Crosstalk of proteins, miRNAs involved in metastatic and epithelial–mesenchymal transition pathways

ABSTRACT

Metastasis is an intricate process which involves the proliferation of a tumour to distant parts of the body from its original site. To successfully colonize a distant area in the body, a cancer cell must complete a series before it becomes clinically detectable. These steps involve a large number of proteins indulging in various pathways. Proteins such as matriptase require serine protease for activation, processing and degradation of any signal. Stim1/Orai1 controls the Ca²⁺ channel which is important for cell migration. Sox protein plays a vital role in various cellular activities and the disruption of its gene plays a role in instigating invasion. Unlike the above three, Metadherin has an inhibitory role to play. Protein inhibits the epithelial–mesenchymal transition (EMT); thus the loss of metadherin evokes metastasis. These proteins play a role in amalgamation with various pathways such as the AKT E-cadherin and EMT pathway, PI3K/AKT pathway, integrin-linked kinase (ILK) – integrin signalling pathway, PI3K/AKT and notch signalling pathway. In this article, we have combined various proteins and pathways that work in coordination to result in a metastatic colony. There are two major events that occur during metastasis, that is epithelial–mesenchymal transition and mesenchymal–epithelial transition. Both these events are an indispensable part for metastasis.

KEYWORDS:

• Metastasis
• cancer
• epithelial–mesenchymal transition (EMT)
• pathways
• tumour
• proteins
• mesenchymal–epithelial transition (MET)

Cancer

The burden of cancer is projected to be more than twice of its current value globally. This situation has raised the prospect of an enormous public health. Change in distributions of risk factors as well as a rapidly changing pattern of ageing is contributing largely to the absolute burden of cancer (Siegel et al. 2014). According to the data of GLOBOCONN data3, an International Agency for Research on Cancer (IARC), 14.1 million cases were diagnosed and also 8.2 million deaths were reported worldwide in 2012 alone. These data did highlight the urgent need for expanding cancer-preventing efforts. In preventive measures, include population-based intervention like those that aim at controlling tobacco use, decreasing ultraviolet radiation exposures, reducing alcohol consumption, vaccinating against Human Papilloma Virus infection (Tarver 2012), etc. There are a number of treatments and medications available for cancer but the number of diseased people is still rising. One major issue with the treatment of cancer is its highly effective technique of transforming normal cells into metastatic ones. According to the study conducted by a group of researchers from Harvard-affiliated Brigham Women’s Hospital (BWH), cancer cells have the capability to extend their reach, transforming normal cells into metastatic and cancerous ones via ‘metastatic hijacking’. According to Shiladitya Sengupta, Bioengineering Division in the Department of Medicine and the corresponding author of the above study, metastasis will remain the final frontier in adopting and searching for a better cure for cancer. This research has been published in Nature Communications and gives an insight into the metastatic process. Thus, it is the need of the hour to search for loopholes in the metastatic pathway and protein functioning so that we can come up with a better cure.

Hallmarks of cancer

A review article authored by Douglas Hanahan ‘The hallmarks of cancer’ published in the Cell in 2000 categorized cancer on the basis of hallmarks. In total, six hallmarks were reported in the paper and later on new markers were also added to the list, making it 10. The main characteristics include the self-sufficiency of cancerous cells to initiate growth via growth signals (such cells are generally insensitive to the antigrowth signals), have the capability to evade apoptosis, have effective replicative potential, have sustained angiogenesis, which is a remarkable feature, invade new tissues and metastasis, have instability in the genome and being prone to mutation, cause inflammation in the affected tissue, reprogramme the energy metabolism and so on (Hanahan & Weinberg 2000).

EMT, metastasis and cancer

Despite the investments of huge resources, only incremental improvement has been observed in mortality. Most of the patients succumbed to either complicated treatment or metastatic disease. These huge data of reoccurrence suggest that the understanding of the metastatic machinery may lead to the development of effective anti-metastatic therapies that may provide an additional support in combating the morbidity and mortality of patients. The metastatic pathway involves a complex procedure of cell invasion from primary tumour, intravasation, extravasation of the circulatory system and all this followed by growth of new blood vessels, which is called angiogenesis (Gupta & Massagué 2006; Steeg 2006). The number and size of large lesions are indications of metastatic lesions, which can be detected by imaging. Known proteins are reported for the process of metastasis. Metastasis is a complex process that involves the spread of a tumour or cancer to distant parts of the body from its original site (Leber & Efferth 2009). However, this is a difficult process. To successfully colonize a distant area in the body, a cancer cell must complete a series of steps before it becomes a clinically detectable lesion. Metastasis comprises multiple complex pathway proteins and other microenvironmental factors that are interlinked. It encompasses various characteristics: intravasation proliferation in competent organs leading to colonization, have an ability to survive in the circulatory system, infiltration of tumour cells into the adjacent tissue (Eger & Mikulits 2005). The process of metastasis includes very efficient, well-coordinated multiple processes that are involved in making the whole phenomenon successful. If the process fails at any step, the complex machinery proceeds to the elimination of emigrating cancer cells at any of the multiple steps (Chambers et al. 2002; Mehlen & Puisieux 2006). There are large numbers of proteins involved in a cascade manner to lead to metastasis. In this article, we focus on the various RNAs, proteins, pathways and other molecules involved in the metastatic process, via elucidating the various cellular and molecular mechanisms, recognizing new targets to combat metastasis.

MET and its role in metastasis

Embryonic development leads to the switching of various cellular processes from one form to another. Epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) are two of them. Interswitching of both these phenomena results in the formation of a tumour at various cellular levels (Nakajima et al. 2000 ; Nakaya et al. 2004; Li et al. 2014). MET is a completely reversible phenomenon which involves various processes such as transiting cells from motile to planer arrays. There is a whole lot of differences in epithelial and mesenchymal cells in terms of interactions and biochemical nature. There are no mature cell–cell contacts. The cells have the ability to invade the extracellular matrix, and also possess the ability to express markers such as Snail, Twist, Fibronectin, Vimentin and N-cadherin (Thiery 2002; Baum et al. 2008). Determination of cell fate is a major issue that needs to be solved in developmental biology. Molecular mechanisms of reprogramming have led researchers to have an insight into decisions regarding the fate of cells. Reprogramming of the cells generally starts with MET, which suggests that the procedure is a complete reversal of EMT. This kind of switching generally occurs during the embryogenetic phase of the cell. Recent research has found two processes for the switching between these mechanisms. Since MET has been found to play a vital role in reprogramming it would be of greater use to study it in deciding the fate of the cell. It is a multistep procedure that involves an enormous number of proteins in the process of transforming cells from one type to another. Now the whole MET procedure is required for reprogramming but it also plays a significant role in the developmental event of carcinogenic modification. Now from various experimental studies it has been confirmed that MET is an important event along with EMT in order to complete the whole procedure of metastasis. The reprogramming procedure consists of various events such as initiation, maturation and the final step of stabilization (Samavarchi-Tehrani et al. 2010). Further analysis of gene expression has confirmed that MET is composed of downregulation of mesenchymal markers and transcriptional factors such as Zeb1, Snail and Cadherin (Li et al. 2010; Samavarchi-Tehrani et al. 2010). Thus MET is a definite first response of somatic reprogramming. Figure 1 shows the various pathways of conversion from MET to EMT.

