes with Rab5 at the early endosome in cells, and coimmunoprecipitation experiments confirmed their interaction. Together, these data establish the essential role of Beclin1 in cellular homeostasis as a central regulator of endosomal activity.

Nuclear Localisation of Autophagic p62 and Associated Cytoplasmic Beclin-1 and Bcl-2 Expressions in Adenomas and Adenocarcinomas of the Colorectal Regions

Background: Autophagy is an important biological process that is involved in cellular homeostasis and survival. Derailment of some cellular autophagic processes affects normal cellular function, resulting in cancers and other disorders. Autophagy related proteins are Beclin-1, a human tumour suppressor, Bcl-2 and p62 have been characterised in most cancers. Particularly, a number of studies have reported a loss of Beclin-1 and up regulation of Bcl-2 and p62 in breast cancers. However, studies regarding the expression of these proteins in colorectal adenomaadenocarcinoma transformation sequence are yet to be described. In this study, we examined the expression patterns of Beclin-1, Bcl-2 and p62 in both colorectal adenomas and adenocarcinomas. Methods: Immunohistochemistry was performed on formalin-fixed paraffin embedded tissue sections from 14 patients with colorectal tumours and the expression patterns were semi-quantitatively evaluated based on the intensity of staining and the percentage of tumour cells stained. Results: Cytoplasmic Beclin-1 and p62 expression patterns ranged from moderate to high in both tubular adenomas and adenocarcinomas as compared to normal colonic mucosa. Cytoplasmic Bcl-2 expression was moderately expressed in tubular adenomas but negative to low expression was observed in the adenocarcinomas. This study also provided, for the first time, nuclear localization of p62 in only the colorectal adenocarcinomas. Conclusion: Beclin-1, Bcl-2 and p62 may be up regulated in the transition of colorectal adenomas to adenocarcinomas.

Loss of beclin 1 expression in ovarian cancer: A potential biomarker for predicting unfavorable outcomes.

The clinicopathological significance and prognostic value of the expression of proteins associated with autophagy, beclin 1 (BECN1), 1A/1B-light chain 3 (LC3) and high mobility group box-1 protein (HMGB-1), were investigated in patients with ovarian carcinoma, receiving combination chemotherapy with a platinum agent and a taxane. Immunohistochemical staining was performed for autophagy-associated proteins in tumor tissues from 141 patients with ovarian carcinoma. Clinical data were collected retrospectively by reviewing medical charts, and the association between protein expression, clinicopathological features and survival was investigated. Amongst 141 ovarian carcinoma samples, the loss of BECN1, LC3, and HMGB-1 expression was identified in 59 (41.8%), 35 (24.8%), and 66 (46.8%) samples, respectively. Clinicopathological factors were not significantly associated with the loss of BECN1 expression. However, significant associations were demonstrated between the expression of BECN1, LC3, and HMGB-1. In addition, loss of BECN1 expression demonstrated a significant association with poor progression-free and poor overall survival. Multivariate analysis demonstrated that loss of BECN1 expression and postoperative residual tumor were significant independent predictors of poor progression-free survival and poor overall survival. These results indicated that loss of BECN1 expression in ovarian carcinoma is a negative prognosticator in patients receiving platinum-based chemotherapy. Assessment of BECN1 expression may be useful for predicting an unfavorable response to platinum-based chemotherapy in ovarian carcinoma.

