[9] Debnath, J, Mills, K. R, Collins, N. L, Reginato, M. J, Muthuswamy, S. K, & Brugge, J. S. The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell (2002). , 111, 29-40.

**Author details**

**References**

(2008).

4013-4022.

Malgorzata Gajewska\*

of Life Sciences, Warsaw, Poland

460 Autophagy - A Double-Edged Sword - Cell Survival or Death?

, Katarzyna Zielniok and Tomasz Motyl

Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University

[1] Reginato, M. J, & Muthuswamy, S. K. Illuminating the center: mechanisms regulating lumen formation and maintenance in mammary morphogenesis. Journal of Mamma‐

[2] Capuco, A. V, & Ellis, S. Bovine mammary progenitor cells: current concepts and fu‐

[3] Mailleux, A. A, Overholtzer, M, & Brugge, J. S. Lumen formation during mammary epithelial morphogenesis: insights from in vitro and in vivo models. Cell Cycle.

[4] Humphreys, R. C, Krajewska, M, Krnacik, S, Jaeger, R, Weiher, H, Krajewski, S, Reed, J. C, & Rosen, J. M. Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis. Development (1996). , 122(12),

[5] Barcellos-hoff, M. H, Aggeler, J, Ram, T. G, & Bissell, M. J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted base‐

[6] Li, M. L, Aggeler, J, Farson, D. A, Hatier, C, Hassell, J, & Bissell, M. J. Influence of a reconstituted basement membrane and its components on casein gene expression and secretion in mouse mammary epithelial cells. Proceedings of the National Acad‐

[7] Debnath, J, Muthuswamy, S. K, & Brugge, J. S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement mem‐

[8] Kozlowski, M, Wilczak, J, Motyl, T, & Gajewska, M. Role of extracellular matrix and prolactin in functional differentiation of bovine BME-UV1 mammary epithelial cells.

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[34] Monks, J, Geske, F. J, Lehman, L, & Fadok, V. A. Do inflammatory cells participate in mammary gland involution? Journal of Mammary Gland Biology and Neoplasia

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[35] Capuco, A. V, & Akers, R. M. Mammary involution in diary Animals. Journal of

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[37] Sobolewska, A, Gajewska, M, Zarzynska, J, Gajkowska, B, & Motyl, T. IGF-I, EGF and sex steroids regulate autophagy in bovine mammary epithelial cells via the

[38] Mulder, K. M. Role of Ras Mapks in TGF-beta signaling. Cytokine and Growth Fac‐

[39] Gajewska, M, & Motyl, T. IGF-binding proteins mediate TGF-βinduced apoptosis in bovine mammary epithelial BME-UV1 cells. Comperative Biochemistry and Physiol‐

[40] Kolek, O, Gajkowska, B, Godlewski, M. M, & Motyl, T. Colocalization of apoptosisregulating proteins in mouse mammary epithelial HC11 cells exposed to TGF-β1. Eu‐

[41] Daniel, C. W, Robinson, S, & Silberstein, G. B. The transforming growth factors beta in development and functional differentiation of the mouse mammary gland. Advan‐

[42] Weigelt, B, & Bissell, M. J. Unraveling the microenvironmental influences on the nor‐ mal mammary gland and breast cancer. Seminars in Cancer Biology (2008).

[43] Petersen, O. W, Rønnov-jessen, L, Howlett, A. R, & Bissell, M. J. Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proceedings of the National

[44] Debnath, J, & Brugge, J. S. Modelling glandular epithelial cancers in three-dimen‐

[45] Chen, N, & Karantza-wadsworth, V. Role and regulation of autophagy in cancer. Bio‐

[46] Qu, X, Yu, J, Bhagat, G, Furuya, N, Hibshoosh, H, Troxel, A, Rosen, J, Eskelinen, E. L, Mizushima, N, Ohsumi, Y, Cattoretti, G, & Levine, B. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. Journal of Clinical Investi‐

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[34] Monks, J, Geske, F. J, Lehman, L, & Fadok, V. A. Do inflammatory cells participate in mammary gland involution? Journal of Mammary Gland Biology and Neoplasia (2002).

[21] Wullschleger, S, Loewith, R, & Hall, M. N. TOR signaling in growth and metabolism.

[22] Pattingre, S, Espert, L, Biard-piechaczyk, M, & Codogno, P. Regulation of macroau‐

[23] Liang, J, Shao, S. H, Xu, Z. X, Hennessy, B, Ding, Z, Larrea, M, Kondo, S, Dumont, D. J, Gutterman, J. U, Walker, C. L, Slingerland, J. M, & Mills, G. B. The energy sensing LKB1-AMPK pathway regulates kip1) phosphorylation mediating the decision to en‐

[24] Fleming, J. M, Desury, G, Polanco, T. A, & Cohick, W. S. Insulin growth factor-I and epidermal growth factor receptors recruit distinct upstream signaling molecules to

[25] Yanochko, G. M, & Eckhart, W. (2006). Type I insulin-like growth factor receptor over-expression induces proliferation and anti-apoptotic signaling in a three-dimen‐

[26] Liu, H, Radisky, D. C, Wang, F, & Bissell, M. J. Polarity and proliferation are control‐ led by distinct signaling pathways downstream of PI3-kinase in breast epithelial tu‐

[27] Lamote, I, Meyer, E, Massart-leen, A. M, & Burnvenich, C. Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation and involu‐

[28] Atwood, C. S, Hovey, R. C, Glover, J. P, Chepko, G, Ginsburg, E, Robison, W. G, & Vonderhaar, B. K. Progesterone induces side-branching of the ductal epithelium in

[29] Nilsson, S, Mäkelä, S, Treuter, E, Tujague, M, Thomsen, J, Andersson, G, Enmark, E, Pettersson, K, Warner, M, & Gustafsson, J. A. Mechanisms of estrogen action. Physio‐

[30] Fox, E. M, Andrade, J, & Shupnik, M. A. Novel actions of estrogen to promote prolif‐ eration: integration of cytoplasmic and nuclear pathways. Steroids (2009). , 74,

[31] Levin, E. R. Bidirectional signaling between the estrogen receptor and the epidermal

[32] John, S, Nayvelt, I, Hsu, H. C, Yang, P, Liu, W, Das, G. M, Thomas, T, & Thomas, T. J. Regulation of estrogenic effects by beclin1 in breast cancer cells. Cancer Research

[33] Monks, J, & Henson, P. M. Differentiation of the mammary epithelial cell during in‐ volution: implications for breast cancer. Journal of Mammary Gland Biology and Ne‐

growth factor receptor. Molecular Endocrinology (2003). , 17, 309-317.

the mammary glands of peripubertal mice. Journal of Endocrinology (2000).

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[47] Yue, Z, Jin, S, Yang, C, Levine, A. J, & Heintz, N. Beclin 1, an autophagy gene essen‐ tial for early embryonic development, is a haploinsufficient tumor suppressor. Pro‐ ceedings of the National Academy of Sciences U S A. (2003).

**Chapter 21**

**Integrin and Adhesion**

Eric A. Nollet and Cindy K. Miranti

http://dx.doi.org/10.5772/55398

**1. Introduction**

Additional information is available at the end of the chapter

and its role in cellular differentiation and survival.

**Regulation of Autophagy and Mitophagy**

Cell differentiation is a dynamic process that generates a functionally distinct cell from its progenitor. For example, human erythrocytes lack most organelles - including a nucleus while erythrocyte precursors have a complete set of organelles. Autophagy plays a critical role in organelle elimination in differentiating erythrocytes. On the other hand, most other differentiated cells in the body do not lose their organelles, and would not seem to heavily depend on autophagy during differentiation. However, evidence indicates that these cells require regulated autophagy during the differentiation process. For example, during keratinocyte differentiation a basal cell detaches from the basement membrane and is pushed to the upper strata, and as the cell ascends its intracellular contents are replaced with copious amounts of keratin. Drastic changes such these require cells to eliminate or turn over a large amount of biomass. Differentiation also causes a shift in signaling and survival pathways. An example of this is the prostate gland where the luminal cells require PI3K for survival while the undifferentiated basal cells do not; they depend on MAPK signaling for their survival. As we will discuss below, several different cell types use and require the autophagic pathway to properly differentiate and survive. While links be‐ tween autophagy and differentiation are rapidly being identified, the mechanisms that trigger autophagic processes during differentiation are poorly understood. Genetics has served as a powerful tool for identifying the components of the autophagy machinery, but how they are integrated with cellular cues that trigger differentiation need further charac‐ terization. We will discuss the recently identified link between autophagy and cell adhesion,

> © 2013 Nollet and Miranti; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 Nollet and Miranti; licensee InTech. This is a paper distributed under the terms of the Creative Commons

## **Chapter 21**

## **Integrin and Adhesion Regulation of Autophagy and Mitophagy**

Eric A. Nollet and Cindy K. Miranti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55398

## **1. Introduction**

[47] Yue, Z, Jin, S, Yang, C, Levine, A. J, & Heintz, N. Beclin 1, an autophagy gene essen‐ tial for early embryonic development, is a haploinsufficient tumor suppressor. Pro‐

[48] Jin, S, & White, E. Role of autophagy in cancer: management of metabolic stress. Au‐

[49] Degenhardt, K, Mathew, R, Beaudoin, B, Bray, K, Anderson, D, Chen, G, Mukherjee, C, Shi, Y, Gélinas, C, Fan, Y, Nelson, D. A, Jin, S, & White, E. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer

[50] Qadir, M. A, Kwok, B, Dragowska, W. H, To, K. H, Le, D, Bally, M. B, & Gorski, S. M. Macroautophagy inhibition sensitizes tamoxifen-resistant breast cancer cells and en‐ hances mitochondrial depolarization. Breast Cancer Research and Treatment (2008).

[51] Samaddar, J. S, Gaddy, V. T, Duplantier, J, Thandavan, S. P, Shah, M, Smith, M. J, Browning, D, Rawson, J, Smith, S. B, Barrett, J. T, & Schoenlein, P. V. A role for mac‐ roautophagy in protection against hydroxytamoxifen-induced cell death and the de‐ velopment of antiestrogen resistance. Molecular Cancer Therapeutics (2008). , 4.

[52] Debnath, J. Detachment-induced autophagy during anoikis and lumen formation in

[53] White, D. E, Kurpios, N. A, Zuo, D, Hassell, J. A, Blaess, S, Mueller, U, & Muller, W. J. Targeted disruption of betaintegrin in a transgenic mouse model of human breast cancer reveals an essential role in mammary tumor induction. Cancer Cell. (2004). , 1.

[54] Lock, R, & Debnath, J. Extracellular matrix regulation of autophagy. Current Opinion

epithelial acini. Autophagy (2008). , 4, 352-335.

in Cell Biology (2008). , 20, 583-588.

ceedings of the National Academy of Sciences U S A. (2003).

tophagy. (2007).

464 Autophagy - A Double-Edged Sword - Cell Survival or Death?

Cell. (2006).

