**Abstract**

Genetic alterations can cause cancer, including pancreatic cancer (PC) as well as certain neurodegenerative diseases. Our lab has recently identified genes that are modulated during pancreatic cancer liver metastasis, and some are known to have a role in neurobiology or neurodegenerative diseases. Autophagy or self-eating portrays the lysosomaldependent degradation and recycling of protein aggregates and defective organisms in eukaryotic cells. Deregulation of autophagy as a cellular mechanism is common in neurodegenerative diseases as well as cancer and may represent a platform by which some genes can affect both disorders. This is exemplified for optineurin, which is an autophagy receptor that was found among genes with intensive modulation of expression in PC liver metastasis. Our results on this autophagy receptor draw the attention to the expression status of this and other autophagy genes in pancreatic cancer progression.

**Keywords:** pancreatic cancer, nervous system, neurodegenerative diseases, autophagy, optineurin

## **1. Introduction**

Recent findings from microarray analyses of cancer cells have shown a growing list of genes with modulated expression, which are known to have importance in diseases other than cancer. This is in particular true for genes or gene families that have been identified to play a role in some neurodegenerative diseases or are a factor in cells of the nervous system, which may have a controlling role on the growth and development of cancer in general and of pancreatic cancer in particular. One cellular property, the alteration of which seems to be related to both types of diseases, is autophagy. In the lines below, we will discuss whether deregulation of autophagy could be a mechanism, which is in common between certain neurodegenerative diseases and pancreatic cancer, and thus may represent a link between two extremely different diseases.

## **2. Disrupted autophagy links cancer and neurodegenerative diseases**

Autophagy is a Greek term that means self-eating and it portrays the lysosomaldependent way of degrading and recycling cytosolic components in eukaryotic

cells. Autophagy is categorized into three different types, that is, microautophagy, macroautophagy, and chaperone-mediated autophagy (**Figure 1**), which are different in terms of their cargo and the mechanism of their occurrence [1, 2].

*Micro-autophagy* refers to the process where minute parts of the cytoplasm become sequestered and later on completely engulfed by lysosomal invaginations [3].

*Chaperone-mediated autophagy* is a selective pathway of autophagy, where proteins are targeted by the presence of a pentapeptide motif in the amino acid sequence of the substrate proteins, which is recognized by a chaperone protein. This complex is then delivered to the lysosomes for degradation in a receptor-dependent manner [2]. The motif should be accessible to the chaperone protein regardless of its position in the protein, but under normal conditions, it is concealed within the core when the protein is appropriately folded. Substrates for CMA are recognized by chaperones as heat shock cognate protein of 70 kD (hsp70) and cochaperones, which may be responsible for the unfolding of the protein before substratechaperone interaction can take place, but without direct interactions in some cases. Cochaperones include proteins PINK as hsp90, hsp40, and Bcl-2–associated athanogene 2 (Bag-1). Then, the substrates bind to the cytosolic tail of lysosomeassociated membrane protein type 2A (LAMP-2A) and through its multimerization are translocated toward the lysosomal lumen, and this is seen as a limiting step for the pathway [2, 4, 5].

*Macroautophagy*, referred to below as autophagy, is the most widely investigated type of autophagy and entails the formation of what is called an autophagosome, which is a double membrane structure that is used to deliver cargos later through fusion to lysosomes or endosomes [6]. It is formed through several chronological steps starting from nucleation, elongation, to closure of a phagophore or isolation membrane that leads to the autophagosome formation. Proteins responsible to drive the autophagy process were basically discovered through analyzing the yeast genome and are named autophagy-related (ATG) proteins [6, 7].

Once stimulated, the autophagic process starts with the assembly of the initiation complex (ULK1 complex) and the nucleation complex (BECN1 complex) at the phagophore assembly site and this forms the basis for recruiting other ATG proteins and the elongation of the phagophore membrane [1]. ATG8/LC3 becomes bound to the inner and outer membrane of the phagophore following cleavage by the ATG4 protease and following conjugation to phosphatidylethanolamine (PE). Before

#### **Figure 1.**

*Schematic representation of the three different types of autophagy showing the underlying difference in the mechanisms, microautophagy by invaginations of the lysosomes, macroautophagy through the formation of autophagosomes, and CMA with the targeted protein recognized by chaperones and delivered to the lysosomes through the LAMP2 protein.*

**43**

disorders [1].

*Autophagy-Related Gene Expression Changes Are Found in Pancreatic Cancer…*

closure, all ATGs dissociate and are recycled except for ATG8/LC3, which is located on the outer membrane. This protein is recycled after closure with the assistance of ATG4, while lysosomal enzymes in the autophagosome lumen degrade the ATG8/

Autophagosomes later unite with late endosomes and lysosomes to proceed for degradation [1]. LAMP proteins again control the fusion step and protect against

Autophagy can be either selective or nonselective. During the selective process, specific cargos as aggregated proteins, damaged mitochondria, excess peroxisomes, and invading pathogens undergo degradation after being recognized via autophagy receptors. These autophagy receptors have the ability to recognize degradation signals on cargo proteins and also bind LC3/GABARAP proteins on the forming autophagosome. Among the identified autophagy receptors are p62/SQSTM1 (p62/ Sequestosome 1), OPTN (optineurin), NBR1 (neighbor of BRCA1), and NDP52 (nuclear dot protein 52 kDa). All of them possess an ubiquitin-binding domain and

