Autophagy is a homeostatic process that degrades long-lived or damaged proteins and organelles. By recycling intracellular constituents, it is buffering metabolic stress under starvation conditions. The autophagy role in cancer remains unclear and complicated as it appears to be involved in tumorigenesis, cancer development and treatment outcome in different ways. Autophagy can act as both tumor-promoting and tumor-suppressing agent depending on the stage of cancer progression. During the initiation of cancer, autophagy prevents cells from further DNA damage and genomic instability. It could also be a cell death mechanism in cancer cells with apoptotic defect. Autophagy can also promote tumor growth by facilitating oncogene-induced senescence or protecting tumors against necrosis and inflammation. Once the cancer is formed, autophagy can contribute to tumor progression (by allowing cells to survive in stressful conditions) and metastasis. There is evidence that breast cancer could also be controlled by autophagy. Regulation of this process, correlated proteins and active factors are currently under scientific study in the aspect of breast cancer effective therapeutic strategies.
- breast cancer
Breast cancer (BC) is a potentially life-threatening malignant tumor that still causes high mortality among women. One of the mechanisms through which cancer development could be controlled is autophagy. This process exerts different effects during the stages of cancer initiation and progression due to the occurring superimposition of signaling pathways of autophagy and carcinogenesis. Chronic inhibition of autophagy or autophagy deficiency promotes cancer due to instability of the genome and defective cell growth as a result of cell stress. However, increased induction of autophagy can become a mechanism which allows tumor cells to survive the conditions of hypoxia, acidosis or chemotherapy. Therefore, in the development of cancer, autophagy is regarded as a double-edged sword. There are different ways of autophagy process control under scientific research for potential therapeutic treatment in anti-cancer strategies.
2. The process of autophagy
Autophagy is a process regulated genetically and/or controlled by a group of evolutionarily conserved genes (ATGs; autophagy-related genes). Initially, autophagy was identified as a cell survival mechanism protecting from nutrient deprivation. It ensures homeostasis by maintaining proteins and organelles turnover. Removing excess or damaged intracellular components in response to stress as well as microorganisms allows cells to restrain damage (including genome instability which limits initiation and progression of cancer) and subsequent inflammation. Cellular stress can be caused by a variety of chemical and physical agents like nutrient starvation, pro-inflammatory state, hypoxia, oxidants, infectious agents and xenobiotics [1, 2]. Under the influence of autophagic pathway, biological and morphological changes have been observed . In certain developmental conditions like in cell’s response to metabolic stress or under cytotoxic stimuli, autophagy results in a form of cell death described as programmed cell death type II .
Currently, over 35 proteins are believed to be essential for autophagy occurrence and progression . The complete macroautophagy (referred to hereafter as autophagy) is generally divided into the following stages: induction, vesicle nucleation, vesicle elongation and completion, docking and fusion, degradation and then recycling [1, 6]. Vesicle nucleation is the initial step in which proteins and lipids are recruited for construction of the autophagosomal membrane. Nucleation consists of the formation of the phagophore or isolation membrane. In mammalian cells, this process is initiated by activation of the class III PI3K/Beclin-1 complex including the core members hVps34/PIK3C3, Beclin-1 (BECN1) and p150. Numerous additional binding partners of this complex function as either positive or negative regulators and include BAX-interacting factor-1 (BIF-1), Atg14L, UVRAG (UV irradiation resistance-associated gene), Ambra1 (activating molecule in Beclin-1-regulated autophagy protein 1) and Rubikon [1, 5, 7-9]. Rubicon (RUN domain Beclin 1-interacting cysteine-rich-containing protein) has also been shown to negatively regulate autophagy. Subsequently, the phagophore is elongated by several ATG proteins. During this elongation step, microtubule-associated protein 1 light chain 3 (LC3)-I is lapidated to LC3-II. Then, the phagophore is maturated primarily upon the action of LC3-II and BECN1 proteins. Maturation leads to the formation of autophagosome (enclosed vesicle). The regulation of the maturation process of the autophagosome is multi-factorial and involves Rab GTPase, SNAREs (soluble N-ethylmaleimide-sensitive fusion attachment protein receptors) and ESCRT (endosomal sorting complexes required for transport) proteins, molecules of the acidic lysosomal compartment (e.g. v-ATPase, LAMP proteins- lysosome-associated membrane glycoprteins; lysosomal carriers and hydrolases) and Beclin-1. Finally, the autophagosome fuses with the lysosome to form an autolysosome. The internal material of the autophagic vacuole is degraded by the lysosomal hydrolases.