Abstract 2491: Beclin1 promotes DNA double-strand breaks repair

Abstract Beclin1 is a well-established core mammalian autophagy protein that has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Since its discovery, studies have also shown that Beclin1 interacts with an increasing number of cofactors. However, the tumor suppressive roles of Beclin1 remain a major research focus, and the exact mechanism by which Beclin1 acts as a tumor suppressor has remained largely obscure. Our confocal analysis showed that in embryonic and neonatal mice, Beclin1 is primarily located in the cytoplasm in hepatocytes, with a very small portion located in the nucleus. When the mice were 15d, roughly half of the total Beclin1 was located in the nucleus. Thereafter, Beclin1 progressively redistributed from cytoplasma to nucleus within a few weeks after birth. This pattern, was similarly sustained not only in hepatocytes but also in other tissues. And we found both 0-50 and 254-278 residues are required for Beclin1 nuclear localization. Furthermore, loss of Beclin1 resulted in reduced DNA damage repair. We found that the beclin1-/- cells showed a marked increased γ-H2AX foci that are considered as a biomarker of DNA DSBs. Moreover, beclin1-/- cells showed approximately about 60-fold and 30-fold reductions in DSB repair by NHEJ and HR pathways. A series of key DSBs repair proteins were significantly reduced in beclin1-/- cells. Interestingly, Beclin1 regulates DSB repair independent of autophagy. Autophagic flux was maintained in the beclin1-/- cells. Perhaps an alternative autophagy may be activated when beclin1 is deleted as ULK1 was upregulated. We further found that Beclin1 promotes DSB repair via a direct interaction with DNA topoisomerase IIβ (Top2b). Pull down results showed that Top2b is one of the major Beclin1-interacting proteins in the nucleus. And we found that this interaction was intensified after exposure to IR. Top2b and Beclin 1 were found to be colocalized to the DSB sites, after exposure to IR. Strikingly, depletion of Top2b by gene silencing completely prohibited Beclin1 localized to the DNA break sites. We constructed a beclin1-/- cell line using CRISPR/Cas9 system. The spot counts of protein foci were measured by imaging flow cytometry after exposure to 6Gy IR. HR and NHEJ reporter assays were used to detect the activity of DSBs repair pathways. We pulled down nuclear proteins with ectopically expressed Flag-tagged Beclin1. The identities of the Beclin1-interacting proteins were determined using mass spectrometry. Our work establishes that Beclin1 plays an autophagy-independent role in maintaining genomic integrity by promoting DNA damage repair. The finding challenge the long-standing paradigm of the mechanisms that are involved in the autophagy-mediated tumor suppressive role of Beclin1. This finding suggests that when manipulating cytoplasmic Beclin1 that is involved in the autophagy pathway in order to explore targeted tumor therapies, the non-autophagy cytoprotective role of Beclin 1 in the nucleus should also be considered. Citation Format: Fei Xu, Lili Yan, Jianrong Wang, Jinming Yang. Beclin1 promotes DNA double-strand breaks repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2491. doi:10.1158/1538-7445.AM2017-2491

Low expression of MAP1LC3B, associated with low Beclin-1, predicts lymph node metastasis and poor prognosis of gastric cancer.

Since the roles of autophagy in gastric cancer remain unclear, we aim to investigate the expression of autophagy-related proteins MAP1LC3B and Beclin-1 in human gastric cancer and discuss their clinical significance and correlation with prognosis of patients with gastric cancer. A total of 160 consecutive patients with gastric cancer who had undergone gastrectomy were enrolled in this study. The expressions of MAP1LC3B and Beclin-1 were assessed by immunohistochemistry. The protein expression rates were analyzed with χ 2 and Fisher's exact tests. Survival analysis (overall survival (OS) and relapse-free survival (RFS)) was determined using the Kaplan-Meier method and Cox's proportional hazard regression model. Both the expressions of MAP1LC3B and Beclin-1 were lower in gastric cancer tissues than adjacent normal tissues (57 vs. 82%, p=0.007; 72 vs. 88%, p=0.046, respectively). Relativity analysis indicated MAP1LC3B expression was positively correlated with Beclin-1 expression (r=0.424, p<0.001). Both the MAP1LC3B-high-expression patients and Beclin-1-high-expression patients have longer OS time and RFS time than MAP1LC3B-low-expression patients and Beclin-1-low-expression patients (MAP1LC3B: both p<0.001; Beclin-1: p=0.014, p=0.015, respectively). High simultaneous MAP1LC3B and Beclin-1 expressions were associated with longer OS and RFS compared with low simultaneous MAP1LC3B and Beclin-1 expressions (56.77 vs. 24.42months, p<0.001; 53.56 vs. 22.33months, p<0.001, respectively). Multivariate survival analysis showed both MAP1LC3B and Beclin-1 were independent prognostic factors for OS time (p=0.016, p=0.041, respectively). However, MAP1LC3B (p=0.022) was an independent prognostic factor for RFS. Moreover, low expressions of MAP1LC3B and Beclin-1 were significantly associated with lymph node metastasis (p=0.007, p=0.030, respectively). The loss of MAP1LC3B, correlated with loss of Beclin-1, was observed in gastric cancer and correlated with poor prognosis and lymph node metastasis of gastric cancer patients.