Cell differentiation is a dynamic process that generates a functionally distinct cell from its progenitor. For example, human erythrocytes lack most organelles - including a nucleus while erythrocyte precursors have a complete set of organelles. Autophagy plays a critical role in organelle elimination in differentiating erythrocytes. On the other hand, most other differentiated cells in the body do not lose their organelles, and would not seem to heavily depend on autophagy during differentiation. However, evidence indicates that these cells require regulated autophagy during the differentiation process. For example, during keratinocyte differentiation a basal cell detaches from the basement membrane and is pushed to the upper strata, and as the cell ascends its intracellular contents are replaced with copious amounts of keratin. Drastic changes such these require cells to eliminate or turn over a large amount of biomass. Differentiation also causes a shift in signaling and survival pathways. An example of this is the prostate gland where the luminal cells require PI3K for survival while the undifferentiated basal cells do not; they depend on MAPK signaling for their survival. As we will discuss below, several different cell types use and require the autophagic pathway to properly differentiate and survive. While links be‐ tween autophagy and differentiation are rapidly being identified, the mechanisms that trigger autophagic processes during differentiation are poorly understood. Genetics has served as a powerful tool for identifying the components of the autophagy machinery, but how they are integrated with cellular cues that trigger differentiation need further charac‐ terization. We will discuss the recently identified link between autophagy and cell adhesion, and its role in cellular differentiation and survival.

© 2013 Nollet and Miranti; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Nollet and Miranti; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Role of autophagy during differentiation**

#### **2.1. Erythrocytes**

Erythrocytes are especially unique cells; they lack a nucleus, internal membrane-bound organelles, and ribosomes, and they are packed with the oxygen transporter hemoglobin. The cell arises from reticulocytes, a nucleated progenitor capable of generating the needed surplus hemoglobin and the erythrocyte itself [1,2]. Generating such a simple cell requires processes that eliminate organelles not essential for fully differentiated erythrocytes. A series of publi‐ cations from 2008 to present not only elucidated the involvement of autophagy during reticulocyte differentiation, but also discovered that the mitochondria are specifically targeted for autophagy by a protein called Nix [3-7].

**2.2. Lymphocytes**

**2.3. Adipocytes**

In the same Atg7-/- transplantation model, there was a four-fold reduction in the number of white blood cells and nine-fold reduction of lymphoid cells [7]. This observation was further validated in hematopoietic-specific stem, fetal, and adult cell Atg7 knockout mouse models [9,

cells succumbed to apoptosis in vitro more readily than the wild type controls [11]. T-cell specific knockouts of Atg5 and Atg7 also displayed increased amounts of mitochondria and were more susceptible to apoptosis [12,13]. Considering that the overall population of mature T lymphocytes decreased and sensitivity to apoptosis increased in the autophagy-deficient T lymphocyte models, it seems that the lymphopenia occurs due to excess cell death. Puo et al tested this by stimulating T-cell proliferation in wild type and Atg5-/- T-cells and quantifying the amount of daughter cells produced by using the stable dye, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE diffuses into both cells during mitosis, causing a decrease in individual cell fluorescent intensity with every division. The analysis demonstrat‐ ed that stimulated Atg5-/- T-cells did not produce daughter cells after three days and the authors concluded that Atg5 is necessary for T-cell proliferation. However, CFSE cannot differentiate between a dead or permeabilized cell and a non-proliferating cell, and over the three day period there was a moderate increase in apoptosis [14,15]. In this model, it appears that autophagy is required during T-cell hematopoiesis, at least in part, to prevent death of the newly differentiated cells. It would be interesting to investigate this in an inducible model, to test whether the dependence on autophagy is transient, i.e. only during early differentiation

Evidence also supports a role for autophagy in adipocyte differentiation. Treatment of mouse embryonic fibroblasts (MEFs) with a cocktail of differentiation-inducing factors over the course of two weeks causes accumulation of lipid analogous to mature adipocytes. In the absence of Atg5 the MEFs fail to accumulate lipid droplets or display the molecular or structural pheno‐ type of differentiated adipocytes. Furthermore, Atg5-/- mouse pups have far fewer adipocytes [16]. Knocking down Atg7 in 3T3-L1 preadipocytes attenuated the accumulation of triglycer‐ ides and several markers of mature adipocytes as well. This was further validated in vivo in an adipose-specific Atg7 knock out mouse; the total fat in the knock out mouse weighed less than half that of the wild type mice. Interestingly, the adipose-specific Atg7-/- mouse had increased amounts of brown adipose tissue (BAT) and less white adipose tissue (WAT) relative to the wild type mouse, and both the lipid droplets and adipocytes were smaller and more densely packed than normal adipocytes [17,18]. The brown appearance of BAT is due to high quantities of densely packed mitochondria. BAT can uncouple the mitochondrial ATPase and continue oxidizing nutrients to generate thermal energy in place of the chemical energy provided by ATP. Zhang et al. validated these results, but went further and confirmed an increased presence of mitochondria [18]. Thus, while autophagy deficient adipocytes fail to express differentiation markers and lack lipid droplet morphology, some of the effects seen in autophagy-deficient adipocytes may result from the accumulation of mitochondria. Indeed,

and CD4+

Integrin and Adhesion Regulation of Autophagy and Mitophagy

T-cells, and these

http://dx.doi.org/10.5772/55398

467

10, 11]. Mitochondrial accumulation was observed in both CD8+

stages, or required to maintain the differentiated population.

Nix and its related family member, Bnip3, are unique mitochondrial-localized BH3-only proteins. Although they can induce apoptosis when over expressed like most of the BH3-only proteins, Nix and Bnip3 function to stimulate autophagy and mitophagy as well. The mito‐ phagic function of Nix was discovered in differentiating reticulocytes. Researchers knew that Nix expression dramatically increases in the terminal stage of reticulocyte differentiation, but the purpose for this remained unknown for several years [8]. In a relatively simple experiment, researchers harvested erythrocytes from Nix-/- mice and using mitotrophic dyes quantified mitochondria-containing erythrocytes. A significant population of the Nix-/- erythrocytes still contained mitochondria. The absence of Nix did not affect LC3 levels, and autophagosomes were still present. However, the autophagosomes in Nix-/- erythrocytes contained significantly less mitochondria than the wild type controls [4].

Erythrocytes from Ulk1-/- mice retained mitochondria and ribosomes, indicating the necessity of the general autophagy program in reticulocyte differentiation. Inhibiting autophagy with the class III-PI3K inhibitor 3-methyladenine (3-MA) in wild type reticulo‐ cytes prevented mitochondrial clearance as well. The peripheral blood in Ulk-/- mice contained an increased number of reticulocytes indicating a reduced capacity to be converted to erythrocytes [3]. Transplantation of fetal liver cells from Atg7-/- mice into irradiated wild type mice resulted in overall fewer erythrocytes, and these erythrocytes contained mitochondria.[7]. Thus, both general macroautophagy, and organelle-specific autophagy is required for erythrocyte differentiation. The above studies focused on mitochondria, but whether specific autophagic targeting of other organelles, such as ribosomes and ER, is mediated by Nix or other organelle-specific factors during reticulo‐ cyte differentiation remains unanswered. Bnip3, like Nix, localizes to the ER, and research‐ ers recently demonstrated that Bnip3 targets the ER for autophagy [9]. This raises the possibility that Nix may also target the ER for autophagy. Targeted autophagic degrada‐ tion of ribosomes, termed ribophagy, occurs in S. cerevisiae. However, whether selective ribophagy occurs in higher eukaryotes remains unknown [10]. These questions would be interesting to answer in the reticulocyte differentiation model.

#### **2.2. Lymphocytes**

**2. Role of autophagy during differentiation**

466 Autophagy - A Double-Edged Sword - Cell Survival or Death?

for autophagy by a protein called Nix [3-7].

less mitochondria than the wild type controls [4].

interesting to answer in the reticulocyte differentiation model.

Erythrocytes are especially unique cells; they lack a nucleus, internal membrane-bound organelles, and ribosomes, and they are packed with the oxygen transporter hemoglobin. The cell arises from reticulocytes, a nucleated progenitor capable of generating the needed surplus hemoglobin and the erythrocyte itself [1,2]. Generating such a simple cell requires processes that eliminate organelles not essential for fully differentiated erythrocytes. A series of publi‐ cations from 2008 to present not only elucidated the involvement of autophagy during reticulocyte differentiation, but also discovered that the mitochondria are specifically targeted

Nix and its related family member, Bnip3, are unique mitochondrial-localized BH3-only proteins. Although they can induce apoptosis when over expressed like most of the BH3-only proteins, Nix and Bnip3 function to stimulate autophagy and mitophagy as well. The mito‐ phagic function of Nix was discovered in differentiating reticulocytes. Researchers knew that Nix expression dramatically increases in the terminal stage of reticulocyte differentiation, but the purpose for this remained unknown for several years [8]. In a relatively simple experiment, researchers harvested erythrocytes from Nix-/- mice and using mitotrophic dyes quantified mitochondria-containing erythrocytes. A significant population of the Nix-/- erythrocytes still contained mitochondria. The absence of Nix did not affect LC3 levels, and autophagosomes were still present. However, the autophagosomes in Nix-/- erythrocytes contained significantly

Erythrocytes from Ulk1-/- mice retained mitochondria and ribosomes, indicating the necessity of the general autophagy program in reticulocyte differentiation. Inhibiting autophagy with the class III-PI3K inhibitor 3-methyladenine (3-MA) in wild type reticulo‐ cytes prevented mitochondrial clearance as well. The peripheral blood in Ulk-/- mice contained an increased number of reticulocytes indicating a reduced capacity to be converted to erythrocytes [3]. Transplantation of fetal liver cells from Atg7-/- mice into irradiated wild type mice resulted in overall fewer erythrocytes, and these erythrocytes contained mitochondria.[7]. Thus, both general macroautophagy, and organelle-specific autophagy is required for erythrocyte differentiation. The above studies focused on mitochondria, but whether specific autophagic targeting of other organelles, such as ribosomes and ER, is mediated by Nix or other organelle-specific factors during reticulo‐ cyte differentiation remains unanswered. Bnip3, like Nix, localizes to the ER, and research‐ ers recently demonstrated that Bnip3 targets the ER for autophagy [9]. This raises the possibility that Nix may also target the ER for autophagy. Targeted autophagic degrada‐ tion of ribosomes, termed ribophagy, occurs in S. cerevisiae. However, whether selective ribophagy occurs in higher eukaryotes remains unknown [10]. These questions would be

**2.1. Erythrocytes**

In the same Atg7-/- transplantation model, there was a four-fold reduction in the number of white blood cells and nine-fold reduction of lymphoid cells [7]. This observation was further validated in hematopoietic-specific stem, fetal, and adult cell Atg7 knockout mouse models [9, 10, 11]. Mitochondrial accumulation was observed in both CD8+ and CD4+ T-cells, and these cells succumbed to apoptosis in vitro more readily than the wild type controls [11]. T-cell specific knockouts of Atg5 and Atg7 also displayed increased amounts of mitochondria and were more susceptible to apoptosis [12,13]. Considering that the overall population of mature T lymphocytes decreased and sensitivity to apoptosis increased in the autophagy-deficient T lymphocyte models, it seems that the lymphopenia occurs due to excess cell death. Puo et al tested this by stimulating T-cell proliferation in wild type and Atg5-/- T-cells and quantifying the amount of daughter cells produced by using the stable dye, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). CFSE diffuses into both cells during mitosis, causing a decrease in individual cell fluorescent intensity with every division. The analysis demonstrat‐ ed that stimulated Atg5-/- T-cells did not produce daughter cells after three days and the authors concluded that Atg5 is necessary for T-cell proliferation. However, CFSE cannot differentiate between a dead or permeabilized cell and a non-proliferating cell, and over the three day period there was a moderate increase in apoptosis [14,15]. In this model, it appears that autophagy is required during T-cell hematopoiesis, at least in part, to prevent death of the newly differentiated cells. It would be interesting to investigate this in an inducible model, to test whether the dependence on autophagy is transient, i.e. only during early differentiation stages, or required to maintain the differentiated population.