Basal levels of macroautophagy are kept in check by mTORC1 (mammalian target of rapamycin complex 1) which phosphorylates Atg13 and ULK1 (uncoordinated 51-like kinase 1/Atg1) or ULK2. This activity in consequence is giving the inhibition of FIP200 (focal adhesion kinase interacting protein of 200 kD/Atg17) phosphorylation by ULK1 . The mTORC1 complex is an important component of a network that accordingly maintains homeostasis by controlling the levels of anabolism and catabolism. For example, high levels of amino acids maintain mTORC1 in an active state by enhancing its binding to regulatory GTPases, Rag (Ras-related GTPase) and Rheb (Ras homolog enriched in brain) . mTORC1 activity could be indirectly induced by insulin and IGF1 (insulin like growth factor 1) . Low glucose levels or high AMP levels (adenosine 5’-monophosphate), indicators of low cellular energy status or stress, could activate AMPK (AMP-activated protein kinase) which in turn inhibits mTORC1 and stimulates autophagy [2,7].
The role of the PI3K/Akt pathway is to suppress autophagy. This pathway activation was shown to decrease autophagy through mTOR activation. It has been considered for cancer treatment. The MAPK pathway also plays a significant role in autophagy. Ras may play a dual role in autophagy . When Ras activates PI3KCA, autophagy is inhibited; however, when it selectively activates the MAPK pathway, autophagy is stimulated.
3. Breast cancer
Breast cancer (BC) is the most common and fatal cancer in women worldwide. Decreasing mortality rates can be observed that result mostly from efficient screening strategies  but still BC is ranked on the second place in mortality among cancer types . It has been estimated that approximately 1.3 million females develop BC each year with around 465.000 expected to succumb to the disease [15,16]. It is causing death of about 350.000 women in both developed and developing countries every year (with slightly more cases in less developed than in more developed regions) . According to another data presented by DeSantis et al., there are still 500.000 breast cancer deaths per year worldwide . More than 90% of lethality in patients is caused by metastasis and the occurrence of distant metastases (distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver) severely limits the prognosis [19,20]. The 5-year survival rate for patients with BC drops sharply from 98% for individuals with localized disease to 23% for those with metastatic disease (cancer statistics from 2012) . A significant subpopulation of patients with metastasis risk has a median survival time of 18–30 months .
In the pathogenesis and progression of BC are involved many factors including genetic, biological and environmental factors as well as a lifestyle . For example, Bcl-2 protooncogene is overexpressed in half of all human malignancies and more than 60% of BC and exerts its oncogenic role by preventing cells from undergoing apoptosis . Still, the disease background is not fully clear because it has been estimated that 75% of women with sporadic invasive BC have no known epidemiological risk factors .
4. Autophagy regulation in breast tumors development
In tumor genesis and treatment responsiveness, autophagy role is complicated and context-dependent. It presumably differs in different stages of cancer development. At the initial stages, autophagy may represent a protective role thanks to its catabolic functions by degrading and/or recycling cell components (e.g. damaged organelles and misfolded proteins) [25-27]. It also protects against the deleterious effects of ROS (reactive oxygen species) in the cells. Proliferation of cells with cancer-linked mutations may be retarded by autophagy. It can also limit propagation of this type of mutations and consequently suppress tumorigenesis by facilitating the cellular senescence phenomenon (biological aging). However, once a tumor develops, the cancer cells can utilize autophagy for their own cytoprotection and use enhanced autophagy to survive under metabolic and therapeutic stress . Autophagy might increase oxidative stress, hence promoting genome instability and malignant transformation [25-27]. As an example, autophagy has been shown to be required for the transformation of mouse embryonic fibroblasts by the Ras oncogene and this effect is linked to its role in nutrients recycling, such as glucose uptake and increased glycolytic flux . What's more, it has been suggested that metastatic cancer cells can escape from anoikis (process of apoptosis induced by lack of correct cell-ECM attachment) through the autophagy induction [29, 30]. The AMP-activated protein kinase (AMPK) stress response pathway is involved in mediating anoikis resistance by inhibiting mTOR and suppression of protein synthesis. The Ras/MAPK and PI3K/Akt pathways are common mechanisms utilized by cancer cells to evade anoikis. Autophagy is necessary for cancer cells survival in hypoxic conditions during the later stages of
The ability of cancer cells to invade and metastasize is closely correlated with the process of epithelial-mesenchymal transition (EMT). It was recently demonstrated that ectopic expression of the DEDD gene in the MDA-MB-231 metastatic BC cell line led to the degradation of the EMT inducers Snail and Twist through autophagy activation . Reversely, knock-down of DEDD in the MCF7 non-metastatic BC cell line led to autophagy reduction and EMT promotion .