Autophagy is a highly conserved process in mammals and is strictly regulated. One of the major regulators is mTOR through its complex 1 (mTORC1) and its activation exerts an inhibitory effect on autophagy induction. A similar effect is shown with PI3 kinases class I, whereas PI3 kinase class III activity is required for autophagosome formation. Starvation and amino acid depletion result in stimulation of the process. Other regulatory and autophagy inducers are based on an increase in cytosolic calcium, inhibition of inositol triphosphate, or starvation-induced autophagy [6, 11, 12]. Starvation also positively regulates autophagy by activation of AMPK that directly phosphorylates ULK1 or by inhibition of mTORC1 activity, or through inhibitory phosphorylation of nonautophagic BECN1 complexes [1]. Oxidative stress [13], DNA damage [14], and hypoxia [15] are all among the cellular

Autophagy has several cellular functions; it provides nutrients, eliminates damaged proteins and organelles, combats against invading microorganisms/ pathogens, and in general keeps the homeostasis and balance in cells [1]. Because of its important role and significant cellular implications, defects or deregulation of this process was detected in different human diseases [1]. Infections and pathogens modulate autophagy according to their requirements to secure their survival in host cells. Certain steps of autophagy are hindered in neurodegenerative disorders and proteinopathies are a feature of these disorders. Autophagy is cytoprotective in cardiovascular tissue under physiological conditions, but it is induced in many cardiovascular diseases and is also deregulated in cancer, diabetes, and immune

In cancer, autophagy can either inhibit or promote cancer development and progression, and this varies according to the genetic lesions, tumor type, and stage. Combating mutagenic reactive oxygen species (ROS) accumulation, DNA damage, genomic instability, and oncogenic proteins are part of the protective functions of autophagy against tumor induction, as they induce autophagy when initiated [16, 17]. With regard to its onco-stimulatory role, downregulation of some autophagy-

related genes, as Beclin-1 or ATG5, results in reduced growth of metastatic carcinoma cell lines, while that of ATG7 will promote apoptosis of colon cancer cells. Autophagy also permits tumors to resist stress and apoptotic signals and is connected in advanced cancer to poor prognosis and invasiveness. It increases ATP levels that support cell survival during hypoxia and starvation. Thus,

*DOI: http://dx.doi.org/10.5772/intechopen.80981*

LC3 attached to the inner membrane.

LC3-interacting regions (LIRs) [9, 10].

mechanisms that can induce autophagy.

**2.1. Role of autophagy in cancer**

degradation of the lysosomal membrane [1, 8].

*Autophagy-Related Gene Expression Changes Are Found in Pancreatic Cancer… DOI: http://dx.doi.org/10.5772/intechopen.80981*

closure, all ATGs dissociate and are recycled except for ATG8/LC3, which is located on the outer membrane. This protein is recycled after closure with the assistance of ATG4, while lysosomal enzymes in the autophagosome lumen degrade the ATG8/ LC3 attached to the inner membrane.

Autophagosomes later unite with late endosomes and lysosomes to proceed for degradation [1]. LAMP proteins again control the fusion step and protect against degradation of the lysosomal membrane [1, 8].

Autophagy can be either selective or nonselective. During the selective process, specific cargos as aggregated proteins, damaged mitochondria, excess peroxisomes, and invading pathogens undergo degradation after being recognized via autophagy receptors. These autophagy receptors have the ability to recognize degradation signals on cargo proteins and also bind LC3/GABARAP proteins on the forming autophagosome. Among the identified autophagy receptors are p62/SQSTM1 (p62/ Sequestosome 1), OPTN (optineurin), NBR1 (neighbor of BRCA1), and NDP52 (nuclear dot protein 52 kDa). All of them possess an ubiquitin-binding domain and LC3-interacting regions (LIRs) [9, 10].

Autophagy is a highly conserved process in mammals and is strictly regulated. One of the major regulators is mTOR through its complex 1 (mTORC1) and its activation exerts an inhibitory effect on autophagy induction. A similar effect is shown with PI3 kinases class I, whereas PI3 kinase class III activity is required for autophagosome formation. Starvation and amino acid depletion result in stimulation of the process. Other regulatory and autophagy inducers are based on an increase in cytosolic calcium, inhibition of inositol triphosphate, or starvation-induced autophagy [6, 11, 12]. Starvation also positively regulates autophagy by activation of AMPK that directly phosphorylates ULK1 or by inhibition of mTORC1 activity, or through inhibitory phosphorylation of nonautophagic BECN1 complexes [1]. Oxidative stress [13], DNA damage [14], and hypoxia [15] are all among the cellular mechanisms that can induce autophagy.

Autophagy has several cellular functions; it provides nutrients, eliminates damaged proteins and organelles, combats against invading microorganisms/ pathogens, and in general keeps the homeostasis and balance in cells [1]. Because of its important role and significant cellular implications, defects or deregulation of this process was detected in different human diseases [1]. Infections and pathogens modulate autophagy according to their requirements to secure their survival in host cells. Certain steps of autophagy are hindered in neurodegenerative disorders and proteinopathies are a feature of these disorders. Autophagy is cytoprotective in cardiovascular tissue under physiological conditions, but it is induced in many cardiovascular diseases and is also deregulated in cancer, diabetes, and immune disorders [1].

