Metabolic Disorders and Cancer: Store-Operated Ca²⁺ Entry in Cancer: Focus on IP₃R-Mediated Ca²⁺ Release from Intracellular Stores and Its Role in Migration and Invasion

Abstract

Store-operated calcium entry (SOCE) plays important roles in a multitude of cellular processes, from muscle contraction to cellular proliferation and migration. Dysregulation of SOCE is responsible for the advancement of multiple diseases, ranging from immune diseases, myopathies, to terminal ones like cancer. Naturally, SOCE has been a focus of many studies and review papers which, however, primarily concentrated on the principal players localized to the plasma membrane and responsible for Ca²⁺ entry into the cell. Much less has been said about other players participating in the entire SOCE event. This review aims to address this shortcoming by discussing the accumulated scientific knowledge focused on the inositol trisphosphate receptors (IP₃Rs), principal player responsible for emptying intracellular Ca²⁺ stores in a majority of cells, and their involvement in regulation of cell migration and invasion in cancer.

George Shapovalov and Natalia Prevarskaya shared senior co-authorship.

1 Introduction

Cancer is one of the most common terminal diseases in developed countries. It is among the main causes of death in the world with more than 100 different types of cancer identified at present, each represented by a specific genotype. Cancer progression development follows three typical steps: initiation, promotion, and progression. The initiation is the step when one or more errors appear in the original DNA sequence, so-called driver mutations, by lesions due to endogenous or exogenous factors. These driver mutations lead to the transformation of the cells which thus become abnormal. The promotion is the identical multiplication of transformed cells. These cells form an abnormal mass called neoplasm or tumor. We talk about cancer once transformed cells have acquired the capacity to trigger the formation of blood vessels (neo-angiogenesis) allowing, in particular, their feeding in nutrients and oxygen ensuring the tumor growth. The last stage is the progression during which cancer cells cross the blood vessel and lymphatic basal lamina in order to migrate to other tissues. Once the cells have arrived at the new tissue, they cross the vessels’ basal lamina for the second time to insert themselves into the tissues. This process is called metastasis, and it is the main cause of death by cancer (Hanahan and Weinberg 2011; Jin et al. 2017).

Among the proteins of particular importance that can be targeted by genetic alterations, we find ion channels which are implicated in various carcinogenesis processes such as proliferation, migration, or cancer cell survival. This importance of ion channels stems from their role in maintaining the intracellular ion equilibrium which is crucial for most physiological functions. An imbalance in any of these processes, caused by modification of expression and/or functionality of an ion channel, is called a channelopathy and can be a root cause of a severe disease, such as cancer (Prevarskaya et al. 2010).

One of the more important ions in cellular homeostasis is Ca²⁺ which is a key second messenger, regulating numerous physiological and pathological processes ranging from muscle contraction to much more fatally important ones such as cell motility and death. While, classically, ion channels have been considered to be localized to the plasma membrane (PM), many ion channels have recently been found in the membranes of various intracellular organelles, such as the endoplasmic reticulum (ER), mitochondria, or the nuclear membrane (Leanza et al. 2013). Interestingly, while plasma membrane-localized ion channels are best known to participate in fast-paced cellular processes like muscle contraction or action potential propagation, the majority of intracellular ion channels are known to play important roles in regulating cellular function and fate. For instance, the IP₃Rs and the ryanodine receptors (RyRs), acting as Ca²⁺ release channels in the ER, were shown to regulate various important cellular functions, such as apoptosis, contraction, cell motility, proliferation and migration (Prevarskaya et al. 2011; Vervloessem et al. 2014).

In general, increase of intracellular Ca²⁺ concentration happens by Ca²⁺ entering the cell via the Ca²⁺-permeable ion channels of the plasma membrane or by the release of Ca²⁺ from internal stores, typically the endoplasmic or sarcoplasmic reticulum. The release of Ca²⁺ from the ER correspondingly requires the presence of Ca²⁺-permeable ion channels with the two principal and best studied such protein classes being IP₃Rs and RyRs. The basal cytoplasmic Ca²⁺ concentration is regulated also by specialized transport proteins, such as PMCA (plasma membrane Ca²⁺ ATPase), responsible for the transport of excessive Ca²⁺ out of the cell through the plasma membrane, and SERCA, present in the ER membrane and responsible for Ca²⁺ reuptake by the internal stores. Additionally, store-operated channels (SOC) are known to be present in the plasma membrane, where they are responsible for Ca²⁺ entry into the cells when internal stores are exhausted, and implicate Orai and STIM (stromal interaction molecule) proteins (Yang et al. 2012). The mechanism of such replenishment of Ca²⁺ upon store depletion is called store-operated Ca²⁺ entry (SOCE). In the majority of studies, SOC response is stimulated by blocking SERCA chemically (Michelangeli and East 2011). A classical irreversible SERCA inhibitor is thapsigargin, which blocks Ca²⁺ reuptake into the ER and leads to gradual decrease of its Ca²⁺ content and eventual opening of SOCs.