Figure 1. Interaction of few factors in MET and EMT conversion.

Major proteins involved in interchange of MET and EMT

As already mentioned in various literature search and papers, it is evident that for the occurrence of metastasis, primary tumours must disassociate from the primary location and undergo de-differentiation through various activations and deactivations of the embryonic programme that involves EMT (Thiery et al., 2009). De-differentiated cancer cells in combination with EMT lead to the migration of the stem cells (Brabletz et al. 2005). Now TWIST1 is such a protein which is significant in co-inducing EMT and other stemness properties, thus leading to the linkage of EMT and the cancer stem cell property (Mani et al. 2008; Morel et al. 2008) (Shimono et al. 2007). Metastasis is not only governed by the EMT phenomenon but most of the metastatic cancers show re-differentiation in terms of MET. Here the one question that arises is why do the cells undergo re-differentiation? The probable answer to this is that in metastatic and invasive cells growth is arrested and the proliferation can only occur in re-differentiated cells (metastatic cells), leading to the conclusion that there should be a reverse phenomenon of EMT for the proliferation of cells and to make colonization possible. Many clinical experiments have reported evidence of the fact that cells undergo EMT–MET switching in the metastatic phenomenon but still the evidences provided are very less in number (e.g. Chaffer et al. 2006; Korpal et al., 2011). In studies it has been found that downregulation of TWIST1 is linked directly with the reversal of an EMT-related growth arrest. It has been considered that EMT plays a vital role in dissociation of the tumorous cells from the primary tumour site and that MET is required for proliferation and colonization of the disseminated cells; the sequence is shown in Figure 2(a). Furthermore, it has been found that miR-200 plays an important role in re-differentiation. It also impacts SEC23A directly. SEC23A has been found to be involved in the production of protein, which leads to the suppression of metastatic progress (Korpal et al., 2011). The other protein that significantly affects the EMT phenomenon is prrx1 ‘paired-related homeobox transcription factor1’. In comparison to other factors affecting the metastasis, prrx1 leads to the suppression of stemness, thus negatively regulating the EMT phenomenon. Further, the protein works in combination with TWIST1, leading to the relevant changes of migration and invasion. In a study carried out by Tsai et al. (2012), it was shown that loss of TWIST1 might lead to the disruption of the metastatic phenomenon, which was later disproved by conducting various experiments. Depletion of TWIST1 has only a marginal effect which indicates the indispensable role of prxx1 as a prime factor in regulating the whole event of metastasis. Further studies have shown that Prxx1 is responsible for negative regulation of stemness. Results are significant for a biologist in this field because they support the phenomenon of EMT/MET switching of metastasis and also they recognize a new mechanism for colonization of metastatic cells via incoupling of EMT with stemness, which leads to growth arrest in order to favour the maintenance of stemness, MET and proliferative phenotype (Figure 2(b)). EMT and MET switching gets affected by many factors and proteins. The works of Tsai et al. (2012) and Ocaña et al. (2012) provide ample evidence that there is an upregulation of Twist1 which is an EMT activator in invasive cells of primary tumour. It is required to downregulate the EMT inducer in order to facilitate MET which is a redifferentiation process at various distant places. This helps in colonization of the tumours at various distant places. The peculiar characteristics of Twist1 include arrest of growth and induction of stemness. Twist leads to two occurrences: it provides the cell with stemness but leads to arrest of growth, which provides the cell with mobile but non-proliferating cancerous cells. Prxx1 is activated by EMT; this is a newly formed protein (Ocana et al. 2012). This protein leads to the suppression of stemness in EMT. It is required to downregulate the expression of prrx1 to activate stemness of the cells from the primary tumour and also to allow the colonization of the same. Thus now it is evident from all the literature search and experimental data that both EMT and MET are required for metastasis. EMT is required for dissociation of the cell and MET is required for colonization.

Figure 2. (a) Effect of twist1 on the metastatic property of cancerous cells. (b) Effect of prxx on the metastatic property of cancerous cells.

miRNA and its role in metastasis

The miRNA-200 family

It is a small noncoding RNA usually of 22 nucleotides. Generally, it is found in viruses, plants and animals and plays a role in silencing RNA and in regulation of gene expression (Ambros 2004; Bartel 2004). In plants and animals, it is encoded as eukaryotic nuclear DNA and in viruses b viral DNA. It functions by pairing with the complementary sequence of the mRNA molecules (Bartel 2009). This resembles siRNAs marginally except for the fact that miRNAs are derived from a longer region of RNA. The human genome might encode over 1000 miRNAs (Bentwich et al. 2005). These are abundant in almost all the cell types (Lagos-Quintana 2002; Lim et al. 2003). They target approximately 60% of the genes of humans (Lewis 2005; Friedman 2008). The family of miRNA-200 consists of at least five members and they are found to form two clusters which are located at two different genomic regions. Further studies have found that the first cluster, I miR-200 in humans, is constituted of miR-200b, -200a and -429(miR-200b/200a/429) which are located at chromosome number 1. Cluster II miR-200s contains miR-200c and 141(miR-200c/141) which are located at chromosome number 12 (Michael et al. 2003; Altuvia et al. 2005). Apart from their distinction on the basis of location, they can also be distinguished on the basis of their functional aspects. They only differ in seed sequence by a single nucleotide that is AAUACUG for the first group and AACACUG for the second one. Conserve sequences have been found in the miR-200 family in the vertebrate species. ZEB1 and ZEB2 have been found to be bound to the promoter regions of the miR-200 family members (Kim et al. 2011; Lesnikoff et al. 2014). Studies have shown that the miR-200 family is the most studied and researched miRNAs in cancer and metastasis. Its role has been found in all the stages of cancer that is initiation, invasion and movement of tumorous cells.

Role of the miR-200 family in transformation of cells and in tumourigenesis

Initiation of tumour progression is a complicated procedure that leads to the transformation of the normal cells into malignant ones. During the whole mechanism of tumour formation, the molecular profile of the cell changes in order to make it tumorous.

The miR-200 family in cancer metastasis

The multistep signalling mechanism in a cascade manner helps in the occurrence of metastasis, where cells move from the primary site of the tumour to colonize at distant organs and form secondary metastatic tumours (Chaffer et al. 2013). Cells that attain metastatic movement generally undergo an extensive epigenetic and genetic modification in their molecular mechanism. Broadly, the whole event of metastasis has been divided into six steps (Weihua et al. 2008): (a) formation of tumour at the primary site, (b) detachment of the cancerous cells and their migration away from the primary site, (c) intravasation into the blood stream and lymph nodes of the tissues, (d) circulation in the blood stream, (e) extravasation, (f) colonization at some distant organs and formation of secondary tumours. Research has shown that molecules have the potential to affect every step of metastasis. There is an effect of miR-200 on various events of metastasis.

Effect of the miR-200 family on tumour growth, angiogenesis and nearby tissue invasion

After getting transformed into malignant cells, the checkpoints are bypassed, which results in uncontrolled growth. It has been found from studies that miR-200 has an inhibitory effect on tumour growth (Senol et al. 2014). On the contrary, many researches have shown that it has a very small role to play in tumour growth. Some researchers say that it plays a vital role in inhibiting tumours; others suggest that it has a small or no role to play in altering the metastatic pathways (Iliopoulos et al. 2010; Williams et al. 2013; Humphries et al. 2014; Li et al. 2014). Hence in light of these evidences, it is clear that more extensive work is needed in the study of miR-200.