Regulation of autophagy in tumors is governed by principles similar to normal cells only in a much more complicated manner. Abnormal PI3K activation in cancer cells is frequently observed. The multitude of interactions between the PI3K/Akt/mTOR pathway and other cell signaling cascades could often be deregulated . For example, Ras ⁄Raf ⁄ERK pathway, indicated as one of the most commonly deregulated pathways identified in tumors, frequently are observed activating mutations in Ras or B-Raf oncogenes . ERK activity has been associated with autophagy and autophagic cell death in many cellular models in response to different stresses . It also happens in TNFalpha treatment in MCF-7 cells. A deregulated PI3K/Akt/mTOR axis not only suppresses autophagy process but also induces protein translation, cell growth and proliferation thereby can force tumorigenesis. Tumors with constitutively active PI3K mutations, PTEN loss or Akt activation would be expected to be dependent on autophagy for energy homeostasis and survival. Suppression of autophagy by the PI3K cascade is disadvantageous for rapidly proliferating tumor cells and there are theses that compensatory mechanisms (like deregulated apoptosis and/or metabolism) might be concurrently activated to prevent the negative implications of defective autophagy on tumor cell survival.
Many proteins and active factors correlated with autophagy are reported to be associated with human cancers . Various tumor suppressors (e.g. PTEN, TSC1/2, p53, and DAPK) are autophagy inducers whereas some inhibitors of autophagy (e.g. Akt and Ras) possess oncogenic activity . Some studies [37, 38] showed that the more advanced stages of breast cancer over-express several other oncogenic and signaling proteins such as IGF-1R, Cyclin D1, c myc, pERK, Stat3 and Pak4. Some are known activators of Akt/mTOR pathway. Several other autophagy regulators like mitogen-activated kinases (BNIP3)  and HSpin 1 (human homologue of the Drosophila spin gene product)  play a critical role in cancer cells. Deletion of the essential autophagy gene
PTEN, a critical regulator of the PI3K pathway, has a stimulatory effect on autophagy by downregulating PI3K/Akt signaling through inhibition of the Akt/PKB activation. Akt inhibition leads to mTOR signaling suppression and the induction of autophagy . AMPK (AMP-activated protein kinase) pathway has a negative effect on mTOR signaling and promotes autophagy (e.g. upon starvation conditions by activation of Tuberin -TSC2 and/or mTOR signaling inhibitor).
mTORC1, class I PI3K, Akt, class III PI3K, Beclin-1 and p53 are critical components of the autophagic pathway that have become major targets of autophagy-related drug design. As an example, rapamycin and its derivatives (e.g. rottlerin, PP242 and AZD8055) target the PI3K/Akt/mTOR signaling pathway to induce autophagy. Spautin-1 and tamoxifen regulate Beclin-1 activity to respectively inhibit and promote autophagy. Oridonin and metformin trigger p53-mediated autophagy and cell death .