#### **2.3. Adipocytes**

Evidence also supports a role for autophagy in adipocyte differentiation. Treatment of mouse embryonic fibroblasts (MEFs) with a cocktail of differentiation-inducing factors over the course of two weeks causes accumulation of lipid analogous to mature adipocytes. In the absence of Atg5 the MEFs fail to accumulate lipid droplets or display the molecular or structural pheno‐ type of differentiated adipocytes. Furthermore, Atg5-/- mouse pups have far fewer adipocytes [16]. Knocking down Atg7 in 3T3-L1 preadipocytes attenuated the accumulation of triglycer‐ ides and several markers of mature adipocytes as well. This was further validated in vivo in an adipose-specific Atg7 knock out mouse; the total fat in the knock out mouse weighed less than half that of the wild type mice. Interestingly, the adipose-specific Atg7-/- mouse had increased amounts of brown adipose tissue (BAT) and less white adipose tissue (WAT) relative to the wild type mouse, and both the lipid droplets and adipocytes were smaller and more densely packed than normal adipocytes [17,18]. The brown appearance of BAT is due to high quantities of densely packed mitochondria. BAT can uncouple the mitochondrial ATPase and continue oxidizing nutrients to generate thermal energy in place of the chemical energy provided by ATP. Zhang et al. validated these results, but went further and confirmed an increased presence of mitochondria [18]. Thus, while autophagy deficient adipocytes fail to express differentiation markers and lack lipid droplet morphology, some of the effects seen in autophagy-deficient adipocytes may result from the accumulation of mitochondria. Indeed, increased lipid catabolism in the form of β-oxidation occurred in the Atg7-/- adipocytes [17]. These findings points to a potentially functionally significant role for mitophagy during adipocyte differentiation. WAT primarily stores fatty acids for use under starvation conditions. Perhaps mitophagy is a necessary step for preventing wasteful energy expenditure through lipid catabolism in adipocytes. However, loss of the pro-mitophagic protein Bnip3 causes hepatocytes to accumulate mitochondria like adipocytes, but at the same time actually increase anabolic processes such as fatty acid synthesis [19]. It would be interesting to identify the cause of these differences.

cells of the EB display increased amounts of autophagic vacuoles similar to the breast epithelia 3D acini. However, unlike what happens in the breast acini, autophagy inhibition resulted in the accumulation of dead cell bodies in the EB lumen, suggesting that in this case increased autophagy is responsible for clearing out the dead cell debris accumulating at the center of the EB spheroid. In EB, cavitation occurs in response to increased hypoxia. Qi et al concluded that the hypoxic environment increased apoptosis inducing factor (AIF), which in turn increased ROS production and HIF2α signaling, and subsequently up-regulated pro-autophagic Bnip3. Knocking down any of the aforementioned proteins delayed cavitation of the EB. However, unlike the 3D breast acini model, inhibiting autophagy delayed caspase cleavage [29].

Integrin and Adhesion Regulation of Autophagy and Mitophagy

http://dx.doi.org/10.5772/55398

469

The examples in breast and embryoid bodies indicate that one of the roles of autophagy is to control when and how cell death occurs during differentiation, whereas autophagy is required for organelle clearing and survival in granulocytic keratinocytes and erythrocytes [21]. Thus, depending on the situation or cell state, autophagy can play distinct roles in differentiation.

A key question that remains in many of these models is why autophagy is important during differentiation. Mitochondrial clearance is likely important for the functional aspect of erythrocytes; if erythrocytes still contained mitochondria and carried out oxidative phosphor‐ ylation they would probably deplete much of their own oxygen in circulation before reaching tissues needing oxygen. Failure to eliminate mitochondria during adipocyte differentiation results in increased catabolism and a poor ability to store fat. However, in many systems autophagy seems to allow the cells to simply survive differentiation. Perhaps the metabolic stress of differentiating takes a toll on the integrity of the mitochondria and other intracellular components and autophagy increases just to keep up with the increased demand for mainte‐ nance. On the other hand, autophagy may have specific tumor suppressive effects during differentiation. Loss or reduction in autophagy appears to be required for tumor initiation and it is well established that tumorigenesis is linked with aberrant differentiation. One possibility is that autophagy actually promotes terminal differentiation, creating an antagonist to tumorigenesis. The sacrifice the tumor cells make is to create a greater dependence on antiapoptotic survival pathways to compensate for the lack of the survival benefit of autophagy. However, as cancer progresses, some aspects of the autophagy pathway re-emerge as a mechanism to escape death under stress, but this must be balanced by preventing differentia‐

In the case of multilayered epithelia, such as skin, bladder, prostate, and breast, differentiation occurs as the cells detach from the basement membrane and ascend apically. Such major changes require extensive reprogramming of signaling networks and gene expression, and cells must eliminate or inhibit the former cellular programming machinery. Failure to effi‐ ciently modify the programming can cause cellular stress and/or oncogenesis. Perhaps an immediate increase in autophagy aids in temporal separation of the undifferentiated and differentiated cell signaling and programming pathways. Coincident with this process is the

tion and reduced dependence on mitochondria for energy.

**3. Adhesion and regulation of autophagy**

#### **2.4. Epithelial cells**

Autophagy plays a role in different aspects of epithelial differentiation. One aspect resembles that seen in erythrocytes. Keratinocyte basal cells begin differentiation by detaching from the basement membrane and moving apically toward their fate of cornification and exfoliation. In the granulation stage, keratinocytes lose their organelles and nucleus [20]. Increased protea‐ some activity accompanies granulation and accounts for some of the degradative action, but entire organelles may require the autophagy pathway. Granulating keratinocytes are packed with lysosome bodies, and examination of expression patterns during differentiation revealed increased expression of the pro-autophagic proteins Beclin and Sirt1 [21,22]. Whether these events are more specifically controlled by organelle-specific autophagy mechanisms as is observed in erythrocytes has yet to be determined.

A question that remains is whether there is a role for autophagy in earlier epithelial differen‐ tiation stages, such as during the first emergence of suprabasal cells in striated epithelium. In one study, 3-MA-treated keratinocytes failed to express the differentiation marker involucrin when stimulated to differentiate in low glucose and high calcium [22]. However, this study should be interpreted with caution, because the reduction in glucose used to induce differen‐ tiation is a metabolic stressor. Because the metabolic state of cells regulates rates of autophagy, the increase in autophagy seen here may be an independent coincidence caused by the metabolic stress used to induce differentiation. Additionally, the sole use of 3-MA to inhibit autophagy brings up the question of selectivity. 3-MA also inhibits class I PI3K [23], an important regulator of keratinocyte differentiation [24,25].

The most striking link between autophagy and early epithelial differentiation comes from studies in the mammary gland. Autophagy promotes the survival of differentiated breast epithelial cells in 3D models of breast acinar formation [26]. Like keratinocytes, the breast epithelial cells lose adhesion to the ECM and push away from the basement membrane as differentiation occurs. Autophagy is increased in the differentiating cells in the center of the acini. However, inhibiting autophagy with 3-MA during differentiation increases luminal cell caspase-3 cleavage and death rather than blocking differentiation per se [27]. This suggests autophagy controls the rate of lumen cell loss, but the reason for needing this intermediate step is not clear.

Another example of autophagy in epithelial differentiation is in development of the embryoid body (EB). Embryoid bodies develop spontaneously from cultured embryonic stem cells, forming a spherical body of cells that can differentiate into various cell types [28]. The inner cells of the EB display increased amounts of autophagic vacuoles similar to the breast epithelia 3D acini. However, unlike what happens in the breast acini, autophagy inhibition resulted in the accumulation of dead cell bodies in the EB lumen, suggesting that in this case increased autophagy is responsible for clearing out the dead cell debris accumulating at the center of the EB spheroid. In EB, cavitation occurs in response to increased hypoxia. Qi et al concluded that the hypoxic environment increased apoptosis inducing factor (AIF), which in turn increased ROS production and HIF2α signaling, and subsequently up-regulated pro-autophagic Bnip3. Knocking down any of the aforementioned proteins delayed cavitation of the EB. However, unlike the 3D breast acini model, inhibiting autophagy delayed caspase cleavage [29].

The examples in breast and embryoid bodies indicate that one of the roles of autophagy is to control when and how cell death occurs during differentiation, whereas autophagy is required for organelle clearing and survival in granulocytic keratinocytes and erythrocytes [21]. Thus, depending on the situation or cell state, autophagy can play distinct roles in differentiation.

A key question that remains in many of these models is why autophagy is important during differentiation. Mitochondrial clearance is likely important for the functional aspect of erythrocytes; if erythrocytes still contained mitochondria and carried out oxidative phosphor‐ ylation they would probably deplete much of their own oxygen in circulation before reaching tissues needing oxygen. Failure to eliminate mitochondria during adipocyte differentiation results in increased catabolism and a poor ability to store fat. However, in many systems autophagy seems to allow the cells to simply survive differentiation. Perhaps the metabolic stress of differentiating takes a toll on the integrity of the mitochondria and other intracellular components and autophagy increases just to keep up with the increased demand for mainte‐ nance. On the other hand, autophagy may have specific tumor suppressive effects during differentiation. Loss or reduction in autophagy appears to be required for tumor initiation and it is well established that tumorigenesis is linked with aberrant differentiation. One possibility is that autophagy actually promotes terminal differentiation, creating an antagonist to tumorigenesis. The sacrifice the tumor cells make is to create a greater dependence on antiapoptotic survival pathways to compensate for the lack of the survival benefit of autophagy. However, as cancer progresses, some aspects of the autophagy pathway re-emerge as a mechanism to escape death under stress, but this must be balanced by preventing differentia‐ tion and reduced dependence on mitochondria for energy.