Impact of miR-200 on EMT and migration of tumorous cells

Metastasis is one of the most lethal properties of tumour cells. They disseminate from the primary site and move to the site of secondary infection. One important process that has been explored extensively that results in this phenomenon is EMT. The EMT mechanism is a prime process during embryogenesis and now with all the findings it has been found to be a major contributor to the metastatic event. ZEB1 and ZEB2 are homeobox factors that play a vital role in maintaining the cell in its mesenchymal phenotype by keeping the transcription level of miR-200 and E-cadherin low. Thus, the interplay between the protein and various transcription factors, that is, ZEB1/ZEB2 and the miR-200 family, drives the cell into an EMT state. Furthermore, the study conducted by Gregory et al. (2008) has shown that it helps in suppression of the cell movement (Gregory et al. 2008). This group has used the potent inhibitor of miR-200 and found an enormous increase in cell migration in the Madin-Darby canine kidney epithelial cells, which suggests that the miR-200 family does play an important role in suppressing metastasis (Park et al. 2008). There are various proteins and factors involved in certain pathways and mechanisms via which miR-200b works. It acts on moesin, thus regulating the migration and invasion. Upregulation of moesin facilitates the metastatic movement, whereas its downregulation leads the miR-200b to show its function and inhibit the migration. Further, it has also been found that miR-200b negatively regulates the level of PKCα; it alters its expression by targeting the 3′UTR of PKCα (Wang et al. 2014). Moreover, knockdown of PKCα reduces the migration significantly and also forced expression of the protein leads to the reversion of an inhibitory effect of miR-200b. These studies indicate that miR-200b inhibits migration of metastatic cells by targeting PKCα. Further, in the study it was found that miR-200 is involved in Rho-ROCK signalling, focal adhesion, MMP activity, invadopodia formation, etc.

Role of miR-200b in tumour intravasion

It is a complicated step that involves the entrance of cancerous cells into blood vessels/lymphatic system. Intravasion involves many signalling pathways in its completion. The two that are extensively involved are Notch and tumour-associated macrophages (Wyckoff et al. 2007; Sonoshita et al. 2011). Blood vessels of the primary tumour are somewhat leaky and facilitate the moving cell with the appropriate environment for intravasation (Jain 2005). In studies it has been found that miR-200 affects intravasion negatively but the mechanism of the same is still not known (Carmeliet & Jain 2011).

Role of miRNA34 in metastasis

The molecular determinant of metastasis is still very much under wraps or not known completely in spite of the fact that it causes the largest percentage of deaths due to cancer (Fidler 2003; Parkin et al. 2005; Thorgeirsson & Grisham 2002). Various researches have revealed the crucial role played by miRNAs in the process of metastasis. miRNAs regulate a large number of genes post-transcriptionally, which plays an important role in metastasis (Lim et al. 2005; Dalmay & Edwards 2006; Kim et al. 2008). In mammals, miRNAs are inculcated in RNA-inducing silencing complex and also they are associated with the 30 untranslated regions of a specific target, which suppresses the translation and also induces degradation (Pillai 2005; Zamore & Haley 2005). It has also been found that they play a significant role in differentiation of cells and proliferation (Chen et al. 2004; Croce & Calin 2005). They can behave both ways, as a suppressor of tumour or as an oncogene depending on their target (Cimmino et al. 2005; Johnson et al. 2005). It has been found that oncogenic miRNAs only express in tumorous growth such as miR-520c, miR-21, miR-373 and miR-10b (Zhu, et al. 2008). On the contrary, the suppressive ones are downregulated and these include miR-29c, miR-126, miR-335 and miR-146a, which lead to the invasion (Lin, Chinag, et al. 2008; Musiyenko et al. 2008; Sengupta et al. 2008; Tavazoie et al. 2008). Furthermore, in the study it has been found that miRNAs are overexpressed in all the tumours. There are peculiar miRNAs, for example, miR-529c, miR-373, miRNA-21, miRNA-10b, overexpressed in tumours (Asangani et al. 2008; Lin, Chiang, et al. 2008; Zhu et al. 2008). miR-34a works via targeting apoptosis, leads to arrest of G1 phase and also leads to the senescence of the cell by regulating cell cycle cyclin and cyclin dependent kinase such as cyclin D1 (CCND1), cyclin E2 (CCNE2) and Bcl-2. It also leads to the suppression of the tyrosine kinase receptor c-Met (oncogenic) (Kim et al. 2008). From these evidences, it can be deduced that miR-34a plays a role in metastatic moment and also helps in invasion. In one study, it was found that miRNA leads to the regulation of invasion and migration, and that it also helps in scattering of cells, in HCC tissues in HepG2 cell lines. C-Met is considered to be one of the prominent receptors for the hepatocyte growth factor and it is inhibited by miR-34a (Leelawat et al. 2006; Kim et al. 2008). This growth factor leads to the activation of the c-Met which triggers the phosphorylation of the signalling pathway such as ERK1/2, which influences the migration and invasion (Sipeki et al. 1999; Ma et al. 2007). c-Met also plays a role in scattering of the cells. These evidences indicate that miR-34a plays a role in metastasis. Constitutive instigation of the components of the downstream pathway of the MAPK pathway leads to the migration (Ma & Tretiakova et al. 2007). All the above studies along with data predict the role of the component, that is miR-34, could be investigated for therapeutic application of the metastatic movement.

Proteins involved in metastasis and invasion

Various proteins and pathways play a significant role in metastasis. These proteins interact with each other intensively to make cells metastatic. There are various proteins whose chemical or natural inhibitors have been reported while others may or may not have known inhibitors. These proteins have been classified and discussed under two categories, one category having chemical or natural inhibitors reported and the other without any known inhibitor, but in vitro experiments have proved their role in metastasis. A list of a few of the important proteins with their known inhibitors is presented in Table 1. Another list presented in Table 2 consists of proteins and enzymes along with the area that they are affecting. These proteins may or may not have any effective known inhibitor.

Table 1. Important protein responsible for metastasis and their known inhibitors.

	Protein	Inhibitors
1	Matriptase	CVS-3983, Benothiozole containing peptidomimetic
2	STIM1	OphiobolinA, f96365, ML-9
3	Sox	Polyoxometalase
4	TGF-bR1	Repsox, 2-[5[(6-methylpyridin-2-yl)-1H-Pyrazol-4-yl]-1,5-napthyridine
5	Snail1	Co(III)-Ebox, Co(III)-DNA Conjugate
6	HMGA	Rosuvastatin (crestor), Lovastatin (Mevacor), Atorvastatin (Lipitor)
7	Lysyl oxidase	β-Aminopropionitrile (BAPN)
8	Periostin	SiRA an antisense oligonucleotide or a ribozyme
9	E-cadherin	Curcumin
10	Survivin	Ardisianoe, YM155 in combination with doxorubicin and cisplatin
11	Caspase-3	CrmA, P35 autographa californica, P49 spodoptera litoralis Iodoacetamide
12	Osteopontin	Agelastin
13	Activin A	SB-431542, BMP
14	Integrin B	RGD peptide (GRGDNP), SHARPIN
15	Cofilin	CytochalasinD (cyto D), cucurbitan E
16	Cathepsin	Cystatins, VBY-825
17	Metaadherin	AZD6244
18	STAT3	S3I-201, STATTIC, STA-21
19	Vimetin	Withaferin (WFA)

Table 2. Important protein and enzymes responsible for metastasis.