4.1. Beclin-1 role
The most important evidence linking dysfunctional autophagy and cancer come from studies on mice demonstrating that the inhibition of autophagy by disruption of
Human breast cancer cell lines FISH analysis with the Beclin-1-containing PAC 452O8 as a probe revealed that 9 out of 22 cell lines had allelic BECN1 deletions . Monoallelic deletion of BECN1 has been also detected in 40-75% cases of human breast, ovarian and prostate tumors [2, 26]. Thereby BECN1 is considered as a tumor suppressor gene . Deletions of Beclin-1 have recently been found mostly associated with BRCA1 in breast and ovarian human tumors (suggesting that BRCA1 loss is the mutation driver and that Beclin-1 is lost because of its proximity to it) . Many breast carcinoma cell lines, although polyploidal for chromosome 17 (
Beclin-1 also alters the expression of several autophagy proteins such as Atg5 and UVRAG .
4.2. Adipokines role
Adipokines, auto-/ endocrine and paracrine-acting bioactive molecules secreted by adipose tissue are one of the recently discovered factors correlating with autophagy and BC . Adiponectin (AdipoQ) is the cytokine secreted in greatest abundance. The prevalence correlate low levels of AdipoQ in the blood circulation with higher BC risk and poorer prognosis. In breast tissue, AdipoQ has a direct anti-carcinogenic effect at the site of tumor growth. This cytokine is potentially capable of regulation of autophagy through AMP kinase (5'AMP-activated protein kinase) and its activation has been observed in breast cancer cells . Liu and colleagues observed that AdipoQ caused upregulatation of autophagy in MDA-MB-231 cells
4.3. microRNA role
MicroRNAs (miRNAs) are endogenous ~22 nucleotide RNAs that suppress gene expression via messenger RNA (mRNA) cleavage and/or translational repression. Unregulated miRNAs of lymphoma, prostate, lung and breast cancers have been also detected in blood plasma and serum. Circulating miRNAs are currently assessed as proxy biomarkers for BC . There is evidence that miRNAs can influence autophagy process in BC cells at many points. MiR-20a, miR-101, miR-106a/b and miR-885-3p have been reported to have direct possibility of targeting ULK1/2 . Also, miR-155 might target multiple players in mTOR signaling including Rheb, RICTOR (RPTOR independent companion of mTOR) and RPS6KB2 (ribosomal protein S6 kinase). MiR-30a and miR-519a can directly target Beclin-1 causing negative regulation in the autophagic flow thereby resulting in decreased autophagic activity. Action of miR-30a was shown in the
4.4. Cancer Stem Cells (CSC) and autophagy
Autophagy is thought to be a critical process for cancer stem cells (CSC) or tumor initiating cell maintenance but the mechanisms through which autophagy supports survival of CSCs remain poorly understood . The CSC theory proposes that heterogeneity within a tumor is driven by a small population of cells which have ability to differentiate and/or self-renewal, increased membrane transporter activity, anchorage independence and ability to migrate, tumorigenic capacities and pluripotency [61, 62]. Breast cancer follows this model since it has been shown that the CD44+/ CD24 low/-phenotype of cell surface markers (which can be found also in normal stem cells in the breast), have an increased ability to form tumors in immunosuppressed mice than the bulk of the tumor cells. It has been predicted that a quality control mechanism like autophagy is important for maintaining normal and cancer stem cell homeostasis . Maycotte
CSCs are characteristically resistant to conventional anticancer therapy which may contribute to treatment failure and tumor relapse. CSCs exhibit the potential for regeneration which may promote tumor metastasis . Recently, autophagy has been shown to be a critical factor for CSC survival and drug resistance .
5. Authophagy in anti-breast cancer therapies
There are different ways of autophagy process usage and/or influence recognized according to potential therapeutic treatment in anti-cancer strategies.
Inducing protective autophagy and prosurvival mechanism in human cancer cell lines have been shown in a number of currently used antineoplastic therapies including radiation therapy, chemotherapy (e.g. doxorubicin, temozolomide and/or etoposide), histone deaceltylase inhibitors, arsenic trioxide, TNFα, IFNγ, imatinib, rapamycin and anti-estrogen hormonal therapy (e.g. tamoxifen) [12, 23]. In fact, the therapeutic efficacy of these agents can be increased if autophagy is inhibited .