## **3. Adhesion and regulation of autophagy**

increased lipid catabolism in the form of β-oxidation occurred in the Atg7-/- adipocytes [17]. These findings points to a potentially functionally significant role for mitophagy during adipocyte differentiation. WAT primarily stores fatty acids for use under starvation conditions. Perhaps mitophagy is a necessary step for preventing wasteful energy expenditure through lipid catabolism in adipocytes. However, loss of the pro-mitophagic protein Bnip3 causes hepatocytes to accumulate mitochondria like adipocytes, but at the same time actually increase anabolic processes such as fatty acid synthesis [19]. It would be interesting to identify the cause

Autophagy plays a role in different aspects of epithelial differentiation. One aspect resembles that seen in erythrocytes. Keratinocyte basal cells begin differentiation by detaching from the basement membrane and moving apically toward their fate of cornification and exfoliation. In the granulation stage, keratinocytes lose their organelles and nucleus [20]. Increased protea‐ some activity accompanies granulation and accounts for some of the degradative action, but entire organelles may require the autophagy pathway. Granulating keratinocytes are packed with lysosome bodies, and examination of expression patterns during differentiation revealed increased expression of the pro-autophagic proteins Beclin and Sirt1 [21,22]. Whether these events are more specifically controlled by organelle-specific autophagy mechanisms as is

A question that remains is whether there is a role for autophagy in earlier epithelial differen‐ tiation stages, such as during the first emergence of suprabasal cells in striated epithelium. In one study, 3-MA-treated keratinocytes failed to express the differentiation marker involucrin when stimulated to differentiate in low glucose and high calcium [22]. However, this study should be interpreted with caution, because the reduction in glucose used to induce differen‐ tiation is a metabolic stressor. Because the metabolic state of cells regulates rates of autophagy, the increase in autophagy seen here may be an independent coincidence caused by the metabolic stress used to induce differentiation. Additionally, the sole use of 3-MA to inhibit autophagy brings up the question of selectivity. 3-MA also inhibits class I PI3K [23], an

The most striking link between autophagy and early epithelial differentiation comes from studies in the mammary gland. Autophagy promotes the survival of differentiated breast epithelial cells in 3D models of breast acinar formation [26]. Like keratinocytes, the breast epithelial cells lose adhesion to the ECM and push away from the basement membrane as differentiation occurs. Autophagy is increased in the differentiating cells in the center of the acini. However, inhibiting autophagy with 3-MA during differentiation increases luminal cell caspase-3 cleavage and death rather than blocking differentiation per se [27]. This suggests autophagy controls the rate of lumen cell loss, but the reason for needing this intermediate

Another example of autophagy in epithelial differentiation is in development of the embryoid body (EB). Embryoid bodies develop spontaneously from cultured embryonic stem cells, forming a spherical body of cells that can differentiate into various cell types [28]. The inner

of these differences.

468 Autophagy - A Double-Edged Sword - Cell Survival or Death?

**2.4. Epithelial cells**

step is not clear.

observed in erythrocytes has yet to be determined.

important regulator of keratinocyte differentiation [24,25].

In the case of multilayered epithelia, such as skin, bladder, prostate, and breast, differentiation occurs as the cells detach from the basement membrane and ascend apically. Such major changes require extensive reprogramming of signaling networks and gene expression, and cells must eliminate or inhibit the former cellular programming machinery. Failure to effi‐ ciently modify the programming can cause cellular stress and/or oncogenesis. Perhaps an immediate increase in autophagy aids in temporal separation of the undifferentiated and differentiated cell signaling and programming pathways. Coincident with this process is the need to maintain cell survival. This brings to question what signaling molecules in differen‐ tiation regulate autophagy.

but inhibiting EGFR alone did not. Since a class I-specific inhibitor could not induce apoptosis in PrECs, it is unlikely that off target inhibition of class I PI3K by 3-MA resulted in caspase cleavage. In addition, cell adhesion or inhibition of EGFR had no effect on Akt phosphoryla‐ tion, which suggests that integrin-mediated maintenance of autophagy does not occur through the PI3K/Akt pathway in PrECs [36]. This study demonstrated that integrin adhesion mediates autophagy. While the exact downstream signaling pathway from integrins to autophagy remains unclear, survival was dependent on Erk signaling which is known to promote autophagy. A subsequent study demonstrated that in prostate cancer cells the androgen receptor promoted survival via integrin α6β1 activation of IKK/NF-κB signaling, independent of the PI3K/Akt pathway [37]. A similar dependence on the IKK/NF-κB pathway for survival is mediated by integrin α6β4 in polarized cells of 3D breast acinar structures and over activation of this pathway is sufficient to overcome suspension-induced anoikis and apoptosis [38-40]. Given that activation of the IKK/NF-κB pathway can induce autophagy [41], it is reasonable to suspect that integrins may regulate autophagy in part through the IKK/NF-κB pathway (Figure 1A). Further insight into how integrins regulate NF-κB and autophagy is

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Stimulation of smooth muscle cells with the integrin ligand osteopontin, stimulated autophagy related-genes, autophagosome formation, and ultimately cell death [42]. This effect was mediated by p38-MAPK through integrin and CD44 (Figure 1C). In a Drosophila genetic screen, paxillin, a downstream target of integrins, was found to associate with Atg1 and to be required for starvation-induced autophagy in MEFs [43]. However, in the fly, the ability of paxillin to facilitate autophagy was not linked with integrins. Whether this is true in the mammalian model has not been determined. Thus, several studies implicate integrin-based adhesion in both suppressing as well as stimulating autophagy. Since all these studies were conducted in different model systems and under different culture conditions, it is difficult to draw a definitive conclusion. Therefore, when studying different biological events it is necessary to consider several possible mechanisms and signaling pathways that could

Some consideration should be given to a potential alternative mechanism to explain detach‐ ment-induced autophagy. Although it was quite thoroughly demonstrated that the cells increased the rate of autophagy after losing ECM contact, in both the EB and acini models the cells are undergoing a differentiation program and remaining in contact with neighboring cells. In the MCF-10A breast model where cells were put into suspension and 25% survived anoikis, these cells existed in large aggregates [27]. In light of the fact that increased cell density also increases autophagy in vitro, one could hypothesize that the proteins mediating cell-cell

A relatively new process called entosis, whereby cell-cell adhesion induces the entry of one cell into a neighboring cell, was found to trigger an autophagic response [44] by mechanisms that resemble pathogen engulfment by immune cells. The ability of cells to entose depends on adherens junctions in the absence of integrin adhesion [45]. The major protein that mediates

contact may be signaling to increase autophagy in the detached cells [23].

**3.3. Cell-cell adhesion-induced autophagy**

needed.

influence autophagy.

#### **3.1. Integrins and detachment-induced autophagy**

Integrins, a family of heterodimeric transmembrane proteins, mediate cell adhesion to the extracellular matrix (ECM). Integrins regulate a variety of functions at the cellular and molecular level [30]. In stratified epithelial cell differentiation the integrins holding basal cells to the basement membrane become internalized and eliminated as the cell stratifies. Integrins form focal points with complexes of signaling molecules to maintain the connection between the matrix and the contracting cytoskeleton [31]. Cell survival is a key function of integrins. Loss of cell adhesion due to disengagement of integrins from their ECM ligand simultaneously activates extrinsic and intrinsic apoptotic cell death termed anoikis [32]. This signaling pathway prevents exfoliated or damaged cells from surviving detachment and adhering at an improper location.

Detachment and anoikis also aids in lumen clearing in secretory epithelium. As epithelial cells become contact inhibited, they force some cells off the basement membrane [33]. In undiffer‐ entiated epithelium this loss of adhesion would normally trigger anoikis and apoptosis. However, in differentiating epithelium, autophagy is triggered which delays the rapid onset of anoikis. This was demonstrated in both 3D acini and in suspended cells. Autophagy was induced in a subpopulation of human mammary epithelial MCF10A and canine kidney epithelial cells when they were placed in suspension, which allowed them to survive anoikis. Inhibiting autophagy by knocking down the autophagy proteins ATG5, ATG6, and ATG7 in suspended MCF-10A cells increased cleaved caspase-3 positive cells and decreased replating efficiency [27]. Further studies demonstrated that the decision to undergo autophagy, as opposed to anoikis, is controlled in part by up-regulation PERK to suppress ROS, through activation of the ER stress pathway, and resulting in increased expression of ATG6 and ATG8 [34]. When squamous cell carcinomas (SCCs) were isolated from FAK-/- mice (which presum‐ ably have altered integrin-based adhesion), or when SCCs isolated from wild type mice were placed in suspension, the rate of autophagy was significantly increased. This resulted in the targeting of Src to autophagosomes through c-Cbl in an E3-ligase-independent mechanism [35]. The major conclusion drawn from these studies is that loss of matrix adhesion induces autophagy.

#### **3.2. Attachment-induced autophagy**

This latter conclusion is partially contradicted by several studies demonstrating that adhesion to matrix is required to promote autophagy. In a study on human primary basal prostate epithelial cells (PrEC) researchers found that blocking integrin interactions with the ECM inhibited autophagy induced by starvation. In culture, PrECs secrete and adhere to a laminin 5, via integrins α3β1 and α6β4. These integrins were necessary for maintaining cell survival through Src and ligand-independent EGFR activation. Blocking integrin α3, α6, or β4 with antibodies inhibited both survival and autophagy in these cells. Particularly interesting is that inhibiting autophagy with 3-MA or blocking integrin α3 antibodies induced caspase cleavage, but inhibiting EGFR alone did not. Since a class I-specific inhibitor could not induce apoptosis in PrECs, it is unlikely that off target inhibition of class I PI3K by 3-MA resulted in caspase cleavage. In addition, cell adhesion or inhibition of EGFR had no effect on Akt phosphoryla‐ tion, which suggests that integrin-mediated maintenance of autophagy does not occur through the PI3K/Akt pathway in PrECs [36]. This study demonstrated that integrin adhesion mediates autophagy. While the exact downstream signaling pathway from integrins to autophagy remains unclear, survival was dependent on Erk signaling which is known to promote autophagy. A subsequent study demonstrated that in prostate cancer cells the androgen receptor promoted survival via integrin α6β1 activation of IKK/NF-κB signaling, independent of the PI3K/Akt pathway [37]. A similar dependence on the IKK/NF-κB pathway for survival is mediated by integrin α6β4 in polarized cells of 3D breast acinar structures and over activation of this pathway is sufficient to overcome suspension-induced anoikis and apoptosis [38-40]. Given that activation of the IKK/NF-κB pathway can induce autophagy [41], it is reasonable to suspect that integrins may regulate autophagy in part through the IKK/NF-κB pathway (Figure 1A). Further insight into how integrins regulate NF-κB and autophagy is needed.

Stimulation of smooth muscle cells with the integrin ligand osteopontin, stimulated autophagy related-genes, autophagosome formation, and ultimately cell death [42]. This effect was mediated by p38-MAPK through integrin and CD44 (Figure 1C). In a Drosophila genetic screen, paxillin, a downstream target of integrins, was found to associate with Atg1 and to be required for starvation-induced autophagy in MEFs [43]. However, in the fly, the ability of paxillin to facilitate autophagy was not linked with integrins. Whether this is true in the mammalian model has not been determined. Thus, several studies implicate integrin-based adhesion in both suppressing as well as stimulating autophagy. Since all these studies were conducted in different model systems and under different culture conditions, it is difficult to draw a definitive conclusion. Therefore, when studying different biological events it is necessary to consider several possible mechanisms and signaling pathways that could influence autophagy.

Some consideration should be given to a potential alternative mechanism to explain detach‐ ment-induced autophagy. Although it was quite thoroughly demonstrated that the cells increased the rate of autophagy after losing ECM contact, in both the EB and acini models the cells are undergoing a differentiation program and remaining in contact with neighboring cells. In the MCF-10A breast model where cells were put into suspension and 25% survived anoikis, these cells existed in large aggregates [27]. In light of the fact that increased cell density also increases autophagy in vitro, one could hypothesize that the proteins mediating cell-cell contact may be signaling to increase autophagy in the detached cells [23].