Expression, modulation, and clinical correlates of the autophagy protein Beclin-1 in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is characterized by rapidly increasing incidence and mortality rates and poor survival. Efficacious preventive and treatment options are urgently needed. An increasing number of pharmacologic agents targeting cancer cell death via autophagy mechanisms are being evaluated in hopes of circumventing apoptotic and therapeutic resistance. We report for the first time, loss of Beclin-1, a key mediator of autophagy, was significantly linked to prognostic factors in EAC. Specifically, Beclin-1 expression loss occurred in 49.0% of EAC patients versus 4.8% of controls. There was a significant inverse correlation between loss of Beclin-1 with histologic grade and tumor stage supporting a tumor suppressive role for Beclin-1. Autophagy modulation linked to cell death was examined in EAC cell lines following treatment with a proanthocyanidin-rich cranberry extract, C-PAC, and the commonly used autophagy inducer, rapamycin. C-PAC induced Beclin-1-independent autophagy in EAC cells characterized by reduced phosphorylation at serine 15 and 93, and significant cell death induction. In contrast, rapamycin-induced autophagy resulted in concomitant, increases in total Beclin-1 levels as well as Beclin-1-phosphorylation in a cell line specific manner, leading to long-term cell survival. Furthermore, autophagic LC3-II was induced by C-PAC following siRNA suppression of Beclin-1 in EAC cells. Together these data support a prognostic role of Beclin-1 in EAC with evidence that Beclin-dependent autophagy induction is agent specific. Future studies are necessary to fully interrogate the role autophagy plays in the progression of normal tissue to EAC and how specific agents targeting autophagic mechanisms can be efficaciously applied for cancer prevention or treatment. © 2015 Wiley Periodicals, Inc.

Loss of autophagy-related protein Beclin 1 may define poor prognosis in ovarian clear cell carcinomas.

The aim of the present study was to clarify the role of autophagy in cisplatin (CDDP) sensitivity in OCCCs and the role of Beclin 1 in OCCC progression. Autophagy was measured using: i) western blot analysis of LC3 and p62 and ii) microscopic observation of GFP-LC3 puncta. Autophagy was suppressed using chloroquine and Beclin 1 siRNA. Surgical specimens were examined for Beclin 1 protein expression by immunohistochemistry. The correlations between the loss of Beclin 1 expression and clinicopathological characteristics, prognosis and chemosensitivity were investigated. Inhibition of autophagy by chloroquine or Beclin 1 siRNA did not enhance the sensitivity of the ES2 and TOV-21G OCCC cell lines to CDDP. Loss of Beclin 1 expression was observed in 38.3% (23/60) of the analyzed tumors. There was no significant correlation between loss of Beclin 1 expression and FIGO stage, CA125 levels, patient age, status of endometriosis, Ki-67 labeling index, chemotherapy regimen or status of residual tumor. However, negative expression of Beclin 1 was associated with a shorter progression-free survival in comparison to positive Beclin 1 expression in OCCC who received cytoreductive surgery, followed by a standard platinum-based chemotherapy regimen (P=0.027, log-rank test). Beclin 1-negative tumors were no more resistant to primary adjuvant chemotherapy than were Beclin 1-positive tumors (50.0 vs. 66.7%, P=0.937). Beclin 1 knockdown using siRNA increased cell growth but not cell migration and invasion in ES2 and TOV-21G OCCC cell lines. Autophagy defects caused by loss of Beclin 1 are not related to chemoresistance and metastasis, but may be associated with malignant phenotype and poor prognosis of OCCC.