S. no.	Name of protein
1	PRXX
2	SYNTENIN
3	TWIST
4	P2Y2
5	LIVIN
6	MMP2
7	MMP9
8	VIMENTIN
9	ZEB1 AND ZEB2
10	LOX
11	TGB-b
12	E-CADHERIN
13	CATENIN
15	SURVIVIN
16	PERIOSTIN

Matriptase

Matriptase is a member of the family of type II transmembrane serine proteases. It is also known by different names such as EPITHIN, ST14 and TADG (Bugge et al. 2009). It is a protein with a complex structure and regulatory mechanism (Leland & Wright 2006). Its weight is approximately 80–90 kDa. This protein is synthesized as an inactive zymogen and travels via the golgi apparatus to the plasma membrane. It has a short transmembrane part and a large C-terminal region containing a catalytic serine-protease domain and several non-catalytic domains. It also consists of an N-terminus to the cytoplasmic site of unknown function. Activation of matriptase is triggered by shedding of the extracellular part from the cell surface into the surrounding microenvironment (List et al. 2006). Studies have showed that the activation of protein matriptase can occur on the cell surface as well as inside the cells. Action of matriptase is important for activation, processing and degradation of cellular protein. There is considerable evidence indicating the presence of matriptase in tumour formation and metastasis. Even the slightest of overexpression of matriptase is enough to trigger the formation of tumours in mice (List 2009). There are several evidences that link matriptase to carcinogenesis in various cancer types, which include ovarian, prostate and cervical cancers (Lin, Tseng et al. 2008). Consequently, considerable efforts have been made in the development of matriptase inhibitors and methods to put a check over its activity in tumours (Shi et al. 1993; Galkin et al. 2004; Farady et al. 2007; Li et al. 2007; Napp et al. 2010). Matriptase was discovered originally as a matrix-degrading protease in breast cancer cells (Shi et al. 1993). Since matriptase plays a vital role in cell migration and invasion it could be explored as a target for inhibiting invasion.

STIM1/Orai1

Ca²⁺ is one of the critical regulators of cell migration and it is ubiquitous in nature (Pettit & Fay 1998). A store-operated Ca²⁺ entry mechanism is predominant in the mechanism of non-excitable cells (Lewis 2007; Parekh & Penner 1997). Ca²⁺ influx is an important and essential phenomenon for migration of normal as well as tumorous cells (Marks & Maxfield 1990; Komuro & Rakic 1993; Lee et al. 1999; Nishiyama et al. 2003; Li et al. 2005). Two genes have been identified from recent studies, STIM1 (stromal interaction molecule 1) and Orai1 (ORAI calcium release-activated calcium modulator 1), which are responsible for store-operated Ca²⁺ entry (Liou et al. 2005; Roos et al. 2005; Vig et al. 2006; Feske et al. 2006; Zhang et al. 2006). Orai1 is an essential pore-forming component of the store-operated Ca²⁺ entry channel (Yeromin et al. 2006), while STIM1 serves as a sensor of Ca²⁺ (Prakriya et al. 2006). Store-operated calcium channels are reconstituted by coexpression of Orai1 and STIM1 (Mercer et al. 2006; Peinelt et al. 2006; Soboloff et al. 2006). A variety of physiological and pathological processes are controlled by store-operated calcium influx (Fanger et al. 1995; Mogami et al. 1997; Dolmetsch et al. 1998; Lewis 2001; Feske et al. 2005; Yoo et al. 2000). Both the proteins STIM1 and Orai1 have been found to play a role in breast tumour cell migration, invasion and metastasis. The dynamic structure that is under spatial control at the subcellular level is termed as focal adhesion. Focal adhesion turnover consists of proteolysis of proteins and phosphorylation of tyrosine (Webb et al. 2004). An increase in cellular Ca²⁺ could increase the activity of the tyrosine kinase FAK (focal adhesion kinase) and the calcium-dependent protease calpain in focal adhesions (Siciliano et al. 1996; Huttenlocher et al. 2011; Achison et al. 2001; Dourdin et al. 2001). It has been found in studies that in FAK-deficient fibroblasts, migration is decreased with an increased number of focal adhesions (Ilic et al. 1995; Ren et al. 2000). Rac is activated by FAK through p130Cas, Crk and the DOCK180/ELMO complex (Brugnera et al. 2002). Calcineurin and myosin light-chain kinase could also mediate the Ca²⁺ effect on focal adhesion turnover (Lawson & Maxfield 1995; Eddy et al. 2000). Invadopodia has a role to play in cancer invasion, and podosome and invadopodia are also considered to be adhesive in structure (Linder 2009). Blocking store-operated Ca²⁺ influx slows down focal adhesion turnover, resulting in larger focal adhesions and consequently stronger adherence. Strong adherence could lead to the fast migration of cells, including metastatic cancer cells. And hence there are agents like SKF96365, siRNAs for Orai1 and STIM1 that can block the store-operated Ca²⁺ channel’s activity of store-operated calcium, and can be considered as potential therapeutics for tumour metastasis.

SOX

A family of transcription factors containing the DNA-binding domain of SRY is encoded by SOX genes (Dong et al. 2004). In total, 20 different SOX proteins are identified in mammals and these proteins are further categorized into 8 subgroups (A, B1, B2, C, D, E, F, G, H). This categorization is done on the basis of gene structure, presence of specific functional domains HMG box domains, transrepression and transactivation (Castillo 2012). Expression of a targeted protein is either repressed or activated on the basis of the presence of domains and their specific binding partners (Wegner et al. 2010; Kamachi and Kondoh 2013). SOX proteins are involved in various functions during embryonic development such as development of the central nervous, haematopoietic system, sex determination and cell differentiation (Kamachi and Kondoh 2013). Disruption of these genes plays a crucial role in invasion, as it is involved in many of the cellular activities, cellular programming, regulation and adhesion of cell–cell interaction (Sarkar & Hochedlinger 2013).

Metadherin

Invasion involves many steps before its final execution; these steps are intravasation, survival in circulation, extravasation, micrometastasis and final colonization (Thompson et al. 2005; Chaffer & Weinberg 2011). EMT is considered to be the primary and most important step in cell invasion and metastasis in situ (Yang & Weinberg 2008; Thiery et al. 2009; Gonzalez de Castro et al. 2013). It can be understood as adhesion between two cell “cell-cell” adhesion is interrupted which is followed by transformation of the cytoskeleton of epithelial cells adhesion is interrupted which is followed by transformation of cytoskeleton of epithelial cells into mesenchymal-like cells (Acloque et al. 2009; Chao et al. 2009). These studies suggest that a protein that plays an important role in cell adhesion and inhibits cytoskeletal remodelling can be considered to be an important target for inhibiting EMT and tumour metastasis. Overexpression of MTDH leads to inhibition of EMT by reversing the pro-metastatic actin and remodelling cytoskeleton. And loss of or inhibitory expression of MTDH leads to reversal of action and facilitates the invasion and metastasis of proteins. It was demonstrated that MTDJ co-localized with tight junction protein ZO-1 and occludins in polarized epithelial cells (Thompson et al. 1994; Kleinerman et al. 1995; Britt et al. 2004). The occurrence leads to disruption at junction complexes, MTDH dissociates from ZO-1 and it is again reloaded during maturation of the tight junction complex (Britt et al. 2004).