The scientific evidence suggests that autophagy leads to cell death in response to several compounds including etoposide, rottlerin, cytosine arabinoside and staurosporine as well as deprivation of growth-factors. A link has been demonstrated between autophagy and related autophagic cell death with usage of pharmacological inhibitors (e.g. 3-MA (3-methyl adenine), CQ (chloroquine), bafilomycin A1 or ammonium chloride) and genetic silencing or knockdown (silencing of
Autophagy has also been shown to protect against cellular stress induced by the anti-cancer chemotherapeutic drugs (
Such disclosures have led to several clinical trials involving the use of the autophagy flux inhibitors as a combination therapy  to radiotherapy efficacy improvement in BC patients. For example, to such inhibitors could be included hydroxychloroquine (HCQ). HCQ is a less toxic version of CQ and the best autophagy inhibitor currently commercially available for clinical trials . Irradiated cancer cells can induce damage in neighboring un-irradiated cells by intracellular gap-junction communication or signals released outside of the cells . Huang
As Ras⁄Raf⁄ERK pathway belongs to the most commonly deregulated pathways identified in tumors and is currently the target of new antitumor strategies based on the inhibition of upstream ERK regulators. Inhibiting ERK activity in combination therapy with classical antitumor compounds might affect the efficiency of such compounds. For example, in MCF-7 human breast adenocarcinoma cell line such combined therapies with: doxorubicin , tamoxifen , taxol  or Δ Raf1  and, TNFα  were used. For example, tamoxifen, the most commonly used antiestrogen, exerts its pharmacological action by binding to estrogen receptor alpha (ERα) and blocking the growth promoting action of the estrogen in BC cells. However, the development of antiestrogen resistance has become a major impediment in the treatment of ER-positive BC. It was reported that autophagy plays a critical role in the development of antiestrogen resistance and overexpression of Beclin-1 downregulated estrogenic signaling and growth response .
5.1. Autophagic genes silencing
Some studies using gene silencing to receive therapeutic effect via cell death induction could represent genetic therapeutic approaches. For example, the Bcl-2 protooncogene (preventing cells from undergoing apoptosis) is overexpressed in half of all human malignancies and more than 60% of BC. Bcl-2 overexpression not only leads to the resistance of cancer cells towards chemotherapy, radiation and hormone therapy but also causes an aggressive tumor phenotype in patients with a variety of cancers . Recent findings suggested that silencing Bcl-2 expression (by siRNA) in MCF-7 cells led to significant autophagic, not apoptotic, cell death . It has been demonstrated that the knockdown of autophagy genes (e.g.
There are studies connecting autophagy genes profile with BC prognosis. For example, Gu
5.2. Pharmacological approach to the autophagic therapies
In pharmacological approach to the anti-tumor autophagic therapies, the aim is to activate or inhibit autophagy. Many drugs and compounds that modulate autophagy are currently receiving considerable attention [26,35]. For example, autophagy inducers such as rapamycin (mTORC1 inhibitor)  and its analogs called rapalogs (such as Everolimus; RAD001) are also often used as tools to study autophagy process . Everolimus was shown to enhance the sensitivity of tumors to radiation by induction of autophagy .
Also, natural products are considered as potential anti-cancer candidates being direct or indirect sources of new chemotherapy adjuvants to enhance the efficacy of chemotherapy and/or to ameliorate its side effects [87, 88].
The more challenging issue is the monitoring of autophagic activity in humans, in tissue and blood samples. It seems to be more important to measure autophagic flux than autophagosome number. However, measurements of autophagic flux in paraffin-embedded tissue samples have been unsuccessful till now and even the detection of endogenous LC3-II (commonly used marker for autophagosomes) is problematic in tissue sections 
6. Concluding remarks
There has been a tremendous amount of progress in our understanding of the role of autophagy in cancer. But still the molecular mechanisms underlying the regulation of autophagy and the role of autophagy in cancer cells are not fully understood but are progressively revealed. Overall, the data support a dynamic role of autophagy in cancer - both as a tumor suppressor early in progression and later as a pro-tumorigenic process critical for tumor maintenance and therapeutic resistance. The specification of the autophagic cargo in tumors with increased autophagy is important for understanding the changes in metabolism between normal and malignant cells. Undoubtedly, progress in genomics, proteomics and metabolomics will be helpful in this scope. Induction of autophagic cell death may be an ideal approach in resistant cancers therapies. But most experiments regarding BC are carried out on cell lines
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