#### **2.1. Role of autophagy in cancer**

In cancer, autophagy can either inhibit or promote cancer development and progression, and this varies according to the genetic lesions, tumor type, and stage. Combating mutagenic reactive oxygen species (ROS) accumulation, DNA damage, genomic instability, and oncogenic proteins are part of the protective functions of autophagy against tumor induction, as they induce autophagy when initiated [16, 17].

With regard to its onco-stimulatory role, downregulation of some autophagyrelated genes, as Beclin-1 or ATG5, results in reduced growth of metastatic carcinoma cell lines, while that of ATG7 will promote apoptosis of colon cancer cells. Autophagy also permits tumors to resist stress and apoptotic signals and is connected in advanced cancer to poor prognosis and invasiveness. It increases ATP levels that support cell survival during hypoxia and starvation. Thus,

*Gene Expression and Control*

the pathway [2, 4, 5].

proteins [6, 7].

cells. Autophagy is categorized into three different types, that is, microautophagy, macroautophagy, and chaperone-mediated autophagy (**Figure 1**), which are differ-

*Micro-autophagy* refers to the process where minute parts of the cytoplasm become

ent in terms of their cargo and the mechanism of their occurrence [1, 2].

sequestered and later on completely engulfed by lysosomal invaginations [3]. *Chaperone-mediated autophagy* is a selective pathway of autophagy, where proteins are targeted by the presence of a pentapeptide motif in the amino acid sequence of the substrate proteins, which is recognized by a chaperone protein. This complex is then delivered to the lysosomes for degradation in a receptor-dependent manner [2]. The motif should be accessible to the chaperone protein regardless of its position in the protein, but under normal conditions, it is concealed within the core when the protein is appropriately folded. Substrates for CMA are recognized by chaperones as heat shock cognate protein of 70 kD (hsp70) and cochaperones, which may be responsible for the unfolding of the protein before substratechaperone interaction can take place, but without direct interactions in some cases. Cochaperones include proteins PINK as hsp90, hsp40, and Bcl-2–associated athanogene 2 (Bag-1). Then, the substrates bind to the cytosolic tail of lysosomeassociated membrane protein type 2A (LAMP-2A) and through its multimerization are translocated toward the lysosomal lumen, and this is seen as a limiting step for

*Macroautophagy*, referred to below as autophagy, is the most widely investigated type of autophagy and entails the formation of what is called an autophagosome, which is a double membrane structure that is used to deliver cargos later through fusion to lysosomes or endosomes [6]. It is formed through several chronological steps starting from nucleation, elongation, to closure of a phagophore or isolation membrane that leads to the autophagosome formation. Proteins responsible to drive the autophagy process were basically discovered through analyzing the yeast genome and are named autophagy-related (ATG)

Once stimulated, the autophagic process starts with the assembly of the initiation complex (ULK1 complex) and the nucleation complex (BECN1 complex) at the phagophore assembly site and this forms the basis for recruiting other ATG proteins and the elongation of the phagophore membrane [1]. ATG8/LC3 becomes bound to the inner and outer membrane of the phagophore following cleavage by the ATG4 protease and following conjugation to phosphatidylethanolamine (PE). Before

*Schematic representation of the three different types of autophagy showing the underlying difference in the mechanisms, microautophagy by invaginations of the lysosomes, macroautophagy through the formation of autophagosomes, and CMA with the targeted protein recognized by chaperones and delivered to the lysosomes*

**42**

**Figure 1.**

*through the LAMP2 protein.*

autophagy keeps healthy cells away from malignant transformation through maintaining the cellular homeostasis, but after tumor establishment, it may result in increased tumor progression and invasiveness [16, 17]. Autophagy supports tumor development through improving resistance of cells to endogenous apoptotic signals as well as resistance to chemotherapy and maintains the cancer stem cell compartment [17].

Pancreatic tumors show an elevated level of basal autophagy, more than any other epithelial cancer. The role of autophagy in PDAC needs to be clarified; as some investigations and the effect of anticancer drugs that generate ROS and induce autophagy give rise to the idea that autophagy is protective against cancer and stimulates apoptotic cancer cell death following treatment [18]. On the other hand, a number of reports suggested the role of autophagy in PDAC development. Autophagy was anticipated to be the cellular mechanism responsible for cancer development and progression from pancreatitis to overt carcinogenesis in the presence of K-ras mutations. Different observations supported a harmful role of autophagy, as being strongly induced in the inflammatory process. Inhibition of tumor development was observed after using the autophagy inhibitor chloroquine. Finally, LC3 is overexpressed in pancreatic cancer and downregulation of autophagy genes significantly reduces the growth and colony formation of PDAC cells in vitro [19, 20].

#### **2.2. Role of autophagy in neurodegenerative diseases**

Autophagic removal of proteins and damaged organelles is vital for the appropriate function of neurons, not only under pathological conditions, but also at baseline level under normal circumstances, as denoted by some studies [21].

Autophagosomes are very rarely detected in healthy neurons owing to the fact that the brain has a very low level of basal autophagy, or because the robust efficiency of the autophagy processes prevents the autophagosomes from accumulation [21, 22].

Dysfunction in autophagy affects the neuronal function and results in the occurrence of neurodegenerative diseases [23].

Induction of autophagy was anticipated to be an early stress response in axonal dystrophy and to contribute to axon remodeling [24]. Autophagy was shown to play a role in controlling microtubule dynamics and axon regeneration. Autophagy induction encouraged neurite growth, lessened the inhibitory effects of myelin, and reduced the formation of retraction bulbs after axonal injury in cultured cortical neurons [25].