8-p-Hdroxybenzoyl Tovarol Induces Paraptosis Like Cell Death and Protective Autophagy in Human Cervical Cancer HeLa Cells

8-p-Hdroxybenzoyl tovarol (TAW) is a germacrane-type sesquiterpenoid that can be isolated from the roots of Ferula dissecta (Ledeb.) Ledeb. In this study, the growth inhibitory effects induced by TAW were screened on some types of tumor cells, and the mechanism was investigated on TAW-induced growth inhibition, including paraptosis and autophagy in human cervical cancer HeLa cells. TAW-induced paraptosis involved extensive cytoplasmic vacuolization in the absence of caspase activation. Additionally, TAW evoked cell paraptotic death mediated by endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Autophagy induced by TAW was found to antagonize paraptosis in HeLa cells. This effect was enhanced by rapamycin and suppressed by the autophagy inhibitor, 3-methyladenine (3MA). Loss of beclin 1 (an autophagic regulator) function led to promote ER stress. Taken together, these results suggest that TAW induces paraptosis like cell death and protective autophagy in HeLa cells, which would provide a new clue for exploiting TAW as a promising agent for the treatment of cervical cancer.

Expression of beclin-1 in the microenvironment of invasive ductal carcinoma of the breast: correlation with prognosis and the cancer-stromal interaction.

We examined the pathobiological properties of beclin-1, which is a key regulator of autophagosome formation in invasive ductal carcinoma of the breast, with a particular focus on the cancer microenvironment. Immunohistochemistry demonstrated that cancer cells and stromal mesenchymal cells expressed beclin-1 in 68 and 38 of 115 invasive ductal cancers, respectively. Expression of beclin-1 in cancer or stromal cells alone did not correlate with patient prognosis. In contrast, loss of beclin-1 in cancer cells and overexpression in stromal mesenchymal cells was associated with local cancer recurrence, postoperative lymph node metastasis, and a poor disease-free survival rate. A comprehensive gene expression analysis was performed on a co-culture of breast cancer cells and mesenchymal stromal cells, that latter of which either expressed beclin-1 or was depleted of beclin-1 by siRNA. Notably, siRNA-mediated downregulation of beclin-1 in mesenchymal cells co-cultured with breast cancer cells decreased the levels of various pro-inflammatory cytokines, their receptors, and collagen receptors. Quantitative reverse transcription polymerase chain reaction analysis confirmed that reduction of stromal beclin-1 expression decreased the expression of IL-1β and collagen receptor discoidin domain receptor 2 (DDR2). Microenvironmental IL-1β is believed to play an important role in tumor invasion. Recent work has also indicated that overexpression of DDR2 contributes to breast cancer invasion and lymph node metastasis. Taken together, these findings indicate beclin-1 expression in the stroma might be important for shaping the breast cancer microenvironment and thus could be a potent molecular target in patients with invasive ductal carcinoma of the breast.

Beclin 1 regulates growth factor receptor signaling in breast cancer.

Beclin 1 is a haplo-insufficient tumor suppressor that is decreased in many human tumors. The function of Beclin 1 in cancer has been attributed primarily to its role in the degradative process of macroautophagy. However, Beclin 1 is a core component of the Vps34/Class III PI3K (PI3KC3) and Vps15/p150 complex that regulates multiple membrane trafficking events. In the current study, we describe an alternative mechanism of action for Beclin 1 in breast cancer involving its control of growth factor receptor signaling. We identify a specific stage of early endosome maturation that is regulated by Beclin 1, the transition of APPL1-containing phosphatidyIinositol 3-phosphate-negative (PI3P-) endosomes to PI3P+ endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P-/APPL+ signaling competent compartment. As a result, suppression of BECN1 sustains growth factor stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, Beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Our data identify a novel role for Beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of Beclin 1 expression would enhance breast cancer progression.

Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis.

Physalin A Induces Apoptotic Cell Death and Protective Autophagy in HT1080 Human Fibrosarcoma Cells

Physalin A (1) is a withanolide isolated from Physalis alkekengi var. franchetii. In this study, the selective growth inhibitory effects on tumor cells induced by 1 were screened, and the mechanism was investigated on 1-induced growth inhibition, including apoptosis and autophagy, in human fibrosarcoma HT1080 cells. Apoptosis induced by 1 in HT1080 cells was associated with up-regulation of caspase-3 and caspase-8 expression. However, there were no significant changes in caspase-9, Bid, Bax, and Bcl-2 expression, indicating that 1-induced apoptosis in HT1080 cells occurs mainly through activation of the death receptor-associated extrinsic apoptotic pathways. Autophagy induced by 1 was found to antagonize apoptosis in HT1080 cells. This effect was enhanced by rapamycin and suppressed by the autophagy inhibitor 3-methyladenine (3MA). Loss of beclin 1 (as an autophagic regulator) function led to similar results to 3MA. However, 1 did not show inhibitory effects on normal human cells (human peripheral blood mononuclear cells). Taken together, these results suggest that 1 may be a promising agent for the treatment of cancer.