Since their discovery at the beginning of the twenty-first century, a lot of studies have involved STIM and Orai in various cellular processes from immune cell activation, muscle contraction, and sperm chemotaxis to regulation of gene transcription and cell fate (Kahl and Means 2003; Yoshida et al. 2003). In these studies, Orai1 and STIM1 have been found to be important for cellular migration. For example, suppression of these proteins by siRNA or inhibition of SOC currents by SKF96365 inhibited the migration of the human breast adenocarcinoma cell line MDA-MB-231 and decreased the formation of metastases in mice (Yang et al. 2009). Thus, the importance of SOCE in various cellular processes, including such critical ones as migration and invasion, is well known. The role of STIM and ORAI proteins in hallmarks of cancer and in particular in tissue invasion or metastasis processes has also been extensively studied (Fiorio Pla et al. 2016). However, the majority of associated research focused on immediate players responsible for Ca²⁺ entry into the cells via the PM, with less evidence put forward on how other players, such as IP₃Rs, participate in this big picture. This review aims to close this gap by focusing on the recent findings of how IP₃R activity is regulated by different players and how changes of this activity affect the complex processes of migration and invasion of cancer cells.

2 Cellular Migration and Invasion

During metastasis formation, one of the six initial hallmarks of cancer, the migration of epithelial and endothelial cells is a key step leading to the propagation of the primary tumor and to the invasion of neighboring tissues (Hanahan and Weinberg 2000, 2011). However, cell migration is also a central mechanism in homeostasis of healthy tissues. Important physiological processes such as embryogenesis, immune defense, angiogenesis, and wound healing are closely linked to the capability of cells to migrate.

In general, cell migration is conceptualized as a cyclic process (Lauffenburger and Horwitz 1996). It consists of multiple steps, such as the formation of organized adhesive structures, or focal adhesion sites, containing receptors and integrins linked to the extremities of actin fibers (Westhoff et al. 2004) and membrane protrusions, such as lamellipodes or invadopodes, controlled by small GTPases, Rac and Cdc42, with a finely controlled balance between detachment and adhesion at the focal adhesion sites (Ridley et al. 2003). The lamellipodes are surface-attached structures that are found at the leading edge of migrating cells. In order to become invasive, tumor cells need to acquire abilities that allow increased migration through the extracellular matrix (ECM). In order to do this, tumor cells have specialized membrane protrusions, called invadopodia, that provide important functions for invasion. It should be noted that the term invadopodia is primarily used in conjunction with cancer cells, while the term podosome can often be found in the literature when talking about normal cells. Invadopodia are actin-rich plasma membrane protrusions at the ventral surface of the cell, often situated under the nucleus, that are proteolytically active and responsible for the focal degradation of ECM components through matrix metalloproteinases (MMP) (Nabeshima et al. 2002; Yoon et al. 2003).

Both, Ca²⁺ release and Ca²⁺ influx, have been linked to cell migration depending on cell types and stimuli (Agle et al. 2010; Tsai et al. 2014; Yang and Huang 2005). Thus, migrating cells are polarized and exhibit a transient and steady gradient of intracellular Ca²⁺. That gradient increases from the front of the cell to the rear and is thought to be responsible for rear-end retraction (Brundage et al. 1991; Hahn et al. 1992). The increased cytosolic Ca²⁺ concentration ([Ca²⁺]_i) at the rear end is related among others to myosin II contraction and calpain-mediated release of focal adhesions leading to the retraction of the uropod (Eddy et al. 2000; Palecek et al. 1998). The retraction is accomplished via myosin II contraction, which is regulated by myosin light chain (MLC) phosphorylation through Ca²⁺-dependent MLC kinase (MLCK) and by disassembly of adhesions at the rear of the cell owing to calpain-mediated cleavage of focal adhesion proteins such as integrins, talin, vinculin, and focal adhesion kinase (FAK) (Ridley et al. 2003). However, the cyclic morphological and adherence changes that are observed during cell migration are also supported by repetitive Ca²⁺ signals, which take the form of Ca²⁺ spikes or oscillations (Pettit and Fay 1998).