Neurodegeneration is associated with deterioration of cognitive capabilities as well as motor functions with progressive damage to the nerve structure and functions. Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, and Huntington's diseases represent the most famous neurodegenerative diseases [26]. They share in common the accumulation of toxic misfolded protein aggregates, as extracellular amyloid protein (Aβ) plaques and intracellular accumulation of tau protein in Alzheimer's disease, α-synuclein and ubiquitin in Parkinson's disease, and mutant Huntingtin protein in Huntington's disease [26]. Defective mitochondria, generating excessive reactive oxygen species (ROS), represent another feature of neurodegenerative diseases, as ROS harm the cellular constituents including DNA, lipids, and proteins [26]. The misfolded proteins are responsible for neuronal damage and death, and hence autophagy becomes a crucial target for the management of neurodegenerative diseases [27].

Recent studies revealed that alterations in autophagy-related genes trigger neurodegenerative diseases [28].

**45**

*Autophagy-Related Gene Expression Changes Are Found in Pancreatic Cancer…*

In Alzheimer's disease, transmembrane amyloid precursor protein (APP) is processed by β- and γ-secretases, which thus produce Aβ peptide [28, 29].

Autophagosomes show γ-secretase activity and represent a possibly active compartment for the production of Aβ and contribute to its deposition in affected neurons in Alzheimer's disease [30]. Dysfunction of Presenilin-1, associated with familial Alzheimer's disease, hinders proteolysis in autolysosomes and results in accumulation of Aβ peptides in autophagosomes [28]. Beclin-1 deficiency impedes the autophagic clearance of APP as an autophagy substrate, and it increases the pathol-

Huntington's disease is an inherited neurodegenerative disease and shows a range of declining motor, behavioral, and cognitive functions. Mutations in the huntingtin (HTT) gene are associated with cytosine-adenine-guanine (CAG) expansion, encoding a polyglutamine (polyQ ) at the N-terminus of the gene [32]. This mutation affects the interaction of huntingtin with other proteins leading to all the neuropathological changes observed in the disease [33]. Huntingtin protein was shown to function as a scaffold protein for selective macroautophagy through interaction with autophagy pathway components; it cooperates with the autophagy receptor p62 and enables its connection with the essential autophagosome element LC3 and with ubiquitinated substrates. Huntingtin also binds to ULK1, the initiator kinase for autophagy, and this interaction liberates ULK1 from the negative regula-

Parkinson's disease (PD) is a common progressive neurological disorder characterized by tremors, bradykinesia, rigidity, and loss of postural reflexes. The disease is caused by the loss of dopaminergic neurons in the *substantia nigra* [28, 35]. Different stages of Parkinson's disease showed deregulation of autophagy with the aggregation of α-synuclein in 'Lewy bodies.' Disrupted mitophagy is a major mechanism in the pathogenesis of the disease through mutations of PINK1 and PARKIN, which are essential for mitochondrial biogenesis and recycling [36]. Physiologically, PINK1 is a target of the ubiquitin-proteasome system after it has been processed by the mitochondrial protease presenilin-associated rhomboid-like (PARL). In depolarized mitochondria, this process is inhibited and PINK1 accumulates on the outer mitochondrial membrane, autophosphorylates, and recruits Parkin to damaged mitochondria. Parkin is an enzyme 3 (E3) ubiquitin ligase, and it was suggested that its ubiquitinated proteins recruit the autophagy receptor p62 to be integrated in an autophagosome for the autophagy degradation of damaged mitochondria

The two autophagy receptors previously linked to xenophagy, nuclear dot protein 52 kDa (NDP52) and optineurin (OPTN), were identified in a recent study to be the primary receptors for PINK1- and Parkin-mediated mitophagy. In this study, PINK1 recruits these two autophagy receptors, but not p62, to activate mitophagy directly, independent from Parkin. Upon their recruitment to mitochondria, NDP52 and OPTN recruit other autophagy components as ULK1, DFCP1, and WIPI1, thus revealing a function for these autophagy receptors upstream of LC3. While PINK1 begins mitophagy in the absence of Parkin, mitophagy is significantly boosted in

Amyotrophic lateral sclerosis is a deadly progressive neurodegenerative disease

characterized by the degeneration and deterioration of motor neurons. Beside the association of several genes in ALS, it has been reported that mutations in the Sigma-1 receptor (SigmaR1) are related to the autosomal recessive familial form of ALS [28, 38]. SigmaR1 controls calcium transport and its reduced expression can lead to the increased release of calcium from ER, the depolarization of mitochondrial membrane potential, and apoptosis. It reduces autophagic flux and autophagic degradation [28, 39]. Recent exome sequencing studies identified TANK-binding

*DOI: http://dx.doi.org/10.5772/intechopen.80981*

ogy of Alzheimer's disease [31].

tion exerted by mTOR [34].

(mitophagy) [28].

the presence of Parkin [37].

*Autophagy-Related Gene Expression Changes Are Found in Pancreatic Cancer… DOI: http://dx.doi.org/10.5772/intechopen.80981*

In Alzheimer's disease, transmembrane amyloid precursor protein (APP) is processed by β- and γ-secretases, which thus produce Aβ peptide [28, 29]. Autophagosomes show γ-secretase activity and represent a possibly active compartment for the production of Aβ and contribute to its deposition in affected neurons in Alzheimer's disease [30]. Dysfunction of Presenilin-1, associated with familial Alzheimer's disease, hinders proteolysis in autolysosomes and results in accumulation of Aβ peptides in autophagosomes [28]. Beclin-1 deficiency impedes the autophagic clearance of APP as an autophagy substrate, and it increases the pathology of Alzheimer's disease [31].