Cathepsin

Cathepsin B induction is required for the oesophageal cancer invasion into ECM (Andl et al. 2010). Overexpression of Cathepsin D results in increased fibroblast motility and invasion (Pruitt et al. 2013). Activity of integrin gets affected by Cathepsin H, which further influences the process of migration and invasion (Jevnikar et al. 2013). Regulation of expression of MMP9 and Cathepsin K occurs by coronin 3, which further leads to the expression of invasional characteristics (Ren et al. 2012). It has been revealed by immune-staining that there is a strong expression of Cathepsin K in most of the primary melanomas’ metastasis. Therefore, it could be concluded that Cathepsin K has an important role in invasion and metastasis (Quintanilla-Dieck et al. 2008). Progression of prostate tumour is facilitated in bone by Cathepsin K (Keller et al. 2013). In case of colorectal carcinomas it is said that Cathepsin S plays novel roles in migration and invasion (Burden et al. 2012). And also Cathepsin S has a role in invasion and migration in case of gastric and hepatocellular carcinoma (HCC, Yang et al. 2010; Fan et al. 2015). Upregulation of Cathepsin X was also directly associated with higher invasiveness (Krueger et al. 2005). Inactivation of the tumour-suppressive proteins like profiling 1 by Cathepsin also leads to the migration or invasive feature of cells (Pečar Fonović et al. 2013).

Involvement of factors

Metastasis is a complex process that involves multiple cellular process such as angiogenesis (generation of new blood cells), migration (motility), invasion (movement) and growth. These processes are complicated and involve various factors that either suppress or enhance the activity of specific proteins. All these factors are released in normal as well as cancerous conditions but there are imbalances of certain factors leading to characters such as angiogenesis and extra growth. Change in adhesion is also one of the major factors required for invasion, but these processes are complex and require disruption of local cell–cell interactions on one hand and formation of cell–cell interactions on the other hand. Therefore, here we are proceeding to the individual gene and protein involved in it.

Growth factors and their receptors

Although metastasis is a late event in the natural history of carcinogenesis, early gene changes can influence later events. Growth factors and their receptors are often perceived to be early changes due to their ability to act as oncogenes in some systems, and their ability to induce transition through the cell cycle and so induce proliferation (Jakowlew 2006). The event of metastasis gets affected significantly by signalling through growth factor receptors

However, it has become apparent that these impacts include regulation of the cytoskeleton, effects on cell shape, adhesion of cell–cell and expression of a variety of genes related to cell growth such as proteins that modulate invasion.

PRRX

The PRRX1 gene encodes a DNA-associated protein called prrx. The protein encoded by the gene comes under the family of Homeobox proteins that is localized to the nucleus of a cell. It acts as a coactivator of transcription, thus enhancing the binding affinity of DNA to the serum response factor. Muscle creatin kinase is also regulated by this protein, thus indicating the vital role it plays in the development of various mesodermal muscle types. Splicing leads to the formation of two isoforms of the same protein that variedly differ in their expression and pattern. It has been found during experimental work that the mice that lack both prrx 1 and prrx2 (Suzuki et al. 1997; Wissmüller et al. 2006). PRXX1 regulates various proteins which includes differentiation of mesenchymal precursor. One example of this kind of regulation is inhibition of adipogenesis which occurs by activating the transforming growth factor-beta (TGF-beta) signalling (Gregory et al. 2006). It has also been found in recent studies that it downregulates the tumour necrosis factor-alpha in order to inhibit osteoblast differentiation (Kim et al. 2007). These recent findings have clearly instigated researchers to find more about the role and significance of protein. Furthermore, it has been discovered in in vivo studies that for metastasis to occur efficiently it is required to lose the Prrx1 and this procedure leads to the acquisition of stem cell properties. Unlike traditional EMT transcription factors, Prrx 1 leads to the uncoupling of EMT and stemness and is also considered as a biomarker for patients who have survived the lethal effect of metastasis. Apart from the entire regulatory role that it plays there is a paradox related to the protein functioning in that its downregulation is needed for induction of stem cell property but its expression to a certain level is required for invasion.

E-cadherin and b-catenin

E-cadherin is supposed to help in adhesion of molecules with catenin; when the level of adhesive molecules depletes due to any reason it leads to a disruption of intercellular contacts. And this disruption of cellular contacts is considered to be the prime step towards early metastasis. Only destruction could not guarantee a successful metastatic procedure. The depletion of the E-cadherin protein does via inducing the epithelial to mesenchymal transition and invasiveness. It has been found in studies that loss of E-cadherin induces series of transcriptional changes, thus leading to the activation and deactivation of various proteins of metastasis. Thus, the overall loss of E-cadherin provokes the phenomenon of EMT, which is followed by elevated motility of cells, increased invasiveness and also resistance to apoptosis (Cancer Res 2008). There are two methods employed by tumours cell to deactivate E-cadherin: in one method only the level of E-cadherin gets affected and in the other truncated or inactive protein production occurs. It was found that mutation in the extracellular domain of the protein does not lead to initiation of any metastatic event instead a complete elimination is required for instigation of the event (Berx et al. 1998) are not likely to result in an EMT or to afford functional traits that allow completion of the later steps of metastasis. Complete loss of the protein leads to multiple events in a cascade manner to finally complete the process of metastasis.

Vimentin

The VIM gene is accountable for the production of the vim protein. It is expressed in mesenchymal cells and is considered to be the type III intermediate filament. Protein vimentin play vital role in humans and also it has shown its findings in metazoan and bacteria (Eriksson et al. 2009; Cabeen & Jacobs-Wagner 2010). Since vimentin is one of the major structural constituents of the mesenchymal cell it is often used as a biomarker for cells which are derived mesenchymally or which undergo (EMT) transition. It also plays a significant role in positioning and anchoring of the organelles in the cytosol. Its attachment to the various organelles such as mitochondria, endoplasmic reticulum and nucleus is laterally or terminally (Katsumoto et al. 1990). It provides flexibility to the cell, and integrity (Goldman et al. 1996). Through the various experimental setups it has been found that it is used as a marker for sarcoma tumour to identify the mesenchyme (Leader et al. 1987). Further investigation reveals that it regulates the interaction of various cytoskeletal proteins with that of cell adhesion molecules and thereby plays a pivotal role in migration, invasion, signalling and adhesion. Phosphorylation, polymerization and depolymerization act as regulatory mechanisms for the protein and thus affect cell–cell interaction. These new findings about the protein put it in the category of a potent target for anti-metastatic therapies and research.