It is therefore natural that migration of the tumor, being strongly dependent on these Ca²⁺ signals, is accompanied by changes in expression and activity of ion channels. Below we discuss how IP₃Rs participate in these processes, regulate intracellular Ca²⁺, and work in ensemble with SOCs.

3 Intracellular Ca²⁺ Release Channels: The IP₃Rs

3.1 The Discovery of the IP₃Rs

Involvement of IP₃R in intracellular Ca²⁺ regulation was observed for the first time over 30 years ago, when Streb et al. (1983) found a second messenger that was able to increase [Ca²⁺]_i from a non-mitochondrial store: inositol 1,4,5-trisphosphate (IP₃). This discovery was followed by concentrated efforts of many researchers to discover the target of this second messenger. This goal was achieved first by K. Mikoshiba’s team, which was working on a P400 protein which was absent in Purkinje-neuron-degenerating mutant mice and found that this protein binds IP₃ (Furuichi et al. 1989a; Maeda et al. 1988), and established its complete primary sequence (Furuichi et al. 1989b; Maeda et al. 1990).

One year later the P400 protein was found to form a channel by incorporating it into a planar lipid bilayer and recording its activity and was renamed IP3R. Moreover, in the same paper, it was shown that this channel exhibited a Ca²⁺ conductance (Maeda et al. 1991). These findings were confirmed by the work of other teams that achieved similar results, establishing the identity of the IP₃R at the same time (Sudhof et al. 1991; Supattapone et al. 1988).

The IP₃R is one of the first ion channels that were discovered to be localized in the ER (Ross et al. 1989). The channel is a tetramer, formed from monomers of 260 kDa in size. Each monomer contains binding sites for IP₃ and Ca²⁺ in the N-terminal region, six transmembrane domains forming a pore region and the determinants for tetramer formation in the C-terminus (Fan et al. 2015; Taylor and Konieczny 2016).

The IP₃R is a principal Ca²⁺ release channel from the internal stores in the majority of cells, participating in the regulation of intracellular Ca²⁺ in the cytosol and in Ca²⁺ transfer between ER and mitochondria (Rizzuto et al. 2009). Over the years, a lot of different studies have implicated the IP₃R in plenty of physiological and pathological mechanisms. Indeed, an overexpression of the IP₃R is observed in numerous forms of cancer, typically in correlation with an increase in proliferation or migration (Ivanova et al. 2014).

As mentioned earlier, the first identified physiological trigger for the activation of these channels is known to be IP₃. However, further studies elucidated the specific mechanism of IP₃R activation by IP₃ in a Ca²⁺-sensitive manner. The IP₃R is regulated in a bimodal manner by Ca²⁺ itself—its activity increases with the increase of [Ca²⁺]_i to a certain level, a phenomenon that is known as Ca²⁺-induced Ca²⁺ release (CICR). However, IP₃R activity is inhibited at higher [Ca²⁺]_i, with IP₃ removing this inhibition of IP₃R activity at high [Ca²⁺]_i (Foskett et al. 2007). IP₃ itself is produced in the cells in response to stimulation of surface receptors such as receptor tyrosine kinases (RTK) and G-protein-coupled receptors (GPCR), leading to the activation of phospholipase C (PLC). The PLC is an enzyme that metabolizes phosphatidylinositol 4,5-biphosphate (PiP2) into IP₃ and diacylglycerol (DAG), both of which are second messengers, thus triggering various signaling cascades that can involve IP₃Rs and lead, for example, to cellular growth.

3.2 Role of the IP₃Rs in Regulating Migration and Invasion

While the identity of IP₃R channels and their role in Ca²⁺ release from internal stores have been known for a long time, the question of their involvement in regulating cellular migration and invasion has only recently became a topic of active studies. Publications covering this subject are few, but their number is steadily increasing in the last few years (see Table 31.1).