#### **3.3. Cell-cell adhesion-induced autophagy**

need to maintain cell survival. This brings to question what signaling molecules in differen‐

Integrins, a family of heterodimeric transmembrane proteins, mediate cell adhesion to the extracellular matrix (ECM). Integrins regulate a variety of functions at the cellular and molecular level [30]. In stratified epithelial cell differentiation the integrins holding basal cells to the basement membrane become internalized and eliminated as the cell stratifies. Integrins form focal points with complexes of signaling molecules to maintain the connection between the matrix and the contracting cytoskeleton [31]. Cell survival is a key function of integrins. Loss of cell adhesion due to disengagement of integrins from their ECM ligand simultaneously activates extrinsic and intrinsic apoptotic cell death termed anoikis [32]. This signaling pathway prevents exfoliated or damaged cells from surviving detachment and adhering at an

Detachment and anoikis also aids in lumen clearing in secretory epithelium. As epithelial cells become contact inhibited, they force some cells off the basement membrane [33]. In undiffer‐ entiated epithelium this loss of adhesion would normally trigger anoikis and apoptosis. However, in differentiating epithelium, autophagy is triggered which delays the rapid onset of anoikis. This was demonstrated in both 3D acini and in suspended cells. Autophagy was induced in a subpopulation of human mammary epithelial MCF10A and canine kidney epithelial cells when they were placed in suspension, which allowed them to survive anoikis. Inhibiting autophagy by knocking down the autophagy proteins ATG5, ATG6, and ATG7 in suspended MCF-10A cells increased cleaved caspase-3 positive cells and decreased replating efficiency [27]. Further studies demonstrated that the decision to undergo autophagy, as opposed to anoikis, is controlled in part by up-regulation PERK to suppress ROS, through activation of the ER stress pathway, and resulting in increased expression of ATG6 and ATG8 [34]. When squamous cell carcinomas (SCCs) were isolated from FAK-/- mice (which presum‐ ably have altered integrin-based adhesion), or when SCCs isolated from wild type mice were placed in suspension, the rate of autophagy was significantly increased. This resulted in the targeting of Src to autophagosomes through c-Cbl in an E3-ligase-independent mechanism [35]. The major conclusion drawn from these studies is that loss of matrix adhesion induces

This latter conclusion is partially contradicted by several studies demonstrating that adhesion to matrix is required to promote autophagy. In a study on human primary basal prostate epithelial cells (PrEC) researchers found that blocking integrin interactions with the ECM inhibited autophagy induced by starvation. In culture, PrECs secrete and adhere to a laminin 5, via integrins α3β1 and α6β4. These integrins were necessary for maintaining cell survival through Src and ligand-independent EGFR activation. Blocking integrin α3, α6, or β4 with antibodies inhibited both survival and autophagy in these cells. Particularly interesting is that inhibiting autophagy with 3-MA or blocking integrin α3 antibodies induced caspase cleavage,

tiation regulate autophagy.

improper location.

autophagy.

**3.2. Attachment-induced autophagy**

**3.1. Integrins and detachment-induced autophagy**

470 Autophagy - A Double-Edged Sword - Cell Survival or Death?

A relatively new process called entosis, whereby cell-cell adhesion induces the entry of one cell into a neighboring cell, was found to trigger an autophagic response [44] by mechanisms that resemble pathogen engulfment by immune cells. The ability of cells to entose depends on adherens junctions in the absence of integrin adhesion [45]. The major protein that mediates

Atorva to induce an endoplasmic reticulum stress response, autophagy, and phosphorylation of eIF2α [51]. Interestingly, in the MCF-10A detachment-induced autophagy model, suspend‐ ed cells had increased levels of activated PERK, a well-documented cytoprotective ER stressresponse protein that induces autophagy, and increased phosphorylation of the downstream PERK target eIF2α in conjunction with increased autophagic flux [34]. Considering the overlapping pathways and the correlation of detachment-induced autophagy with cell-cell

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Deficiency of the laminin α2 chain (LAMA2) in laminin 2 causes a form of congenital muscular dystrophy known as MDC1A. MDC1A patients present with severe muscle weakness, peripheral neuropathy, and joint contractures [52]. The LAMA2-deficient mouse model (dy3K/ dy3K) develops muscle weakness two weeks after birth and dies by 5 weeks with severe muscular dystrophy [53,54]. The dy3K/dy3K mice feature increases in myocyte apoptosis, degeneration/regeneration cycles, variable fiber sizes, and connective tissue hyperprolifera‐ tion. dy3K/dy3K mice exhibit significantly increased expression of the autophagy related genes Bnip3, Bnip3l, p62, LC3B, GABARAP1, Vps34, Atg4b, Cathepsin L, Lamp2a, and Beclin. Administration of 3-MA to the dy3K/dy3K mice increased lifespans, increased average muscle fiber diameter, decreased the presence of caspase-3 in myocytes, and partially restored muscular morphology. In addition, immunofluorescent staining of muscle biopsies from MDC1A patients showed an increased amount of LC3B in the myocytes. Thus, patients with MDC1A may essentially suffer from excessive muscular autophagy due to a lack of interaction with the matrix protein laminin 2 (Figure 2A). This example supports a role for matrix

On the other hand, one of the LAMA2 receptors, dystroglycan which binds laminin extracell‐ ularly and dystrophin intracellularly, may promote adhesion-dependent autophagy. One function of the dystroglycan/dystrophin complex is to bind F-actin to anchor the cytoskeleton in place. Like the dy3K/dy3K mice, the dystrophin mutant (mdx) mice display a muscular dystrophy phenotype. However, in this case, the mdx mice accumulate damaged mitochon‐ dria, and inducing autophagy through AMPK activation in these mice ameliorates the disease phenotype, indicating there is a defect in muscle autophagy in mdx mice. This further suggests that adhesion to laminin through the dystroglycan/dystrophin connection is required to maintain autophagy in myocytes [55,56]. It is worth noting that mutations in integrin α7 are associated with muscular dystrophy as well. The integrin dimer α7β1 is another receptor that binds laminin-α2 and resides on myocytes [57]. However, whether α7β1 interactions affect autophagy in these diseases remains unknown. Further evidence supporting the concept that cell adhesion mediates autophagy comes from the study of Ulrich's Congenital Muscular Dystrophy (UCMD) and Bethlem Myopathy. Both diseases involve chronic weakening and degradation of skeletal muscle due to the spontaneous death of myocytes, which ultimately leads to death. Genetic studies discerned that mutations in the gene coding for collagen VI correlate with the condition [58]. Furthermore, genetically modified mice lacking the collagen VI gene (Col6-/-) develop myopathies similar to UCMD and Bethlem Myopathy. The Col6-/-

contact this could be a promising area to investigate.

detachment in inducing autophagy.

**3.4. Genetic evidence for integrin adhesion and autophagy**

**Figure 1.** Cell Adhesion Pathways that Promote Autophagy. (A) Integrin α6β1 activates NF-κB through IKK signaling. Together, IKK and NF-κB can up-regulate and activate Beclin, and NF-κB in turn regulates pro-mitophagic Bnip3 ex‐ pression. (B) AMPK localizes with LKB1 at adherens junctions where LKB1 activates AMPK signaling, which can activate autophagy through Ulk1. (C) Osteopontin activates p38 MAPK-induced autophagy through CD44 and αvβ3.

epithelial cell-cell contact through adherens junctions is E-cadherin. This vital protein holds the epithelium together and promotes the survival of cells [46-48]. Adherens junctions mediate the connection between cells by essentially bridging the actin cytoskeleton of two different cells. A plethora of signaling events are controlled by E-cadherin. In the case of autophagy, one point of interest is regulation of AMPK.

In response to low ATP levels, AMPK directly phosphorylates and activates Ulk1 in parallel to deactivating the autophagy suppressor mTOR [49]. Membrane bound E-cadherin localizes with the AMPK activator LKB1 (Figure 1B), and by doing so increases AMPK activation by bringing it into close proximity with LKB1 [50]. In one study AMPK was required for the drug Atorva to induce an endoplasmic reticulum stress response, autophagy, and phosphorylation of eIF2α [51]. Interestingly, in the MCF-10A detachment-induced autophagy model, suspend‐ ed cells had increased levels of activated PERK, a well-documented cytoprotective ER stressresponse protein that induces autophagy, and increased phosphorylation of the downstream PERK target eIF2α in conjunction with increased autophagic flux [34]. Considering the overlapping pathways and the correlation of detachment-induced autophagy with cell-cell contact this could be a promising area to investigate.

#### **3.4. Genetic evidence for integrin adhesion and autophagy**

epithelial cell-cell contact through adherens junctions is E-cadherin. This vital protein holds the epithelium together and promotes the survival of cells [46-48]. Adherens junctions mediate the connection between cells by essentially bridging the actin cytoskeleton of two different cells. A plethora of signaling events are controlled by E-cadherin. In the case of autophagy,

**Figure 1.** Cell Adhesion Pathways that Promote Autophagy. (A) Integrin α6β1 activates NF-κB through IKK signaling. Together, IKK and NF-κB can up-regulate and activate Beclin, and NF-κB in turn regulates pro-mitophagic Bnip3 ex‐ pression. (B) AMPK localizes with LKB1 at adherens junctions where LKB1 activates AMPK signaling, which can activate autophagy through Ulk1. (C) Osteopontin activates p38 MAPK-induced autophagy through CD44 and αvβ3.

In response to low ATP levels, AMPK directly phosphorylates and activates Ulk1 in parallel to deactivating the autophagy suppressor mTOR [49]. Membrane bound E-cadherin localizes with the AMPK activator LKB1 (Figure 1B), and by doing so increases AMPK activation by bringing it into close proximity with LKB1 [50]. In one study AMPK was required for the drug

one point of interest is regulation of AMPK.

472 Autophagy - A Double-Edged Sword - Cell Survival or Death?

Deficiency of the laminin α2 chain (LAMA2) in laminin 2 causes a form of congenital muscular dystrophy known as MDC1A. MDC1A patients present with severe muscle weakness, peripheral neuropathy, and joint contractures [52]. The LAMA2-deficient mouse model (dy3K/ dy3K) develops muscle weakness two weeks after birth and dies by 5 weeks with severe muscular dystrophy [53,54]. The dy3K/dy3K mice feature increases in myocyte apoptosis, degeneration/regeneration cycles, variable fiber sizes, and connective tissue hyperprolifera‐ tion. dy3K/dy3K mice exhibit significantly increased expression of the autophagy related genes Bnip3, Bnip3l, p62, LC3B, GABARAP1, Vps34, Atg4b, Cathepsin L, Lamp2a, and Beclin. Administration of 3-MA to the dy3K/dy3K mice increased lifespans, increased average muscle fiber diameter, decreased the presence of caspase-3 in myocytes, and partially restored muscular morphology. In addition, immunofluorescent staining of muscle biopsies from MDC1A patients showed an increased amount of LC3B in the myocytes. Thus, patients with MDC1A may essentially suffer from excessive muscular autophagy due to a lack of interaction with the matrix protein laminin 2 (Figure 2A). This example supports a role for matrix detachment in inducing autophagy.