SLUG

Expression of SLUG has been found to be elevated in tumour metastasis in various types of tumours (Zhang et al. 2010). In an experiment conducted on a breast cancer mouse model, it was found that SLUG promotes metastasis by partially inhibiting E-cadherin (Casas et al. 2011). SLUG acts on the metastatic pathway in many ways; apart from inhibiting E-cadherin it regulates CXCL12. Overexpression of this protein leads to the upregulation of CXCL12 and promotes migration and invasion prostate cancer and also reduces the adhesion. In contrast to the above study, knockdown of the same CXCL12 leads to the impairment of SLUG-mediated MMP9 expression and also affects the migration and invasion in PC3 cells. From the above studies, it can be seen that CXCL12 leads to the promotion of invasion by upregulating MMP9 (Shen et al. 2009), which leads to the degradation of extracellular matrix components. SLUG upregulates the expression of both CXCL12 and MMP9. It still has to be determined whether MMP9 is indispensable for SLUG-mediated invasion of prostate cancer cells (Arya et al. 2004). Research has shown that SLUG regulates the expression of the CXCL12/CXCR4 axis positively in human prostate cancer cell lines. Furthermore, in the study it was found that activated SLUG expression leads to the invasion and migration by activating the CXCR4/CXCL12 axis. Thus CXCL12, SLUG and MMP9 can be targeted for the prostate cancer metastasis (Patrussi et al. 2011).

Syntenin

Acquisition of invasiveness is an important feature of progression of a tumour. Mechanism of metastasis involves, primarly a tissue destined for invasion is required to get recognized and interact with the microenvironment that surrounds it. Secondly, it requires undergoing remodelling of the extracellular matrix in such a way so as to achieve the next step. Studies have shown that the migration step is the most important and it is this step that acts as a limiting factor for invasion (Hanahan & Weinberg 2000; Kassis et al. 2001).

It has been found that it is the specific PDZ domain of protein that interacts with other proteins. These proteins may include Syndecans and B ephrin. Eph B-ephrin plays a vital role in regulation of cell migration (Klein 2001) and the PDZ domain of protein colocalize with both Eph receptors and their ephrins. Furthermore, investigation has revealed that the expression of syntenin is elevated in metastatic human breast and gastric cell lines and also that it invokes the migration of the same.

TWIST

TWIST proteins have oncogenic properties which intimate crosstalk between failsafe program escape and cancer cell dissemination. TWIST itself does not lead to metastasis; instead it promotes it by affecting other micro-environmental factors. In further experimental research conducted through oncogenic cooperation assays on epithelial cells, researchers have demonstrated that TWIST works by overriding ERBB2 or RAS-induced senescence, which can promote EMT (Ansieau et al. 2008). It has been found that activation of RAS has a strong impact on various components of the cytoskeleton and that it can synchronize the TWIST proteins. This protein is more likely to put an impact on TWIST’s stabilization, thus effecting its activation significantly. It has generally been assumed that the process of metastatic initiation has been restricted to the later part of the cancer but in recent studies it has been found that some cancer dissemination might get initiated at an early stage, which gives rise to evolving of a single cell independently from a primary tumour. TWIST 1 reactivation leads to early tumour progression and its dissociation (Hüsemann et al. 2008).

P2Y2

Studies have shown that the P2Y receptor helps in stimulation of metastasis by acting on extracellular adenosine 5′-triphosphate (ATP). Various studies have shown the role of P2Y2 in metastasis of tumorous cells. One such receptor is P2Y which has been in focus in the current scenario to investigate its role in metastasis. In an experiment conducted to find out the role of P2Y2, the expression of the protein was silenced via RNAi. Later on in vitro and in vivo experiments were conducted to investigate the role of P2Y2 receptors in cellular metastasis and invasion. Apart from this, a cDNA microarray was also performed to identify the genes which are expressed differentially. The experiment has shown a significant expression of the protein in prostate cancer. Knocking down of the p2y2 receptor leads to suppression of invasion and metastasis in vitro as well as in vivo. Further studies have led to the fact that ATP has an ability to promote IL-8 and snail expression and also regulates Claudin-1 and E-cadherin expression negatively. Further the experiment has shown that knocking down of the protein has affected the expression of EMT/invasion-related genes in vitro and in vivo. In light of these findings, the P2Y2 receptor could be considered to be a potent therapeutic target for treating prostate cancer.

Livin

Livin comes under the category of IAPs (Identical ancestors proteins), which are inhibitors of apoptosis proteins. Livin is the most recently discovered IAP. Previously, it has already been established that IAPs play a significant role in tumour invasion and metastasis. Further research has shown that Livin might play a significant role in cell proliferation by regulating the G1-S cell cycle transition. In one study it was found that the expression of LIVIN is higher in PCa tissues in comparison to non-metastatic tissues. And also its expression is regulated by siRNA, which could inhibit or promote PC-3/LNCaP cell invasion. However, a lot is needed to be discovered regarding regulation of metastasis and invasion. Previous findings indicate that silencing the expression of Livin could reduce the invasive capacity in an SMMc-7721 cell line (Liu et al. 2010), which is an indication that expression of the Livin gene might be associated with tumour cell invasion and metastasis.

MMP9 and MMP2

MMPs are generally concerned with remodelling of a matrix, especially in terms of invasion and angiogenesis. Apart from this, its role has been expanded exclusively to other functions also, which include processing of various substrates related to cancer and its various stages.

These findings clearly make MMPS to the category of potential target against cancer. Despite such an apparent finding for the protein the field of MMP research in cancer is still not as efficient as required because of drastic failure of molecules in multiple phases of clinical trials against MMP (Coussens et al. 2002; Pavlaki & Zucker 2003). There could be various reasons for the failure of these inhibitors, the prime reason being the lack of specificity of the inhibitors and another reason being the side effects caused by dose limitations; the mechanism of dose-limiting effect has still not been understood completely (Pavlaki et al. 2010; Fingleton 2008; Sela-Passwell et al. 2011; Dufour & Overall 2013). Despite the setback one has got in finding potential molecules against MMP inhibitors knowledge and research in field of MMPs have increased tremendously. For inhibiting these MMPs, drugs with novel binding sites are required. In spite of all theextravagant research and findings, it is still challenging to find out ways to find a potent inhibitor molecule and to deliver it perfectly by integrating it with various available techniques such as MRI, PET/SPECT. So it is still a question for many whether to investigate it further or not.

Egfl7

Epidermal growth factor-like domain 7 (Egfl7) is considered to be expressed mainly in endothelial cells. It is one of the secretory proteins. In various studies, elevated levels of protein have been found but the mechanism by which it acts or instigates metastasis and invasion is still unknown. Expression of this factor is predominant in lung cancer, especially HCC cells. For functional characterization of Egfl7 in HCC, its expression in HCCLM3 cells has been depleted by using siRNA. Reduction of the expression of Egfl7 leads to the suppression of migration of HCCLM3 cells. This study reveals that Egfl7 could be taken as a unique marker for metastasis of HCC and also it could be considered as a therapeutic target (Wu et al. 2009)

LOX

Metastasis is an efficient mechanism which involves several steps and which leads to more than 90% death of cancer patients. For development of an effective treatment of cancer, it is required to understand the mechanism and events of the metastasis. The pathway and interaction study will help us to identify the potential drug candidate. One such protein is LOX (lysyl oxidase). Studies have indicated the LOX- dependent cross-linking of collagen which involves in creating a microenvironment which facilitates the metastatic growth by evoking persistence and survival of tumour. Therapeutic targeting of LOX has shown the prevention of metastatic colonization. LOX works in unison with collagen and its cross-linking with the same drastically increases the proliferation of cells and also enhances colonization of metastatic cells. These researches have shown the significance of cross-linking of both the proteins. Thus these proteins could be targeted as a potential target against metastatic tumour.