Table 31.1 Implication of IP₃R in migration and invasion

Treatment with caffeine was found to inhibit the IP₃R-mediated Ca²⁺ release by selectively targeting IP₃R3 in glioblastoma cell lines. Interestingly, the mRNA level of IP₃R3 was significantly increased in glioblastoma cells compared to normal cells (Kang et al. 2010). The inhibition of IP₃R3 by caffeine suppresses the migration and invasion of glioblastoma cell lines when assessed by scrape motility, matrigel invasion, soft agar, and mouse brain slice implantation. To show this, U87MG cells, a glioblastoma cell line known for its high tumorigenicity, were injected into the skin of nude mice, some of which were supplied with caffeine in drinking water. The supplementation with caffeine considerably increased the mean survival rate of mice, proposing IP₃R3 as a promising novel therapeutic target for glioblastoma treatment and showing that caffeine could be a useful adjunct therapy (Kang et al. 2010). Noteworthy, the final concentration of caffeine measured in the mice brain corresponded to levels of caffeine produced by drinking two to five cups of coffee per day by humans. Caffeine is a well-known activator of RyRs but has also been shown to inhibit IP₃R activity (Brown et al. 1992), although ryanodine, a known inhibitor of RyR activity, did not show any effect on migration and invasion of the transformed cells. The type 3 IP₃R was also implicated in peritoneal dissemination of gastric cancer (Sakakura et al. 2003).

IP₃R-mediated Ca²⁺ release has also been linked to metastatic melanoma. Interestingly, Ca²⁺ release from the stores promoted the migration of metastatic epithelial breast adenocarcinoma (MDA-MB-231) and cervical carcinoma (HeLa) cell lines via the activation of S100A4 protein by Ca²⁺ (Mueller et al. 1999). Indeed, the PLC/IP₃/IP₃R1 pathway has been shown to mediate EPAC (exchange protein activated by cyclic AMP)-induced Ca²⁺ elevation that in turn increases melanoma cell migration through the interaction between S100A4 and MHCIIA and the resultant actin assembly (Baljinnyam et al. 2010). This suggests that IP₃R can also be a potential target for the suppression of melanoma cell migration opening a perspective of treatments against the metastasis development (see Fig. 31.1).

Fig. 31.1

This figure represents the cross section of a migrating cell at the leading edge. It demonstrates the known pathways by which the IP₃Rs participate in cell migration/invasion through controlling the release of Ca²⁺ from the ER. Activation of GPCRs by external stimuli is represented via lightning bolts and the activation of further pathways, as well as Ca²⁺ release and production of secondary messengers are indicated by the arrows. The membrane stretch-activated, transient receptor potential cation channel subfamily M member 7 (TRPM7)-mediated Ca²⁺ influx coupled to IP₃R-mediated Ca²⁺ release participates in the guidance of the leading edge toward a chemoattractant (Wei et al. 2010). Formation of focal adhesions is facilitated by IP₃R-mediated Ca²⁺ release that is stimulated through surface GPCRs or EPAC leading to CaMKII-dependent FAK phosphorylation (Fan et al. 2005). IP₃R-mediated Ca²⁺mobilization from the endoplasmic reticulum (ER) stores also promotes cell migration in an S100A4-dependent manner (Baljinnyam et al. 2010)

In addition, the use of the PLC inhibitor U73122 significantly decreased the Ca²⁺ release from the ER and decreased the migration of melanoma cell through monolayers of human umbilical vein endothelial cells (HUVECs) (Peng et al. 2009).

A key constituent involved in the regulation of focal adhesion turnover is FAK. It is a non-receptor kinase that promotes cell migration by transferring signals between growth factor receptors and integrins. FAK is regulated by phosphorylation at tyrosine and serine residues. By using GPCR agonists such as bombesin, bradykinin, and vasopressin stimulates the PLC/IP₃/IP₃R pathway leading to Ca²⁺ mobilization from ER stores in Swiss 3T3 fibroblasts. This released Ca²⁺ associates with CaM, and, subsequentially, the Ca²⁺/CaM complex regulates CaMKII (calmodulin-dependent protein kinase II) which then rapidly phosphorylates FAK at Ser-843. Once FAK is phosphorylated at this and three other locations (Ser-722, Ser-732 and Ser-910), it facilitates the formation of focal adhesion points (Fan et al. 2005) (see Fig. 31.1).