On the other hand, one of the LAMA2 receptors, dystroglycan which binds laminin extracell‐ ularly and dystrophin intracellularly, may promote adhesion-dependent autophagy. One function of the dystroglycan/dystrophin complex is to bind F-actin to anchor the cytoskeleton in place. Like the dy3K/dy3K mice, the dystrophin mutant (mdx) mice display a muscular dystrophy phenotype. However, in this case, the mdx mice accumulate damaged mitochon‐ dria, and inducing autophagy through AMPK activation in these mice ameliorates the disease phenotype, indicating there is a defect in muscle autophagy in mdx mice. This further suggests that adhesion to laminin through the dystroglycan/dystrophin connection is required to maintain autophagy in myocytes [55,56]. It is worth noting that mutations in integrin α7 are associated with muscular dystrophy as well. The integrin dimer α7β1 is another receptor that binds laminin-α2 and resides on myocytes [57]. However, whether α7β1 interactions affect autophagy in these diseases remains unknown. Further evidence supporting the concept that cell adhesion mediates autophagy comes from the study of Ulrich's Congenital Muscular Dystrophy (UCMD) and Bethlem Myopathy. Both diseases involve chronic weakening and degradation of skeletal muscle due to the spontaneous death of myocytes, which ultimately leads to death. Genetic studies discerned that mutations in the gene coding for collagen VI correlate with the condition [58]. Furthermore, genetically modified mice lacking the collagen VI gene (Col6-/-) develop myopathies similar to UCMD and Bethlem Myopathy. The Col6-/-

**Figure 2.** Matrix Adhesion Pathways and Autophagy in Muscle Cells. (A) Laminin α2 is bound by the α- and β-dystro‐ glycan complex, which in turn binds dystrophin, a protein that anchors the complex onto F-actin. Loss of Laminin α2 caused increase expression of Beclin, Bnip3, Bnip3l, Vps34, and several other autophagy related proteins. Both Bnip3 and Bnip3l can induce mitophagy as well. Integrin α7β1 binds laminin α2 as well, but a connection with autophagy has not been established. (B) Loss of Collagen VI increased pro autophagic and pro mitophagic genes and caused de‐ creased autophagy along with decreased expression of Beclin and Bnip3. The collagen receptor integrin α2β1 may be responsible for this.

**Figure 3.** Mechanisms for Cell Adhesion-Induced Autophagy. The intracellular domain of integrins binds to FAK, which activates the Ras/Raf/MEK/Erk signaling cascade. Integrins and dystrophin both can activate p38 MAPK. Both Erk and p38 can phosphorylate JNK and induce autophagy. JNK in turn can either inhibit autophagy through suppres‐ sion of FoxO or promote autophagy through activation of c-Jun, which can positively and negatively regulate autoph‐ agy. Integrins can also activate the IKKα/β–NF-κB pathway. IKKα/β induces autophagy both dependently and independently of NF-κB; however, NF-κB can also inhibit autophagy in some cases. Integrins also activate the PI3K/Akt/TORC1, which inhibit autophagy by phosphorylating and inhibiting FoxO3 and Ulk1. LKB1 binds to E-cad‐ herin and activates AMPK. AMPK both activates Ulk1 and inhibits TORC1, causing an increase in autophagy. Some evi‐

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Adhesion proteins interact with a host of proteins and activate several well characterized pathways that affect autophagy. Recall that the dystrophin glycoprotein complex promotes adhesion dependent signaling in myocytes, and the importance of this was highlighted in the Mdx and dy3K/dy3K mice (Figure 2A). When the dystrophin glycoprotein complex is bound to laminin, an intracellular component of the complex called syntrophin binds and activates the Rac1 signaling complex, leading to activation of JNK, a protein that can both positively and negatively regulate autophagy [63] (Figure 3). JNK can induce autophagy by phosphorylating Bcl-2 and thus preventing Bcl-2 from inhibiting Beclin [64]. JNK also regulates several tran‐ scription factors that in turn mediate expression or repression of autophagy related genes. For example, genetic ablation of all three JNK isoforms results in constitutive activation of the transcription factor FoxO, causing an increase in autophagy and the effector proteins Bnip3 and Beclin. JNK can also activate the transcription factor c-Jun, a protein which when overex‐

dence indicates that cell-cell adhesion may also activate the autophagy-inducer eIF2α.

**3.5. Cell adhesion signaling pathways that regulate autophagy**

 mice have dysmorphic and dysfunctional myofibers characterized by increased apoptosis and reduced muscle strength compared to wild type mice [59]. Electron microscopy of Col6-/ myocytes revealed damaged and misshapen mitochondria and biochemical assays demon‐ strated that the mitochondria are dysfunctional. Inhibiting mitochondrial permeablization with Cyclosporine A in Col6-/- mice and in patients with homologous myopathies ameliorated the diseases effects [60], and genetic ablation of the cyclosporine A target, Cyclophilin D, also mitigated the symptoms in Col6-/- mice [61]

Later research found that Col6-/- mice muscle tissue expressed lower levels of the pro-auto‐ phagic proteins Beclin and Bnip3 and displayed a significant decrease in autophagy (Figure 2B). Forced expression of Beclin and Bnip3 partially reversed the disease phenotype in Col6-/- myocytes, and inducing autophagy by fasting the Col6-/- mice reestablished autophagy and decreased apoptosis in the myocytes. Furthermore, fasting or treating the Col6-/- mice with Rapamycin, to block the autophagy inhibitor mTor, decreased the presence of dysfunctional mitochondria [62]. These latter studies on Col6-related myopathies indicate that myocyte cell adhesion to collagen maintains autophagic flux and cell survival, while unregulated detach‐ ment inhibits autophagy.

Integrin and Adhesion Regulation of Autophagy and Mitophagy http://dx.doi.org/10.5772/55398 475

**Figure 3.** Mechanisms for Cell Adhesion-Induced Autophagy. The intracellular domain of integrins binds to FAK, which activates the Ras/Raf/MEK/Erk signaling cascade. Integrins and dystrophin both can activate p38 MAPK. Both Erk and p38 can phosphorylate JNK and induce autophagy. JNK in turn can either inhibit autophagy through suppres‐ sion of FoxO or promote autophagy through activation of c-Jun, which can positively and negatively regulate autoph‐ agy. Integrins can also activate the IKKα/β–NF-κB pathway. IKKα/β induces autophagy both dependently and independently of NF-κB; however, NF-κB can also inhibit autophagy in some cases. Integrins also activate the PI3K/Akt/TORC1, which inhibit autophagy by phosphorylating and inhibiting FoxO3 and Ulk1. LKB1 binds to E-cad‐ herin and activates AMPK. AMPK both activates Ulk1 and inhibits TORC1, causing an increase in autophagy. Some evi‐ dence indicates that cell-cell adhesion may also activate the autophagy-inducer eIF2α.

#### **3.5. Cell adhesion signaling pathways that regulate autophagy**

 mice have dysmorphic and dysfunctional myofibers characterized by increased apoptosis and reduced muscle strength compared to wild type mice [59]. Electron microscopy of Col6-/ myocytes revealed damaged and misshapen mitochondria and biochemical assays demon‐ strated that the mitochondria are dysfunctional. Inhibiting mitochondrial permeablization with Cyclosporine A in Col6-/- mice and in patients with homologous myopathies ameliorated the diseases effects [60], and genetic ablation of the cyclosporine A target, Cyclophilin D, also

**Figure 2.** Matrix Adhesion Pathways and Autophagy in Muscle Cells. (A) Laminin α2 is bound by the α- and β-dystro‐ glycan complex, which in turn binds dystrophin, a protein that anchors the complex onto F-actin. Loss of Laminin α2 caused increase expression of Beclin, Bnip3, Bnip3l, Vps34, and several other autophagy related proteins. Both Bnip3 and Bnip3l can induce mitophagy as well. Integrin α7β1 binds laminin α2 as well, but a connection with autophagy has not been established. (B) Loss of Collagen VI increased pro autophagic and pro mitophagic genes and caused de‐ creased autophagy along with decreased expression of Beclin and Bnip3. The collagen receptor integrin α2β1 may be

Later research found that Col6-/- mice muscle tissue expressed lower levels of the pro-auto‐ phagic proteins Beclin and Bnip3 and displayed a significant decrease in autophagy (Figure 2B). Forced expression of Beclin and Bnip3 partially reversed the disease phenotype in Col6-/- myocytes, and inducing autophagy by fasting the Col6-/- mice reestablished autophagy and decreased apoptosis in the myocytes. Furthermore, fasting or treating the Col6-/- mice with Rapamycin, to block the autophagy inhibitor mTor, decreased the presence of dysfunctional mitochondria [62]. These latter studies on Col6-related myopathies indicate that myocyte cell adhesion to collagen maintains autophagic flux and cell survival, while unregulated detach‐

mitigated the symptoms in Col6-/- mice [61]

474 Autophagy - A Double-Edged Sword - Cell Survival or Death?

ment inhibits autophagy.

responsible for this.

Adhesion proteins interact with a host of proteins and activate several well characterized pathways that affect autophagy. Recall that the dystrophin glycoprotein complex promotes adhesion dependent signaling in myocytes, and the importance of this was highlighted in the Mdx and dy3K/dy3K mice (Figure 2A). When the dystrophin glycoprotein complex is bound to laminin, an intracellular component of the complex called syntrophin binds and activates the Rac1 signaling complex, leading to activation of JNK, a protein that can both positively and negatively regulate autophagy [63] (Figure 3). JNK can induce autophagy by phosphorylating Bcl-2 and thus preventing Bcl-2 from inhibiting Beclin [64]. JNK also regulates several tran‐ scription factors that in turn mediate expression or repression of autophagy related genes. For example, genetic ablation of all three JNK isoforms results in constitutive activation of the transcription factor FoxO, causing an increase in autophagy and the effector proteins Bnip3 and Beclin. JNK can also activate the transcription factor c-Jun, a protein which when overex‐ pressed can inhibit autophagy under starvation conditions [65]. On the other hand, another study showed that JNK was required for ceramide induced autophagy and expression of Beclin [66]. Furthermore, Jnk1 can phosphorylate Bcl-2 to release Beclin and promote autophagy [67]. Integrins may also regulate autophagy via p38 MAPK. Osteopontin binds integrins and CD44 and induces autophagy through activation of p38 MAPK [42]. Although some evidence indicates that p38 inhibits autophagy, p38 can activate JNK [68], which could provide a potential mechanism for both positive and negative regulation of autophagy by integrins. Finally, integrins stimulate Erk1/2 signaling as well, which activates autophagy through numerous pathways including activation of JNK [68].

mitophagic protein Bnip3, or by starvation-induced autophagy [61,62]. Although not discussed in this chapter, it is interesting to note that Parkinson's disease is associated with mutations in several genes that target damaged mitochondria for mitophagy, and increas‐ ing evidence implicates mitophagy as having a role in retarding aging in general [75,76]. Considering that mitochondria can induce apoptosis and regulate the levels of ROS, one could imagine mitophagy as being a mechanism of survival during detachment or a method

Integrin and Adhesion Regulation of Autophagy and Mitophagy

http://dx.doi.org/10.5772/55398

477

Cell interaction with ECM and other cells of the same organism is imperative for - and essentially defines - multicellular organisms. Aside from being the scaffolding that organizes cells into functional macrostructures, the ECM extensively regulates cellular behavior [30]. The importance of these interactions is well demonstrated in the mounting evidence indicating that varied interactions with the ECM can actually differentially dictate the fate of stem cell differentiation [77]. Understanding the effects of the ECM on cell biology not only increases the understanding of normal organismal development and biology, but also diseases such as cancer or the various forms of muscular dystrophy [78,79]. With the current rise in under‐ standing of autophagy in diseases and development, understanding how extracellular interactions and differentiation affect autophagy is an important avenue to explore in the

of maintaining cell integrity during adhesion.

future.