Huntington's disease is an inherited neurodegenerative disease and shows a range of declining motor, behavioral, and cognitive functions. Mutations in the huntingtin (HTT) gene are associated with cytosine-adenine-guanine (CAG) expansion, encoding a polyglutamine (polyQ ) at the N-terminus of the gene [32]. This mutation affects the interaction of huntingtin with other proteins leading to all the neuropathological changes observed in the disease [33]. Huntingtin protein was shown to function as a scaffold protein for selective macroautophagy through interaction with autophagy pathway components; it cooperates with the autophagy receptor p62 and enables its connection with the essential autophagosome element LC3 and with ubiquitinated substrates. Huntingtin also binds to ULK1, the initiator kinase for autophagy, and this interaction liberates ULK1 from the negative regulation exerted by mTOR [34].

Parkinson's disease (PD) is a common progressive neurological disorder characterized by tremors, bradykinesia, rigidity, and loss of postural reflexes. The disease is caused by the loss of dopaminergic neurons in the *substantia nigra* [28, 35]. Different stages of Parkinson's disease showed deregulation of autophagy with the aggregation of α-synuclein in 'Lewy bodies.' Disrupted mitophagy is a major mechanism in the pathogenesis of the disease through mutations of PINK1 and PARKIN, which are essential for mitochondrial biogenesis and recycling [36]. Physiologically, PINK1 is a target of the ubiquitin-proteasome system after it has been processed by the mitochondrial protease presenilin-associated rhomboid-like (PARL). In depolarized mitochondria, this process is inhibited and PINK1 accumulates on the outer mitochondrial membrane, autophosphorylates, and recruits Parkin to damaged mitochondria. Parkin is an enzyme 3 (E3) ubiquitin ligase, and it was suggested that its ubiquitinated proteins recruit the autophagy receptor p62 to be integrated in an autophagosome for the autophagy degradation of damaged mitochondria (mitophagy) [28].

The two autophagy receptors previously linked to xenophagy, nuclear dot protein 52 kDa (NDP52) and optineurin (OPTN), were identified in a recent study to be the primary receptors for PINK1- and Parkin-mediated mitophagy. In this study, PINK1 recruits these two autophagy receptors, but not p62, to activate mitophagy directly, independent from Parkin. Upon their recruitment to mitochondria, NDP52 and OPTN recruit other autophagy components as ULK1, DFCP1, and WIPI1, thus revealing a function for these autophagy receptors upstream of LC3. While PINK1 begins mitophagy in the absence of Parkin, mitophagy is significantly boosted in the presence of Parkin [37].

Amyotrophic lateral sclerosis is a deadly progressive neurodegenerative disease characterized by the degeneration and deterioration of motor neurons. Beside the association of several genes in ALS, it has been reported that mutations in the Sigma-1 receptor (SigmaR1) are related to the autosomal recessive familial form of ALS [28, 38]. SigmaR1 controls calcium transport and its reduced expression can lead to the increased release of calcium from ER, the depolarization of mitochondrial membrane potential, and apoptosis. It reduces autophagic flux and autophagic degradation [28, 39]. Recent exome sequencing studies identified TANK-binding

*Gene Expression and Control*

cell compartment [17].

cells in vitro [19, 20].

[21, 22].

neurons [25].

**2.2. Role of autophagy in neurodegenerative diseases**

rence of neurodegenerative diseases [23].

neurodegenerative diseases [27].

neurodegenerative diseases [28].

autophagy keeps healthy cells away from malignant transformation through maintaining the cellular homeostasis, but after tumor establishment, it may result in increased tumor progression and invasiveness [16, 17]. Autophagy supports tumor development through improving resistance of cells to endogenous apoptotic signals as well as resistance to chemotherapy and maintains the cancer stem

Pancreatic tumors show an elevated level of basal autophagy, more than any other epithelial cancer. The role of autophagy in PDAC needs to be clarified; as some investigations and the effect of anticancer drugs that generate ROS and induce autophagy give rise to the idea that autophagy is protective against cancer and stimulates apoptotic cancer cell death following treatment [18]. On the other hand, a number of reports suggested the role of autophagy in PDAC development. Autophagy was anticipated to be the cellular mechanism responsible for cancer development and progression from pancreatitis to overt carcinogenesis in the presence of K-ras mutations. Different observations supported a harmful role of autophagy, as being strongly induced in the inflammatory process. Inhibition of tumor development was observed after using the autophagy inhibitor chloroquine. Finally, LC3 is overexpressed in pancreatic cancer and downregulation of autophagy genes significantly reduces the growth and colony formation of PDAC

Autophagic removal of proteins and damaged organelles is vital for the appropriate function of neurons, not only under pathological conditions, but also at baseline level under normal circumstances, as denoted by some studies [21].