Localized Ca²⁺ flickers at the leading lamella permit the decision-making for turning direction of the migrating cells: the cell turns in the direction of higher Ca²⁺ flicker activity. These flickering Ca²⁺ microdomains result in membrane tension via TRPM7 and chemoattractant signal transduction via IP₃R2. However, by themselves, these flickers only determine the turning direction but not the migration speed. Indeed, the use of EGTA to chelate intracellular Ca²⁺ slows the turning but not the migration speed in a dose-dependent manner, showing that spatiotemporal organization of Ca²⁺ microdomains is important in such convoluted cellular processes as cell migration (Wei et al. 2009, 2010) (see Fig. 31.1).

Recently, IP₃Rs have been shown to translocate from cell-cell contacts to the leading edge of migrating pancreatic ductal adenocarcinoma cells (PDAC) which was accompanied by the formation of ER-PM junctions and STIM1 puncta. Xestospongin B, a selective IP₃R inhibitor, and the STIM-Orai inhibitor GSK-79751 suppressed the migration of the cell line PANC-1. Taken together, these data emphasize that both, Ca²⁺ release and Ca²⁺ influx, are important for cell migration (Okeke et al. 2016).

3.3 Role of IP₃Rs in SOCE

The principal pathways by which ion channels regulate cellular migration are associated with their primary functions, such as regulation of cellular adhesion, volume or migration speed, maintenance of cellular potential, and regulation of intra- and extracellular pH. Unfortunately, the link between the expression and function of IP₃Rs and the mechanisms of their involvement in cancer cell migration, invasion, and metastasis is not yet well understood. Nonetheless, there is general consensus that Ca²⁺ signaling alterations play a major role in carcinogenesis (Parkash and Asotra 2011; Wei et al. 2012).

Ion channel activity can essentially be described as a series of transitions between open and closed states, with a particular probability of being open (P _open). The opening/closing of the IP₃Rs can be modulated by different factors such as Ca²⁺, IP₃, and Mg²⁺. For example, the IP₃R activity is regulated by Ca²⁺ in a bimodal manner. P _open of IP₃R, and thus Ca²⁺ release from ER, is very low at low Ca²⁺ levels, gradually increasing with the rise of [Ca²⁺]_i, reaching a plateau at μM concentrations, and finally being again inhibited at higher [Ca²⁺]_i, thus also leading to a decrease in Ca²⁺ release. In the majority of publications, involvement of the IP₃Rs in migration and invasion was found through the use of its major agonist, IP₃. Once the IP₃R is activated, Ca²⁺ is released from the ER and can either stimulate Ca²⁺-dependent proteins such as calpain or Ca²⁺-activated ion channels (Fig. 31.2). In that way, the mobilization of Ca²⁺ from the intracellular stores via IP₃Rs can magnify the Ca²⁺ entry signal via SOCs and result in a strong response capable of inducing cell migration and invasion or even stimulate metastasis formation. The release of Ca²⁺ from the ER via IP₃Rs has also been shown to induce the SOC opening. The Ca²⁺ release induces Ca²⁺ entry via STIM1/ORAI1 resulting in Ras and Rac protein (small GTPases) activation (Yang et al. 2009) which in turn accelerates the turnover of focal adhesions and further increases migration. In support of these observations, there is now considerable evidence that STIM1 activates both, Orai1 and TRPC1, via distinct domains at its C-terminus (Ong et al. 2016).

Fig. 31.2

This scheme illustrates the roles IP3Rs play in SOCE regulation for the promotion of cancer cell metastasis. The black arrows show the sequence of events; the green upward arrows indicate an increase in the expression and/or function of the corresponding protein(s) or augmentation of the corresponding process. The “?” indicates that the associated step has been recently hypothesized in literature (Baljinnyam et al. 2010) but requires further evidence

Concerning the invasion mechanism, there is no evidence that the IP₃Rs can directly affect the activation of metalloproteinases and, thus, the digestion of the extracellular matrix necessary for cell invasion. Otherwise, it has been shown that the induction of MMP2 and MMP9 can happen via Ca²⁺ flux through TRPV2 (Monet et al. 2010). Moreover, S100A4 has been found to control the invasive potential of human prostate cancer cells through the regulation of MMP9 expression, contributing to the proteolysis of ECM by invadopodia (Saleem et al. 2006). It has been suggested that the link between IP₃Rs and ECM degradation can be S100A4 (also known as mts1) which is a Ca²⁺-binding protein associated with invasion and metastasis of cancer cells and can, therefore, be activated by Ca²⁺ release via the IP₃Rs (Baljinnyam et al. 2010). Further studies on this interesting topic are needed to confirm these hypotheses.