**Nomenclature**

3-MA - 3-methyladenine

AIF - apoptosis inducing factor

CFSE - 5,6-carboxyfluorescein diacetate succinimidyl ester

BAT - brown adipose tissue

Col6 - collagen VI gene

EB - embryoid body

ECM - extracellular matrix

LAMA2 - laminin α2 chain

MEF - mouse embryo fibroblast

SSC- squamous cell carcinomas

UCMD - Ulrich's Congenital Muscular Dystrophy

PrEC - prostate epithelial cell

WAT- white adipose tissues

Mdx - dystrophin gene

Signaling through IKKα/β–NF-κB is another node for promoting autophagy (Figure 3). IKKα/ β regulates the expression of autophagy-related genes by activating NF-κB, which in turn has been shown to both positively and negatively regulate autophagy [41,69,70]. IKKα/β can also activate autophagy independently of NF-κB [70]. Cells expressing constitutively active subunits of the IKK complex had increased autophagy and increased levels of phosphorylated active AMPK and JNK [71]. Considering that blocking integrin adhesion in PrECs subsequent‐ ly attenuated autophagy [36], and that integrin mediated adhesion to laminin increased IKKα/ β - NF-κB signaling enough to significantly increase survival in a prostate cancer line [46], signaling through IKKα/β - NF-κB may be a mechanism by which integrin-mediated-celladhesion promotes autophagy. It would be interesting to determine whether this mechanism occurs and whether it is dependent or independent of NF-κB.

PI3K/Akt signaling potently suppresses autophagy [72,73] (Figure 3). Akt activates the TORC1 complex, which in turn phosphorylates and inactivates the autophagy-initiating kinase Ulk1 [73]. Akt itself phosphorylates and inactivates the FoxO3a transcription factor. This prevents FoxO3a from inducing the expression of numerous autophagy- and Mitophagy-related genes [74]. Integrins can activate PI3K/Akt signaling [30], making it possible that integrins could suppress autophagy through this pathway as well. Perhaps this explains the cases in which detachment from the basement membrane actually promoted autophagy. However, it was shown that detachment of breast epithelial cells both increased autophagy and induced phosphorylation of eIF2α, a target of the ER kinase PERK and a protein capable of inducing autophagy [34].

AMPK activates autophagy by inhibiting TORC1 and activating Ulk1 via direct phosphory‐ lation [49]. LKB1 localizes with engaged E-cadherin and activates AMPK signaling when ATP levels are low (Figure 3), providing a potential mechanism for cell-cell-interaction-mediated autophagy that might be relevant in detachment induced autophagy mechanisms in which cell-cell contact is maintained [50].

## **4. Conclusion**

In many of the studies discussed above and in other diseases and models the effects of autophagy are very mitochondria-centric. For example, the Col6-/- mice phenotype was rescued by inhibiting mitochondrial depolarization, re-expressing Beclin or the pro-

mitophagic protein Bnip3, or by starvation-induced autophagy [61,62]. Although not discussed in this chapter, it is interesting to note that Parkinson's disease is associated with mutations in several genes that target damaged mitochondria for mitophagy, and increas‐ ing evidence implicates mitophagy as having a role in retarding aging in general [75,76]. Considering that mitochondria can induce apoptosis and regulate the levels of ROS, one could imagine mitophagy as being a mechanism of survival during detachment or a method of maintaining cell integrity during adhesion.

Cell interaction with ECM and other cells of the same organism is imperative for - and essentially defines - multicellular organisms. Aside from being the scaffolding that organizes cells into functional macrostructures, the ECM extensively regulates cellular behavior [30]. The importance of these interactions is well demonstrated in the mounting evidence indicating that varied interactions with the ECM can actually differentially dictate the fate of stem cell differentiation [77]. Understanding the effects of the ECM on cell biology not only increases the understanding of normal organismal development and biology, but also diseases such as cancer or the various forms of muscular dystrophy [78,79]. With the current rise in under‐ standing of autophagy in diseases and development, understanding how extracellular interactions and differentiation affect autophagy is an important avenue to explore in the future.

#### **Nomenclature**

pressed can inhibit autophagy under starvation conditions [65]. On the other hand, another study showed that JNK was required for ceramide induced autophagy and expression of Beclin [66]. Furthermore, Jnk1 can phosphorylate Bcl-2 to release Beclin and promote autophagy [67]. Integrins may also regulate autophagy via p38 MAPK. Osteopontin binds integrins and CD44 and induces autophagy through activation of p38 MAPK [42]. Although some evidence indicates that p38 inhibits autophagy, p38 can activate JNK [68], which could provide a potential mechanism for both positive and negative regulation of autophagy by integrins. Finally, integrins stimulate Erk1/2 signaling as well, which activates autophagy through

Signaling through IKKα/β–NF-κB is another node for promoting autophagy (Figure 3). IKKα/ β regulates the expression of autophagy-related genes by activating NF-κB, which in turn has been shown to both positively and negatively regulate autophagy [41,69,70]. IKKα/β can also activate autophagy independently of NF-κB [70]. Cells expressing constitutively active subunits of the IKK complex had increased autophagy and increased levels of phosphorylated active AMPK and JNK [71]. Considering that blocking integrin adhesion in PrECs subsequent‐ ly attenuated autophagy [36], and that integrin mediated adhesion to laminin increased IKKα/ β - NF-κB signaling enough to significantly increase survival in a prostate cancer line [46], signaling through IKKα/β - NF-κB may be a mechanism by which integrin-mediated-celladhesion promotes autophagy. It would be interesting to determine whether this mechanism

PI3K/Akt signaling potently suppresses autophagy [72,73] (Figure 3). Akt activates the TORC1 complex, which in turn phosphorylates and inactivates the autophagy-initiating kinase Ulk1 [73]. Akt itself phosphorylates and inactivates the FoxO3a transcription factor. This prevents FoxO3a from inducing the expression of numerous autophagy- and Mitophagy-related genes [74]. Integrins can activate PI3K/Akt signaling [30], making it possible that integrins could suppress autophagy through this pathway as well. Perhaps this explains the cases in which detachment from the basement membrane actually promoted autophagy. However, it was shown that detachment of breast epithelial cells both increased autophagy and induced phosphorylation of eIF2α, a target of the ER kinase PERK and a protein capable of inducing

AMPK activates autophagy by inhibiting TORC1 and activating Ulk1 via direct phosphory‐ lation [49]. LKB1 localizes with engaged E-cadherin and activates AMPK signaling when ATP levels are low (Figure 3), providing a potential mechanism for cell-cell-interaction-mediated autophagy that might be relevant in detachment induced autophagy mechanisms in which

In many of the studies discussed above and in other diseases and models the effects of autophagy are very mitochondria-centric. For example, the Col6-/- mice phenotype was rescued by inhibiting mitochondrial depolarization, re-expressing Beclin or the pro-

numerous pathways including activation of JNK [68].

476 Autophagy - A Double-Edged Sword - Cell Survival or Death?

occurs and whether it is dependent or independent of NF-κB.

autophagy [34].

**4. Conclusion**

cell-cell contact is maintained [50].

3-MA - 3-methyladenine

CFSE - 5,6-carboxyfluorescein diacetate succinimidyl ester

EB - embryoid body

ECM - extracellular matrix

LAMA2 - laminin α2 chain

Mdx - dystrophin gene

MEF - mouse embryo fibroblast

PrEC - prostate epithelial cell

SSC- squamous cell carcinomas

UCMD - Ulrich's Congenital Muscular Dystrophy

WAT- white adipose tissues

## **Acknowledgements**

We wish to thank the Van Andel Research Institute (EAN, CKM) and the Van Andel Institute Graduate School (EAN) for their generous support. This work was also supported in part by an NIH/NCI grant CA154835 (CKM).

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Integrin and Adhesion Regulation of Autophagy and Mitophagy

http://dx.doi.org/10.5772/55398

479

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## **Author details**

Eric A. Nollet1,2 and Cindy K. Miranti1\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Laboratory of Integrin Signaling and Tumorigensis, Van Andel Research Institute, Grand Rapids, Michigan, USA

2 Van Andel Institute Graduate School, Grand Rapids, Michigan, USA

#### **References**

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**Acknowledgements**

**Author details**

Rapids, Michigan, USA

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an NIH/NCI grant CA154835 (CKM).

478 Autophagy - A Double-Edged Sword - Cell Survival or Death?

Eric A. Nollet1,2 and Cindy K. Miranti1\*

\*Address all correspondence to<EMAIL_ADDRESS>
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**Chapter 22**

**Time Flies: Autophagy During Ageing in** *Drosophila*

The process of ageing compromises the age-associated decrease in fertility, gradual loss of function, and increased vulnerability to disease, which progressively diminishes the capability of an organism to survive [1-3]. Unsurprisingly, in the past years it has been of great interest to understand which factors influence this inevitable and complex process. As a result a wide array of molecular and cellular damages has been identified and shown to accumulate during ageing. The lifelong accumulation of such damages will eventually result in frailty and disease [4]. The variety of identified age-dependent damages has given rise to different theories for molecular ageing mechanisms. These mechanisms include decreased cellular capacity to deal with DNA damage, and decline in cellular division capacity, which is linked to the progressive shortening of telomeres upon each cell cycle. Also an increased accumulation of damaged mitochondria and the involved increase in reactive oxygen species (ROS) production and decline in ATP synthesis has been shown to occur over time (reviewed in [5]). One of the phenotypic hallmarks of aged cells is the intracellular accumulation of damaged proteins and therefore protein turnover/protein degradation has attracted attention over the last years [2]. At the same time, forward genetics have allowed to investigate single gene alterations and their influence on lifespan of whole organisms. Even though the ageing process is without doubt influenced by stochastic and environmental factors, single gene mutations were shown to extend lifespan in worms, flies, and mice, suggesting the existence of a central process of ageing [6, 7]. Many of the genetic manipulations that alter longevity affect metabolism, nutrient sensing and stress response pathways. As all these pathways are connected to autophagy (an important player also in protein turnover), the question about the role of autophagy in ageing has come more and more to the fore. In this chapter we will focus on how research conducted

> © 2013 Schultz et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Schultz et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Sebastian Wolfgang Schultz, Andreas Brech and

Additional information is available at the end of the chapter

Ioannis P. Nezis

**1. Introduction**

**1.1. Ageing**

http://dx.doi.org/10.5772/55396

## **Time Flies: Autophagy During Ageing in** *Drosophila*

Sebastian Wolfgang Schultz, Andreas Brech and Ioannis P. Nezis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55396

## **1. Introduction**

#### **1.1. Ageing**

The process of ageing compromises the age-associated decrease in fertility, gradual loss of function, and increased vulnerability to disease, which progressively diminishes the capability of an organism to survive [1-3]. Unsurprisingly, in the past years it has been of great interest to understand which factors influence this inevitable and complex process. As a result a wide array of molecular and cellular damages has been identified and shown to accumulate during ageing. The lifelong accumulation of such damages will eventually result in frailty and disease [4]. The variety of identified age-dependent damages has given rise to different theories for molecular ageing mechanisms. These mechanisms include decreased cellular capacity to deal with DNA damage, and decline in cellular division capacity, which is linked to the progressive shortening of telomeres upon each cell cycle. Also an increased accumulation of damaged mitochondria and the involved increase in reactive oxygen species (ROS) production and decline in ATP synthesis has been shown to occur over time (reviewed in [5]). One of the phenotypic hallmarks of aged cells is the intracellular accumulation of damaged proteins and therefore protein turnover/protein degradation has attracted attention over the last years [2].