Autophagosomes are very rarely detected in healthy neurons owing to the fact that the brain has a very low level of basal autophagy, or because the robust efficiency of the autophagy processes prevents the autophagosomes from accumulation

Dysfunction in autophagy affects the neuronal function and results in the occur-

Induction of autophagy was anticipated to be an early stress response in axonal dystrophy and to contribute to axon remodeling [24]. Autophagy was shown to play a role in controlling microtubule dynamics and axon regeneration. Autophagy induction encouraged neurite growth, lessened the inhibitory effects of myelin, and reduced the formation of retraction bulbs after axonal injury in cultured cortical

Neurodegeneration is associated with deterioration of cognitive capabilities as well as motor functions with progressive damage to the nerve structure and functions. Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, and Huntington's diseases represent the most famous neurodegenerative diseases [26]. They share in common the accumulation of toxic misfolded protein aggregates, as extracellular amyloid protein (Aβ) plaques and intracellular accumulation of tau protein in Alzheimer's disease, α-synuclein and ubiquitin in Parkinson's disease, and mutant Huntingtin protein in Huntington's disease [26]. Defective mitochondria, generating excessive reactive oxygen species (ROS), represent another feature of neurodegenerative diseases, as ROS harm the cellular constituents including DNA, lipids, and proteins [26]. The misfolded proteins are responsible for neuronal damage and death, and hence autophagy becomes a crucial target for the management of

Recent studies revealed that alterations in autophagy-related genes trigger

**44**

kinase 1 (TBK1) as an important protein in both sporadic and familial ALS. TBK1 phosphorylates OPTN [40], and it was reported that a mutation of OPTN is also associated with ALS [41]. Furthermore, neuron-specific Atg5 and Atg7 knockout mice are associated with motor defects [42]. Beclin-1, the major player in the autophagic initiation process, has been shown to promote ALS by interacting with superoxide dismutase 1 (SOD1) and its downregulation was associated with aggregation of amyloid-β tau tangles, which are indicators of Alzheimer's disease (AD).


**Author details**

Biswadeep Das\*, Sayam Ghosal and Swabhiman Mohanty KIIT School of Biotechnology, KIIT University, Bhubaneswar, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2018 The Author(s). Licensee IntechOpen. 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.

**29**

*Aedes: What Do We Know about Them and What Can They Transmit?*

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*Aedes: What Do We Know about Them and What Can They Transmit? DOI: http://dx.doi.org/10.5772/intechopen.81363*


**5. Conclusions**

Based on the described research, it can be concluded that:

An auxiliary cross frame can have various uses in the measurement of durability. It provides advantages when enlarging the width of roads with various pavement structures in the cross section or in pavements with neglected maintenance from the perspective of correct drainage function.

**Author Contributions:** Conceptualization, D.S. and L.M.; methodology, M.V., P.H. and O.D.; software, R.K. (Robert Kaderka), J.S. and P.V.; validation, D.S., P.H., O.D., L.M. and M.V.; formal analysis, M.V. and K.S.; investigation, K.S.; resources, P.H. and O.D.; data curation, M.V. and R.K. (Radek Komenda); writing—original draft preparation, D.S., P.H., O.D., L.M., R.K. (Robert Kaderka), R.K. (Radek Komenda), J.S., P.V., K.S. and M.V.; writing—review and editing, D.S., P.H., O.D., L.M., R.K. (Robert Kaderka), R.K. (Radek Komenda), J.S., P.V., K.S. and M.V.; visualization, R.K. (Radek Komenda), J.S., P.V. and K.S.; supervision, M.V.; project administration, O.D.; funding acquisition, D.S., P.H. and O.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was funded by the Technology Agency of the Czech Republic with Project CK01000158 "Use of higher amounts of reclaimed material in asphalt mixtures with PmB bitumen", Project CK03000169 "Development of road construction layers with optimized grain size replacing insufficient aggregate fractions", and Project TH04030342 "Recycled building materials for transport infrastructure construction".

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.


**7. LIN28-***let-7***-ARID3B Pathway in Trophoblast Cells**

AT-rich interactive domain (ARID) proteins, first recognized in 1997, are a family of 15 proteins which binds to AT-rich regions of DNA [144,145]. ARID proteins play an important role in cell proliferation, differentiation, and development, and are upregulated in tumorous tissues [146]. The subfamilies of ARID proteins include ARID1, ARID2, ARID3, ARID4, ARID5, JARID1, and JARID2. The ARID3 subfamily has three members including ARID3A, ARID3B, and ARID3C. Although most of the ARID proteins act as tumor suppressors, ARID3A and ARID3B promote tumorigenesis [144]. ARID3A inhibits cell differentiation, promotes cell proliferation, and increases survival potential of cells [146,147], whereas ARID3B promotes proliferation, invasion, and migration of cancer cells [148–150]. However, ARID3B is more widely expressed in different tissues compared to ARID3A, suggesting more involvement of ARID3B in biological functions [145]. ARID3A and ARID3B are structurally similar and bind a similar region of DNA. Both ARID3A and ARID3B have an extended central ARID domain and two conserved amino acid domains at the C-terminal, termed REKLES α and REKLES β. Only members of the ARID3 subfamily have REKLES domains [151].

In contrast to ARID3B which is exclusively localized in the nucleus, ARID3A shuttles between the nucleus and cytoplasm. Once in the nucleus, ARID3A interacts with ARID3B through REKLES β domain. Therefore localization of ARID3A in the nucleus is dependent on its interaction with ARID3B in the nucleus, suggesting the dominant role of ARID3B [151,152]. In cancer cells, ARID3A and ARID3B recruit histone demethylase 4C (KDM4C) to make a tri-protein complex, called the ARID3B complex [153]. The ARID3B complex binds in the promoter areas of stemness genes and *let-7* target genes, leading to histone demethylation by KDM4C and increased gene expression by initiation of transcription [153]. Therefore, genes regulated by the ARID3B complex also include *let-7* miRNA target genes. Liao et al. further demonstrated that both ARID3A and ARID3B are targeted by *let-7* miRNAs. Hence, other than directly targeting the mRNAs of target genes, *let-7* miRNA can indirectly regulate their target genes by targeting and reducing the expression of *ARID3A* and *ARID3B* (Figure 4) [11,153].