Periostin

Periostin is found in extracellular matrix which has dual function attachment and it acts as an autocrine, which also helps in adhesion of molecules like integrins α_vβ₃ and α_vβ₅. It also helps in cardiac development, formation of bone, tumour development and wound repair. It is of significant importance to the biologist dealing with metastasis as its overexpression has been ground in almost all the tumorous cells. This involves the maintaining of the naive nature of the stem cells, formation of niche, survival of tumorous cells and angiogenesis. All the above-mentioned mechanisms are indispensable for the EMT and invasion step of tumorous cells. Apart from playing a role in the above-mentioned mechanisms it was also found to activate PI-3K/AKT, Wnt and FAK-mediated signalling pathways to promote metastasis. Since it has a role in multiple pathways and signals it appears to be a potentially good candidate for targeting inhibition.

Survivin

Overexpression of Survivin has been found in various melanomas; it is a mitotic regulator by function. It also acts as a marker for patients with metastatic disease. One study has shown that overexpression of the protein in YUSAC2 human melanoma cells has led to the increased formation of colonies on soft agar. It can further be explored to put it in the category of potent candidate for metastasis (McKenzie et al. 2013)

Pathways involved

Cancer is a complex amalgam of interactions where various factors, proteins and other signalling pathways work together in unison to produce cancer with its peculiar characteristics of metastasis, invasion, extravasion, angiogenesis and so on. Thus, it makes the study of involved pathways very important, so that the overall mechanism of cancer could be understood.

AKT, E-cadherin and EMT

Initially, AKT was classified under the category of oncogene (Staal 1987; Bellacosa et al. 1991). Broadly, three AKTs have been classified and all these have serine-threonine kinase activity (Testa & Bellacosa 2001; Bellacosa et al. 1991). AKT2 is supposed to be upregulated and activated in certain cancers such as ovarian, breast and pancreatic tumours. AKT has many roles to play in cellular processes, which include progression of cell cycle, proliferation of cell, cell survival, EMT and metabolism. AKT induces EMT and it leads to various cellular changes like reduction in cell–matrix adhesion, alteration in production and distribution of specific proteins, decreases cell adhesion, change in morphology, cell motility induction, apicobasolateral loss, there are various examples of alteration of functional proteins for an example desmoplankin is a protein that involves in maintenance of desmosomes is internalized, vimetin is induced in many mesenchymal cells (Figure 3). Akt has also a role to play in synthesis of metalloproteinases and invasion of cells (Kim et al. 2001; Park et al. 2002). In epithelial cells, Akt has two major consequences for E-cadherin; the first is that transcription of the gene responsible for E-cadherin is repressed strongly and the second is that a small amount of protein gets concentrated in the perinuclear region (Grille et al. 2003). These regulations maintain the cell to confine it in a mesenchymal state during exponential growth. The transcriptional repression of E-cadherin is affected by the activation of the Snail gene while its sequestration is related to the activation of Rab5 which is mediated by AKT (Barbieri et al. 1998; Batlle et al. 2000; Cano et al. 2000).

Figure 3. Interaction of various pathways leading to EMT.

Interactions between the PI3K/AKT and other pathways of EMT

Interaction between RAS and ILK, PI3K/AKT plays vital role in instigating EMT. EMT occurs only due to perfectly defined connections between these signal transducers (Boyer et al. 2000; Thiery 2002; Bellacosa et al. 1991). Other EMT pathways are less established and need to be investigated. However, studies have shown some peculiar connections between Wnt/b-catenin and Notch signalling.

Integrin-linked kinase (ILK) and integrin signalling

Focal adhesion requires integrin, which depends on the integrin-linked kinase (ILK) (Wu 1999). Targets of ILK involve AKT by activating phosphorylation on glycogen synthase 3 and S473 (Delcommenne et al. 1998). Overexpression of ILK leads to the nuclear translocation of protein b-catenin, which further increases the invasion and repression of E-cadherin (Novak et al. 1998; Wu et al. 1998) via upregulating Snail transcription (Tan et al. 2001; Barbera et al. 2004). Involvement of ILK was also seen in ILK TGF-b-mediated EMT of human keratinocytes (Lee et al. 2007). Figure 3 shows the various pathways interacting to lead to the EMT phenomenon.

E-cadherin pathways

EMT is a major phenomenon that invokes the event of invasion of EMT and vice versa. Both of these functions are dependent on the E-cadherin molecule. Cadherins are regulated at various levels as at the level of mRNA and at the level of proteins by varying the translational, transcriptional and degradation machinery. It is considered to be a tumour suppressor for the reason that its expression is silenced in various carcinomas (Vleminckx et al. 1991). Production of a defective protein or transcriptional silencing because of hypermethylation may result in the loss of functionality of the E-Cadherin. In addition to its mutation, abnormal post-translational modifications or protein degradation may lead to defective production of E-cadherin. Upregulation of E-cadherin has been reported in tumour progressions, particularly at the time of intravasation (Kang & Massagué 2004; Thiery & Morgan 2004). E-boxes have a peculiar repeating sequence called consensus sequence; in this case it is CANNTG Zeb1; Slug, Sip1 and Sail are the E-boxes that repress the E-cadherin transcription. Transcription of E-cadherin is inversely correlated with the expression of snail. Snail production in large amount is generally associated with the loss of E-cadherin expression (Birchmeier & Behrens 1994; De Craene et al. 2005). The overproduction of Snail or Slug induces EMT in vitro (Batlle et al. 2000). E-cadherin upregulation is associated with the repression of Snail RNA. There is a system of positive and negative regulation of E-cadherin (Palmer et al. 2004). Regulation of E-cadherin occurs via 1, 25(OH) 2D3 along the help of vitamin D receptor and its repression can occur by Snail regulate E-cadherin levels (Palmer et al. 2004). Repressor and nuclear retention activity of Snail are promoted by PAK1 which is activated by phosphorylated p21 (Yang et al. 2005). These examples have shown the complex process of regulation of E-cadherin during EMT. Sip1 helps in modulating the TGF-b.

PI3K/AKT and notch signalling

It has been found in recent studies that Notch ligand expression in human keratinocytes and cancerous cell lines of cervical tissue leads to AKT phosphorylation and also it induces PI3K-dependent EMT that can be characterized by upregulation of vimentin and fibronectin, downregulation of E-cadherin, increased motility and morphological changes (Veeraraghavalu et al. 2005).

RAS pathway

Regulation of activated downstream receptor protein tyrosine kinases of EMT is affected by RAS production. Jun and Fos are involved downstream of RAS Epistatic analysis (Boyer et al. 1996). There are many signalling factors involved in EMT and signalling from RAS to jun, fos, snail, slug and others involves MEK and RAF. Both these are inducers of EMT and MAPK (Edme et al. 2002). Another effector that alters the functioning of RAS is PI3. mRAS also affects the Rho, Rac and GTPases via PI3K. These two molecules play a role in EMT via regulating the adherent junctions, myosin phosphorylation, focal adhesion scattering and motility (Edme et al. 2002; Thiery 2002).