Expression of autophagy-related protein beclin-1 in malignant canine mammary tumors

BackgroundAutophagy is a self-catabolic mechanism that degrades unnecessary cellular components through lysosomal enzymes. Beclin-1, an autophagy-related protein, establishes the first connection between autophagy and tumorigenesis. The purpose of this study is to assess the Beclin-1 expression pattern and to determine its prognostic significance in patients with malignant canine mammary tumor (CMT).ResultsWe examined Beclin-1 expression in 70 cases of malignant CMTs by immunohistochemistry. Cytoplasmic Beclin-1 expression was significantly weaker in cancer cells than in nearby normal mammary glands (p < 0.001). Low cytoplasmic expression (57.14%) was associated with older age, lower degree of tubular formation, increased mitotic activity, higher histologic grade, and extensive necrosis. Low nuclear expression (40%) was connected with older age, lower degree of tubular formation, extensive necrosis, and negative for Her2/neu overexpression. Univariate survival analysis showed that Beclin-1 cytoplasmic expression was a poor prognostic factor for overall survival rate (p < 0.001). Multivariate survival analysis demonstrated that Beclin-1 cytoplasmic expression is an independent prognostic factor (p = 0.016).ConclusionsLoss of Beclin-1 is associated with aggressive clinicopathologic features and poor overall survival. The results suggest that Beclin-1 plays an important role in tumor progression of malignant CMTs.

Beclin 1 is Involved in Regulation of Apoptosis and Autophagy During Replication of Ectromelia Virus in Permissive L929 Cells

Several reports have brought to light new and interesting findings on the involvement of autophagy and apoptosis in pathogenesis of viral and bacterial diseases, as well as presentation of foreign antigens. Our model studies focused on the involvement of apoptosis during replication of highly virulent Moscow strain of ectromelia virus (ECTV-MOS). Here, we show evidence that autophagy is induced during mousepox replication in a cell line. Fluorescence microscopy revealed increase of LC3 (microtubule-associated protein 1 light chain 3) aggregation in infected as opposed to non-infected control L929 cells. Furthermore, Western blot analysis showed that replication of ECTV-MOS in L929 cells led to the increase in LC3-II (marker of autophagic activity) expression. Beclin 1 strongly colocalized with extranuclear viral replication centers in infected cells, whereas expression of Bcl-2 decreased in those centers as shown by fluorescence microscopy. Loss of Beclin 1-Bcl-2 interaction may lead to autophagy in virus-infected L929 cells. To assess if Beclin 1 has a role in regulation of apoptosis during ECTV-MOS infection, we used small interfering RNA directed against beclin 1 following infection. Early and late apoptotic cells were analyzed by flow cytometry after AnnexinV and propidium iodide staining. Silencing of beclin 1 resulted in decreased percentage of early and late apoptotic cells in the late stage of ECTV-MOS infection in L929 cells. We conclude that Beclin 1 plays an important role in regulation of both, autophagy and apoptosis, during ECTV-MOS replication in L929 permissive cells.

Beclin 1 over- and underexpression in colorectal cancer: distinct patterns relate to prognosis and tumour hypoxia