3.4 Role of IP₃Rs Protein Partners in SOCE

It is well known that IP₃R activity is heavily modulated by various partner proteins. Among them are in particular the proteins of the Bcl-2 family that modulate the activity of IP₃Rs directly or indirectly. Multiple studies, such as Rong et al. (2008), have shown that Bcl-2 is present in the ER where it can interact directly with all three IP₃R isoforms, inhibiting their activity via multiple binding sites proposed to explain this interaction (Fouque et al. 2016; Ivanova et al. 2016). Other proteins of the Bcl-2 family such as Bcl-xL or Mcl-1 are also known to interact with the IP₃Rs and modulate its activity (Eckenrode et al. 2010; Monaco et al. 2012).

Interestingly, Bcl-2 and Bcl-xL are found to be overexpressed in many cancers. Overexpression of Bcl-2 is observed in 30–60% of androgen-dependent and nearly 100% of androgen-independent prostate cancers, with similar results observed for Bcl-xL. Unfortunately, the studies carried out so far reported primarily on the changes in the expression levels, with little to no data on the possible impact of the modulation of IP₃R activity by Bcl-2/Bcl-xL on cancer cell migration/invasion. To find this likely link, it could be very interesting to test the effects of different drugs already known to specifically disrupt the interaction between Bcl-2 protein family members and the IP₃Rs, such as ABT-199, a BH3 mimetic that specifically binds Bcl-2’s hydrophobic cleft (Vervloessem et al. 2016), on cancer cell migration and invasion properties.

While the interaction between Bcl-2 family members and IP₃Rs has been known for a while, in the past few years scientists have shown that the IP₃Rs also interact with many other proteins, including the Sigma1 receptor which has also been found to modulate SOCE (Rosado 2016). Additionally, IP₃Rs have been suggested to interact with some of the channels belonging to the family of the TRP channels (Fiorio Pla and Gkika 2013). Thus, for example, TRPC3 was successfully co-immunoprecipitated with the IP₃Rs by glutathione S-transferase (GST) pulldown experiments (Boulay et al. 1999). Moreover, TRPC3 has been shown to be activated by IP₃, whereby the N-terminal IP₃-binding domain of IP₃R was essential (Kiselyov et al. 1999). Similar experiment permits allowed the identification of biochemical interactions between IP₃Rs and TRPC1 (Mehta et al. 2003). It is also interesting to note that there is now considerable evidence that STIM1 activates both, Orai1 and TRPC1, via distinct domains in its C-terminus (Ong et al. 2016).

4 Conclusion and Perspectives

Ca²⁺ plays many important roles in regulating cellular health and migration properties with SOCE being one of the primary mechanisms regulating intracellular Ca²⁺ homeostasis. It is therefore natural that SOCE is at the core of many diseases, including such important ones as cancer, with many publications focusing on the mechanisms and dysregulations of SOCE. This review has focused in particular on a less well-covered area of store-operated Ca²⁺—the IP₃R channels responsible for the depletion of internal stores in a majority of cells and their various roles in modifying SOCE and affecting cellular motility and invasion.

The role of IP₃Rs in SOCE and associated diseases is a relatively novel topic. Recent publications only start to address the question of how IP3Rs modulate cancer progression in particular concerning the questions of migration and invasion. However, IP3R activity is known to be modulated by numerous protein partners. While multitude of studies have been devoted of the topics of this regulation itself, its involvement in SOCE regulation where cancer progression is concerned has not yet been properly addressed in scientific literature. Considering the increasing number of IP₃R protein partners discovered regularly in recent years, the importance of this angle should not be underestimated. However, as one can easily see, there is still a lot of research to be carried out in order to comprehensively understand the mechanisms by which these channels and SOCE regulate migration and invasion of cancer cells. Further research on any of these topics provides a great promise to develop treatments that could counteract or at least arrest progression of various forms of cancer toward metastatic stages.