PI3K/AKT and Wnt/b-catenin signalling

Wnt and PI3/AKT signalling can converge at the level of inhibition of GSK3. It is evident from the studies that two pathways affect the different pools of GSK3 (Weston & Davis 2001; Grille et al. 2003). Other possible pathways may include the stimulation of AKT activity via Wnt/Disheveled signalling (Fukumoto et al. 1997) and b-catenin transcription stimulation (and possibly phosphorylation) by AKT and 14-3-3z (Tian et al. 2004). In spite of establishment of the indirect role of these interactions between PI3K/AKT and Wnt/b-catenin signalling, their direct role in EMT is still needed to be investigated.

Notch pathway leading to EMT

Notch is involved in determination of fate of a cell during development. It has been found from recent studies that notch pathway also plays a major role in inducing EMT. It performs this function by repressing VE-cadherin transcription or by inducing snail. In a same manner if Notch is overexpressed it results in EMT because it leads to the activation of Snail and repressing VE-cadherin (Timmerman et al. 2004). Figure 4 shows the interaction of various proteins with Wnt and B-catenin resulting in invasiveness.

Figure 4. Wnt and b-catenin pathway leading to EMT.

Rac1b and reactive oxygen species (ROS)

ROS has been found to have a role in EMT (Radisky et al. 2005). Production of ROS is stimulated by Rac1b which in turn leads to stimulation of Snail expression and initiation of EMT. When treated with an antioxidant N-acetyl cysteine snail induction and EMT get prevented (Radisky et al. 2005). These findings connect between oxidative damage, extracellular matrix and EMT (Thiery 2002). Figure 5 shows the progression of invasion via the Notch pathway.

Figure 5. Notch pathway leading to EMT.

TGF-b pathway

TGF-b transforming growth factor b imparts its effect on EMT by getting regulated via different factors such as SIP1, Snail and Slug. These factors work in a manner which leads to the suppression of the E-cadherin which is a primary adhesion molecule between two cells.

Its activity is regulated by BMP7 and upregulation of the same leads to production of altered physiological EMT. It does this by inhibiting the Smad-mediated TGF-b signalling and also by reducing the expression of vimentin. It has been shown that the expression of BMP7 is inversely proportional to the tumourigenicity (Buijs et al. 2007; Yang et al. 2011; Buijs et al. 2012; Liu et al. 2012).

TWIST and HIF pathway

TWIST is considered to be the master regulator of the EMT (Van Dusen & Firulli 2012; Xue & Hemmings 2012; Saunders & McClay 2014). It has an evident role to play in type I and type III metastases (reviewed in Khan et al. 2003; Mallini et al. 2004). When the expression of this protein exceeds the normal level, it leads to the metastasis (Croset et al. 2014). It is widely expressed in a variety of metastatic breast cancer cells. It has been found in an experiment that knocking down the TWIST with its siRNA drastically lowers the metastatic movement. Further experiment was carried out on human mammary epithelial cells. Further clinical samples have provided evidences of induction of metastasis by TWIST activity. It has been evident from the research that the epigenetic pathway regulates the mechanism which stimulates the function of migration and invasion by TWIST. It has been evident from the various cell lines that TWIST leads to the expression of the miRNA, miR-10b (Ma et al. 2007). Expression of the same in primary carcinomas is related to the clinical progression (Ma et al. 2007). These findings indicate that both the genetic alteration and epigenetic factors are responsible for the metastatic movement of the cells. Many studies have pointed out that stress conditions like the hypoxic one create an environment that favours metastatic growth and movement (Zhang et al. 2013). Evident proof has been retrieved from the in vitro experiments conducted on MCF7 cells which show that HIF-1A is a driving factor for EMT and metastasis. It has been found to directly regulate the expression of TWIST (Yang et al. 2008). HIF-1a works under a hypoxic condition and it directly interacts with the TWIST, thus activating it efficiently. It consecutively upregulates the endothelial growth factor and also increases the formation of new blood vessels which is followed by an enormous increase of E-cadherin and by loss of vimentin and N-cadherin. The process can totally be reversed via inhibiting HIF-1a with that of siRNA qualifies HIF-1a as a key regulator of EMT (Yang et al. 2008). It can therefore be concluded that the hypoxic condition can lead to induction of HIF-1a which subsequently leads to the activation of TWIST signalling, enabling the molecule to escape into circulation.

E-cadherin pathway

E-cadherin is considered to be one of the most prominent hallmarks of EMT assays and it has been found through various sources that loss of the molecule is very high for initiating the first step of metastasis. Loss of the protein leads to the disruption of the cell–cell adhesion which leads to the cellular polarity, allowing various growth factors which normally would have been removed from the system (Wells et al. 2008). b-Catenin along with cadherin alters many pathways, thus leading to playing a vital role in metastasis. Figure 6 shows the interaction of various pathways, thus leading to the up- or downregulation of various proteins involved in the invasion phenomenon.

Figure 6. Up- and downregulation of different metastatic factors influencing each other.

PI3K AKT fibronectin pathway

Fibronectin is one more ECM protein which plays a pivotal role in migration, adhesion and transformation by instigating the PI3/Akt pathway. It completes the pathway after getting bound to the avb1 integrin molecule (Bae et al. 2013). In vitro experiments on human mammary breast cancer cells have shown that fibronectin plays a vital role in initiating EMT. When in an experiment MCF-10A cells were exposed to fibronectin it has led to the stimulation of the migration and induction of an EMT event which includes the upregulation of the markers of EMT, for example MMP2, Vimentin, Snail, Smad2, N-cadherin, etc. (Park et al. 2011) These studies are a clear indication of the major role of fibronectin in EMT through stimulating the activity of TGF-b. In addition, exogenous fibronectin is able to induce EMT under serum-free conditions; this process could be reversed following addition of a TGF-b-neutralizing antibody (Park et al. 2011). These data suggest that fibronectin can induce EMT in breast cancers by enhancing the activity of endogenous TGF-b.

Conclusion

Tumour metastasis could be explained as a multistep procedure that leads to dissemination from the primary site of infection and forming secondary tumours at a distant site. There is a complete series of events involved in transforming a solid tumour to the metastatic one: invasion, intravasion, transport, extravasations and formation of colony at some distant site. EMT has shown to play an indispensable part in promoting and spreading metastasis. Proteins E-cadherin, b-catenin, Integrin, Syntenin, Survivin, Periostin are some that have shown to be involved in the interplay of metastasis and EMT. Apart from EMT, various studies have shown the role of MET as one of the important factors in re-differentiation of the tumorous cells at distant places. Thus, it is evident from already established data and experiments that all the events are interlinked and inter-conversion of EMT and MET is required for secondary explosion of tumours at a distant place. miRNA 200 and miRNA 34 have also been found to manifest their impact on the process of metastasis. Thus it is evident from the literature and experimental proofs that metastasis is not controlled by a singular factor but by an amalgam of a complex network involving the entire cellular component starting from RNAs, protein and the webbed network of pathways. Thus the process can be explored further as a target against metastatic movement of cancerous tissues. And also therapeutics could be developed targeting the mechanism via utilizing its various components.

Disclosure statement

No potential conflict of interest was reported by the authors.