Introduction:Autophagy enables cells to recycle long-lived proteins or damaged organelles. Beclin 1, the mammalian orthologue of the yeast Apg6/Vps30 gene, functions as a scaffold for the formation of autophagosomes.Materials and method:The immunohistochemical patterns of Beclin 1 expression and their prognostic relevance were studied in formalin-fixed tissues from 155 patients with colorectal adenocarcinoma treated with surgery alone.Results:Using the weak homogeneous expression of Beclin 1 in normal colonic tissues as a basis for assessing tumours, the following grouping/staining patterns were recognised in colorectal carcinomas: a normal-like pattern in 62 of 155 (40%) cases, an underexpression pattern in 24 of 155 (15.5%) cases, extensive overexpression of Beclin 1 in 33 of 155 (21.3%) tumours and limited overexpression of the protein in 36 of 155 (23.2%) tumours. Extensive overexpression of Beclin 1 was significantly linked with overexpression of HIF1α and LDH5, as well as with high histological grade, vascular invasion and nodal involvement. Furthermore, patients with extensive over- or underexpression of Beclin 1 had a significantly poorer overall survival compared with the other two groups (P<0.0001). Beclin 1 had an independent prognostic relevance in multivariate analysis.Conclusions:Beclin 1 has an important role in growth and metastasis of colorectal cancer. Loss of Beclin 1 expression (allelic loss or microRNA regulatory activity, as suggested in the literature) defines poor prognosis presumably by promoting anti-apoptotic pathways, while overexpression of the protein, being linked with tumour hypoxia and acidity, also defines subgroups of tumours with aggressive clinical behaviour.

Chromosome band 17q21 in breast cancer: Significant association between beclin 1 loss and HER2/NEU amplification

Treatment success of breast cancer patients with trastuzumab alone or in combination depends not only on HER2/NEU amplification but also on PTEN and PI3K status and efficient cell death programs. In this pilot study, we found a significant association between loss of beclin 1 and HER2/NEU amplification (both on 17q21) in breast cancers. This finding was confirmed in two public copy number microarray datasets. Furthermore, there is a trend associating beclin 1 loss with TP53 mutations, PI3KCA gene gain, and PTEN mutations. Finally, the observation that beclin 1 gene loss predicted a response to trastuzumab alone or in combination with other drugs is worthy of further confirmation in larger cohorts. Our results suggest that, beclin 1 loss may contribute to genome instability and to a defective autophagy that may lead to tumoral cell death in presence of competent apoptosis or senescence pathways.

Abstract CN05-02: Autophagy and mammary tumorigenesis: role of metabolic and ER stress management

Abstract CN05-02 Autophagy is an evolutionarily conserved catabolic process whereby cellular organelles and bulk cytoplasm are targeted to lysosomes for degradation. Autophagy is a survival pathway required for cell viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin 1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin 1+/- mice are tumor-prone. Thus, autophagy likely suppresses tumorigenesis, but the mechanism has been unknown. We recently showed that allelic loss of beclin 1 compromises the autophagy potential of immortalized mouse mammary epithelial cells (iMMECs) in vitro and in mammary tumors in vivo, sensitizes iMMECs to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. We investigated the mechanism by which autophagy mitigates metabolic stress and limits genome damage in mammary epithelial cells, so as to gain insight into how autophagy suppresses mammary tumorigenesis. A proteomic analysis was performed to determine how autophagy-competent mammary epithelial cells respond successfully to metabolic stress as compared to cells with autophagy defects. Protein extracts from apoptosis-defective beclin 1+/+ and beclin 1+/- iMMECs exposed to metabolic stress for 0, 4 and 7 days were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). More than a hundred proteins with differential expression under metabolic stress were sequenced and mostly represented members of the GRP chaperone system, cellular metabolism, mitochondrial and cytoskeletal proteins. The 2D-DIGE results were validated for a subset of the sequenced proteins on beclin 1+/+ and beclin 1+/- iMMECs under metabolic stress in vitro and on beclin 1+/+ and beclin 1+/- iMMEC-generated mammary tumors in vivo. Expression of several proteins upregulated under metabolic stress was also examined in 3D-morphogenesis assays. For most proteins, expression was higher in the central acinar cells, indicating that the acinar center is a physiologic area of increased metabolic stress. Differential protein expression between wild-type and autophagy-defective iMMECs under conditions of metabolic stress is currently being validated on mammary tissues from beclin 1+/+ and beclin 1+/- female mice. Therefore, failure of autophagy-deficient mammary tumor cells to effectively manage metabolic stress is associated with increased unfolded protein load and endoplasmic reticulum (ER) stress, which may in turn play a pivotal role in promoting genome damage and instability, and thus cancer progression, especially when apoptosis is concurrently inactivated. These studies identify new therapeutic targets and provide valuable clues into how to best modulate autophagy for cancer prevention and treatment. Citation Information: Cancer Prev Res 2008;1(7 Suppl):CN05-02.