Both ARID3A and ARID3B have high expression in human trophoblast cells [154,155]. ARID3A knockout mice have severe structural defects in placenta [156]. In ACH-3P cells, ARID3A, ARID3B, and KDM4C make the tri-protein ARID3B complex [11]. In term human placentas from IUGR pregnancies, LIN28A and LIN28B are low, *let-7* miRNAs are high, and ARID3A and ARID3B are low, which suggest a correlation between LIN28-*let-7* miRNA axis and the ARID3B complex [11]. Due to the well-established pathway of regulation of *let-7* target genes through ARID3B complex and their role in cell proliferation, it is important to understand this phenomenon in early placental development. We recently showed the correlation between LIN28-*let-7* miRNA axis and the ARID3B complex using ACH-3P and Sw.71 cells. Double knockout of LIN28A and LIN28B in ACH-3P cells increases *let-7* miRNAs and decreases the expression ARID3A, ARID3B, and KDM4C. Similarly, double knockin of LIN28A and LIN28B in Sw.71 cells decreases *let-7* miRNAs and increases expression of ARID3A, ARID3B, and KDM4C [11]. In trophoblast cells, the ARID3B complex binds to the promoter areas of proliferation-associated *let-7* target genes including *HMGA1, c-MYC, VEGF-A*, and *WNT1,* facilitating their transcription via KDM4C mediated histone demethylation. ARID3B knockout ACH-3P cells and KDM4C cannot be recruited in the promoter regions of *HMGA1, c-MYC, VEGF-A* and *WNT1*, and expression of these genes is also significantly reduced [73]. Moreover, ARID3B knockout ACH-3P cells have a reduced proliferation rate compared to control cells [11]. Knockdown of LIN28A or LIN28B in sheep trophectoderm increases *let-7* miRNAs and reduces the expression of ARID3A and ARID3B [143], showing regulation of the ARID3B complex by LIN28-*let-7* miRNA axis in vivo. Collectively these findings show that *let-7* miRNAs target the ARID3B complex in trophoblast cells, and the ARID3B complex regulates genes with known importance in placental development.

4) [11,153].

**7. LIN28-***let-7***-ARID3B Pathway in Trophoblast Cells**

AT-rich interactive domain (ARID) proteins, first recognized in 1997, are a family of 15 proteins which binds to AT-rich regions of DNA [144,145]. ARID proteins play an important role in cell proliferation, differentiation, and development, and are upregulated in tumorous tissues [146]. The subfamilies of ARID proteins include ARID1, ARID2, ARID3, ARID4, ARID5, JARID1, and JARID2. The ARID3 subfamily has three members including ARID3A, ARID3B, and ARID3C. Although most of the ARID proteins act as tumor suppressors, ARID3A and ARID3B promote tumorigenesis [144]. ARID3A inhibits cell differentiation, promotes cell proliferation, and increases survival potential of cells [146,147], whereas ARID3B promotes proliferation, invasion, and migration of cancer cells [148– 150]. However, ARID3B is more widely expressed in different tissues compared to ARID3A, suggesting more involvement of ARID3B in biological functions [145]. ARID3A and ARID3B are structurally similar and bind a similar region of DNA. Both ARID3A and ARID3B have an extended central ARID domain and two conserved amino acid domains at the C-terminal, termed REKLES α

In contrast to ARID3B which is exclusively localized in the nucleus, ARID3A shuttles between the nucleus and cytoplasm. Once in the nucleus, ARID3A interacts with ARID3B through REKLES β domain. Therefore localization of ARID3A in the nucleus is dependent on its interaction with ARID3B in the nucleus, suggesting the dominant role of ARID3B [151,152]. In cancer cells, ARID3A and ARID3B recruit histone demethylase 4C (KDM4C) to make a tri-protein complex, called the ARID3B complex [153]. The ARID3B complex binds in the promoter areas of stemness genes and *let-7* target genes, leading to histone demethylation by KDM4C and increased gene expression by initiation of transcription [153]. Therefore, genes regulated by the ARID3B complex also include *let-7* miRNA target genes. Liao et al. further demonstrated that both ARID3A and ARID3B are targeted by *let-7*  miRNAs. Hence, other than directly targeting the mRNAs of target genes, *let-7* miRNA can indirectly

and REKLES β. Only members of the ARID3 subfamily have REKLES domains [151].

**Figure 4.** Gene regulation by the AT-rich interactive domain (ARID)3B complex. ARID3A is imported in the nucleus by importin 9 (IPO9), where it binds ARID3B and histone demethylase 4C (KDM4C) to form the ARID3B complex. The ARID3B complex binds in the promoter regions and activates transcription of *let-7* target genes. Other than directly binding the mRNA of their target genes, *let-7*  miRNAs also target the ARID3B complex and reduce its expression, ultimately leading to reduced **Figure 4.** Gene regulation by the AT-rich interactive domain (ARID)3B complex. ARID3A is imported in the nucleus by importin 9 (IPO9), where it binds ARID3B and histone demethylase 4C (KDM4C) to form the ARID3B complex. The ARID3B complex binds in the promoter regions and activates transcription of *let-7* target genes. Other than directly binding the mRNA of their target genes, *let-7* miRNAs alsotarget the ARID3B complex and reduce its expression, ultimately leading to reduced expression of *let-7* target genes.