At the same time, forward genetics have allowed to investigate single gene alterations and their influence on lifespan of whole organisms. Even though the ageing process is without doubt influenced by stochastic and environmental factors, single gene mutations were shown to extend lifespan in worms, flies, and mice, suggesting the existence of a central process of ageing [6, 7]. Many of the genetic manipulations that alter longevity affect metabolism, nutrient sensing and stress response pathways. As all these pathways are connected to autophagy (an important player also in protein turnover), the question about the role of autophagy in ageing has come more and more to the fore. In this chapter we will focus on how research conducted

© 2013 Schultz et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Schultz et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in the excellent genetic model system *Drosophila melanogaster* has contributed to understand more about the interplay of autophagy and ageing.

subsequently fuse with lysosomes where the captured cytosolic constituents will be degraded. Autophagy can achieve several purposes; it scavenges the cytosol from macromolecules and organelles but also provides a way to supply the cells with amino acids and if necessary with energy once the recycled amino acids are converted into intermediates of the tricarboxylic acid cycle (TCA) [15, 18-20]. It is therefore of little surprise that the autophagic machinery, which under normal conditions is running on low basal levels, can be set in motion by several intraand extracellular stress factors, such as starvation, ER-stress, hypoxia and pathogen invasion [15]. Besides non-selective cytosolic bulk-degradation, autophagy is also implicated in selective turnover in yeast, a pathway known as the cytoplasm-to-vacuole targeting (CVT) pathway [21]. In analogy, cargo selective degradation of aggregated proteins (aggrephagy [22]), mitochondria (mitophagy [23]), ribosomes (ribophagy [24]), peroxisomes (pexophagy [25]), endoplasmic reticulum (reticulophagy [26]) and many more have been reported for mammalian systems [27]. The role of selective autophagy in ageing will be further addressed

Time Flies: Autophagy During Ageing in *Drosophila*

http://dx.doi.org/10.5772/55396

489

Several protein complexes are involved along the path from initiation to completion of autophagy. Induction of autophagy in *Drosophila* requires the Ser/Thr kinase Atg1 that forms a complex with Atg13. Phosphorylation of Atg13 by Atg1 directs phagophore initiation through a complex containing the class III PI(3)-kinase Vps34, the Ser/Thr kinase Vps15 and Atg6 (Beclin1 in mammals). The activation of this complex leads to localized generation of phosphatidylinositol-3-phosphate (PI3P), a critical step in autophagy. In mammalian systems, this core complex has several known interaction partners, e.g. Atg14, Ambra1, UVRAG, or Rubicon, that are all involved in autophagy. Several of these mammalian genes have ortho‐ logues in *Drosophila*, however their involvement in autophagy remains to be shown (reviewed in [28]). UVRAG has recently been found to be important in the regulation of Notch levels in the context of organ rotation during development. This role of UVRAG is coupled to endocytic degradation of Notch and, in this context, not to autophagy [29]. Autophagosome formation requires the ubiquitin-like proteins Atg12 and Atg8 and their respective ubiquitin-like conjugation systems [30]. Atg8 is processed by the cysteine protease Atg4 and covalently linked to phosphatidylethanolamine (PE) through the action of the E1 activating enzyme Atg7, the E2 activating enzyme Atg3 and the E3 like Atg12-Atg5-Atg16 complex, which is found at the phagophore membrane. The E3 like Atg12-Atg5-Atg16 complex itself requires also Atg7 and the E2 activating enzyme Atg10 for its assembly (reviewed in [31]). Once Atg8 is activated and lipid-conjugated it is localized to both sides of the phagophore and Atg4 later only removes the portion residing at the cytosolic side prior to autophagosome-lysosome/endosome fusion. It has also been reported that Atg8 can modulate the size of autophagosomes by influencing membrane curvature. For all these reasons, activation of Atg8/LC3 is widely used to monitor autophagy [15, 32]. The process from autophagy initiation until autolysosome formation is

It is believed that stepwise fusion of autophagosomes with different endosomal populations account for maturation and culminates in the fusion with lysosomes, the organelle responsi‐ ble for degradation [33]. Such stepwise fusion is supported by the findings that impairment of ESCRT machinery results in reduced autolysosome formation, measured as decrease in

in a separate section of this chapter.

schematically illustrated in figure 1.

### **2. Autophagy**

Autophagy, which literally means "self-eating" (coined by Nobel Laureate Christian de Duve in 1963), allows cells to digest cytosolic components via lysosomal degradation. Autophagy and the Ubiquitin Proteasome System (UPS) constitute together the main cellular pathways for protein and organelle turnover [8, 9]. Today, three different classes of autophagy are distinguished: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy.

During microautophagy, which is mainly studied in yeast (containing vacuoles instead of lysosomes), cytoplasmic material is delivered to the vacuolar lumen by direct invagination of the vacuolar boundary membrane and budding of autophagic bodies into the vacuolar lumen [10]. The molecular mechanisms underlying microautophagy in eukaryotic cells are largely unknown. However, Cuervo and colleagues described a microautophagy-like process (named endosomal microautophagy, e-MI) in mammalian cells, whereby soluble cytosolic proteins are selectively taken up by late endosomes/multivesicular bodies (MVBs). The cargo selection in e-MI depends on the chaperone Hsc70 and electrostatic interactions with the endosomal membrane [11]. Hsc70 is also involved in chaperone-mediated autophagy (CMA), in which cytosolic cargo is selectively recognized, bound by the lysosome-associated membrane type protein 2A (LAMP-2A) and finally taken up by the lysosome, thereby allowing for direct lysosomal degradation of cytosolic proteins. The requirement of protein unfolding and the binding of LAMP2-A is characteristic for CMA and thereby distinguishes CMA from e-MI [11, 12]. So far, CMA has not been investigated in *Drosophila melanogaster*. The third common type of autophagy, macroautophagy (henceforth referred to as autophagy), is highly conserved from yeast to mammalian cells [8]. Autophagy allows for cytosolic bulk degradation of longlived macromolecules and organelles. Morphologically this process was already described in the 1960s but it was not before several decades later when genetic screens in *Saccharomyces cerevisiae* identified multiple genes involved in autophagy and thereby allowed to investigate the molecular mechanisms in further detail [13, 14]. Genetic screens in *S. cerevisiae* have since then led to the identification of numerous autophagy-related (ATG) genes and many homologs have been identified and characterized in higher eukaryotes [15]. In general, autophagy can be divided into three steps: 1) induction/nucleation; 2) expansion; and 3) maturation [16].

The formation of a cytosolic double membrane structure called the phagophore (also called isolation membrane) is an important step of autophagy initiation. It is subject of discussion about the origin of this initial autophagic membrane. Independent experiments identified ER, Golgi, or the outer membrane of mitochondria to contribute to the phagophore double membrane [17, 18]. Cytosolic components are enwrapped during the growth of the phago‐ phore. Closure of the phagophore completes this engulfment and gives rise to a new structure called the autophagosome. These newly formed autophagosomes will further mature and subsequently fuse with lysosomes where the captured cytosolic constituents will be degraded. Autophagy can achieve several purposes; it scavenges the cytosol from macromolecules and organelles but also provides a way to supply the cells with amino acids and if necessary with energy once the recycled amino acids are converted into intermediates of the tricarboxylic acid cycle (TCA) [15, 18-20]. It is therefore of little surprise that the autophagic machinery, which under normal conditions is running on low basal levels, can be set in motion by several intraand extracellular stress factors, such as starvation, ER-stress, hypoxia and pathogen invasion [15]. Besides non-selective cytosolic bulk-degradation, autophagy is also implicated in selective turnover in yeast, a pathway known as the cytoplasm-to-vacuole targeting (CVT) pathway [21]. In analogy, cargo selective degradation of aggregated proteins (aggrephagy [22]), mitochondria (mitophagy [23]), ribosomes (ribophagy [24]), peroxisomes (pexophagy [25]), endoplasmic reticulum (reticulophagy [26]) and many more have been reported for mammalian systems [27]. The role of selective autophagy in ageing will be further addressed in a separate section of this chapter.

in the excellent genetic model system *Drosophila melanogaster* has contributed to understand

Autophagy, which literally means "self-eating" (coined by Nobel Laureate Christian de Duve in 1963), allows cells to digest cytosolic components via lysosomal degradation. Autophagy and the Ubiquitin Proteasome System (UPS) constitute together the main cellular pathways for protein and organelle turnover [8, 9]. Today, three different classes of autophagy are distinguished: microautophagy, chaperone-mediated autophagy (CMA),

During microautophagy, which is mainly studied in yeast (containing vacuoles instead of lysosomes), cytoplasmic material is delivered to the vacuolar lumen by direct invagination of the vacuolar boundary membrane and budding of autophagic bodies into the vacuolar lumen [10]. The molecular mechanisms underlying microautophagy in eukaryotic cells are largely unknown. However, Cuervo and colleagues described a microautophagy-like process (named endosomal microautophagy, e-MI) in mammalian cells, whereby soluble cytosolic proteins are selectively taken up by late endosomes/multivesicular bodies (MVBs). The cargo selection in e-MI depends on the chaperone Hsc70 and electrostatic interactions with the endosomal membrane [11]. Hsc70 is also involved in chaperone-mediated autophagy (CMA), in which cytosolic cargo is selectively recognized, bound by the lysosome-associated membrane type protein 2A (LAMP-2A) and finally taken up by the lysosome, thereby allowing for direct lysosomal degradation of cytosolic proteins. The requirement of protein unfolding and the binding of LAMP2-A is characteristic for CMA and thereby distinguishes CMA from e-MI [11, 12]. So far, CMA has not been investigated in *Drosophila melanogaster*. The third common type of autophagy, macroautophagy (henceforth referred to as autophagy), is highly conserved from yeast to mammalian cells [8]. Autophagy allows for cytosolic bulk degradation of longlived macromolecules and organelles. Morphologically this process was already described in the 1960s but it was not before several decades later when genetic screens in *Saccharomyces cerevisiae* identified multiple genes involved in autophagy and thereby allowed to investigate the molecular mechanisms in further detail [13, 14]. Genetic screens in *S. cerevisiae* have since then led to the identification of numerous autophagy-related (ATG) genes and many homologs have been identified and characterized in higher eukaryotes [15]. In general, autophagy can be divided into three steps: 1) induction/nucleation; 2) expansion; and 3) maturation [16].