#### expression of *let-7* target genes. **8. Conclusions**

For a long time, transcription activating proteins were thought to be the main regulators of gene expression in cells. However, in recent years microRNAs have emerged as "regulators of the regulators". miRNAs regulate important processes in trophoblast cells including cell proliferation, differentiation, invasion, and migration. Although identification of widespread *let-7* miRNA target genes makes them an important player in placental development, the role of other miRNAs and molecular pathways in placental development cannot be ignored. Although different studies have reported reduced LIN28 and high *let-7* miRNAs in term human placentas from preeclamptic and IUGR pregnancies, the exact cause of this dysregulation is not clear. Epigenetic modifications due to adverse uterine environment, random genetic mutations, prenatal insults like hypoxia, oxidative stress, maternal malnutrition, and gestational stress are some of the possible factors which can dysregulate LIN28-*let-7* axis in trophoblast cells. LIN28-*let-7*-ARID3B pathway regulates trophoblast cell proliferation by modulating the expression of proliferation associated genes in vitro and in vivo. The trophoblast cells with low LIN28 will have high *let-7* miRNAs and low ARID3B. There are two possible pathways of regulation of proliferation-associated genes by *let-7* miRNAs in trophoblast cells (Figure 5). One pathway involves binding of *let-7* miRNAs in 30 -UTR of their target mRNA leading to mRNA degradation or translational repression. Secondly, *let-7* miRNAs target ARID3A, ARID3B, and KDM4C to inhibit or reduce transcriptional activation of proliferation-associated genes by the ARID3B complex. Therefore, trophoblast cells with high *let-7* miRNAs will have reduced expression of proliferation factors and more of a tendency to differentiate. High *let-7* miRNAs during early placental development reduce cell proliferation, invasion, and migration of trophoblast cells, leading to placental abnormalities. *Let-7* miRNAs might be the major players in pathogenesis of placenta-associated disorders. miRNAs can be readily measured in peripheral blood, tissue biopsies, saliva, cerebrospinal fluid, urine, and other biological samples. High *let-7* miRNAs are upregulated in IUGR and preeclamptic placentas, suggesting that *let-7* miRNAs can be potential biomarkers for early diagnosis of PE and IUGR. It remains to be explored if early stage placentas from compromised

pregnancies will have high *let-7* miRNAs and will the increase in *let-7* miRNAs in placenta be reflected in maternal blood. Animal models of IUGR can be used to answer these questions. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 12 of 20

**Figure 5.** Proposed mechanism for gene regulation in trophoblast cells. Left panel of figure: LIN28 represses the biogenesis of mature *let-7* miRNAs by binding pri-*let-7* and pre-*let-7* miRNAs and inhibiting their processing. Due to the low level of mature *let-7* miRNAs in the cells, there will be less targeting of proliferation-associated genes. Moreover, the ARID3B complex will initiate the transcription of proliferation-associated genes and increase their expression. Increased expression of proliferation-associated genes will lead to increased cell proliferation. Right panel of figure: If LIN28 is knocked-out or knocked-down, there will be no suppression of *let-7* miRNA biogenesis. High *let-7* miRNAs will target and reduce the expression of proliferation associated genes and the ARDI3B complex, driving the cells towards differentiation. **Author Contributions:** A.A. performed research, analyzed the data, and wrote the manuscript; Q.A.W., G.J.B., **Figure 5.** Proposed mechanism for gene regulation in trophoblast cells. Left panel of figure: LIN28 represses the biogenesis of mature *let-7* miRNAs by binding pri-*let-7* and pre-*let-7* miRNAs and inhibiting their processing. Due to the low level of mature *let-7* miRNAs in the cells, there will be less targeting of proliferation-associated genes. Moreover, the ARID3B complex will initiate the transcription of proliferation-associated genes and increase their expression. Increased expression of proliferation-associated genes will lead to increased cell proliferation. Right panel of figure: If LIN28 is knocked-out or knocked-down, there will be no suppression of *let-7* miRNA biogenesis. High *let-7* miRNAs will target and reduce the expression of proliferation associated genes and the ARDI3B complex, driving the cells towards differentiation.

**Funding:** This project was supported by Agriculture and Food Research Initiative Competitive Grant No. 2017- 67015-26460 from the United States Department of Agriculture (USDA) National Institute of Food and **Author Contributions:** A.A. performed research, analyzed the data, and wrote the manuscript; Q.A.W., G.J.B., and R.V.A. edited the manuscript. All authors have read and agree to the published version of the manuscript.

Agriculture. This work was supported by the USDA National Institute of Food and Agriculture, Hatch Project COL00293D, accession number 1021217. **Conflicts of Interest:** The authors declare no conflicts of interest. **Funding:** This project was supported by Agriculture and Food Research Initiative Competitive Grant No. 2017-67015-26460 from the United States Department of Agriculture (USDA) National Institute of Food and Agriculture. This work was supported by the USDA National Institute of Food and Agriculture, Hatch Project COL00293D, accession number 1021217.

**Reference Conflicts of Interest:** The authors declare no conflicts of interest.

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