Эпителиально-мезенхимальный транзит в гепатоцеллюлярной карциноме

Введение

Гепатоцеллюлярная карцинома и насущная необходимость в альтернативных вариантах лечения

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and one of the leading causes of cancer-related mortality worldwide [1]. It is commonly referred to as hepatoma and it is an important public health problem. It occurs more frequently in men than in women and much more frequently in people with a history of liver problems such as hepatitis B and C viral infections [2]. Symptoms are more pronounced and specific during advanced stages of the disease, at which time treatment options and the extent to which these treatment options will be effective are limited. In focal localized hepatocellular carcinoma, partial hepatectomy is possible. In patients with intrahepatic metastasis, complete hepatectomy and liver transplantation (which involves a long wait-list, is surgically invasive, and is not optimal for certain people) are options available [3, 4]. These patients with intrahepatic metastasis as well as those with extrahepatic metastasis may also need systemic therapy with agents such as sorafenib. Unfortunately, targeted therapy with sorafenib only extends life by about 6 months [5]. Currently, the 5-year relative survival rate for patients with liver cancer is only 17%, mainly because of its late diagnosis [6]. As such, better tools are needed for earlier diagnosis and for treatment of advanced HCC.

Эпителиально-мезенхимальный транзит

Diagnostic and therapeutic approaches can exploit biological mechanisms or processes that may be important in normal healthy cells and tissues but may be aberrant in cancer cells and tissues. A biological process that is normal and physiological in certain cells and tissues but that is hijacked by cancer cells is epithelial-tomesenchymal transition (EMT) [7]. EMT is a process during which epithelial cells gradually change to become “mesenchymal-like” in nature. During this process, they also become more motile and invasive [8]. To understand this, it is helpful to distinguish between two distinct types of cells: epithelial and mesenchymal. As shown in Fig. 8.1, epithelial cells are cells that typically line the walls of blood vessels, body cavities, and organs (such as the lungs, stomach, small intestine, pancreas, and kidneys). They are normally polarized, attached to a basement membrane, and closely interconnected with one another. Epithelial cells are held together tightly at junctions via cadherin, catenin, and integrin molecules [9]. In contrast, mesenchymal cells are loosely associated cells that lack polarity and are characterized by greater migratory properties [10]. The transition of cells from epithelial to mesenchymal phenotype requires several biochemical changes and takes place normally during the development of the embryo. It is required for the formation of the mesoderm which occurs during the third week of the developing embryo [11] and neural crest formation which occurs after the formation of the three germ layers, a process otherwise known as gastrulation [12]. There are several things that must happen—specification of the area where EMT will occur, detachment of the epithelial cells from the basement membrane, and conversion to the mesenchymal cell structure/phenotype [13]. In adults, EMT also normally occurs during wound healing [14] and, in women, the formation of the mammary gland [15]. Although EMT is important in embryogenesis and some normal physiological processes in adult humans, EMT has been implicated and demonstrated to be involved in pathological conditions like fibrosis, inflammation, and cancer [16], raising the possibility of exploiting aspects of EMT for diagnostic or therapeutic applications, in HCC.

 

Фиг. 8.1. Эпителиальные против мезенхимальных характеристик

Классификация EMT

Depending on the settings in which EMT occurs and its functional consequences, EMT could be classified into three categories: type I, type II, and type III [8]. Type I EMT occurs in the context of implantation of the embryo to the uterine wall; formation of the placenta, in several instances during embryonic development (including mesoderm and neural crest formation); and organ development. EMT in these contexts results in a diverse population of cells sharing a common mesenchymal phenotype, which is neither induced by inflammation nor results in fibrosis or metastasis [17, 18]. It is important to note that the mesenchymal cells formed retain the ability to revert back to an epithelial state through the process of mesenchymalto-epithelial transition (MET) [19]. Type II EMT occurs in the context of reparation activities such as wound healing, the regeneration of tissue, or organ fibrosis. This type of EMT occurs in response to inflammation and stops when inflammation has subsided [17, 18]. Type III EMT, the type of EMT most pertinent to hepatocellular carcinoma, occurs in the context of carcinoma cells that undergo multiple biochemical and morphological changes, which favor cancer cell invasion, migration, and metastasis, and consequently cancer progression [17, 18].

Понимание сложной оркестровки EMT

To be able to exploit any aspects of EMT for cancer diagnostic or therapeutic purposes, it is essential to fully understand the process, what regulates it, and how EMT—a normal biological process required under certain physiological circumstances—is manipulated by tumorigenic cells. It is important to note that the signals that induce EMT are celland tissue-specific and require the orchestration of multiple regulators and signaling pathways [20]. We will discuss some of these molecular regulators and molecular signaling effects of EMT here, with some focus on HCC.

Молекулы, ответственные за выполнение EMT: эффекторы

The expression profile of a number of specific proteins that are characteristics of either the epithelial or mesenchymal cell phenotype has been established. Furthermore, the types of changes in the expression of these specific proteins during the course of EMT have been established [21]. Proteins associated with an epithelial cell identity include the following: E-cadherin, α-catenin, and γ-catenin [22–24]. These are three of the established molecules associated with and important in cell-cell junctions and adhesion. Among these, loss of E-cadherin is the most widely used molecular characteristic to indicate that EMT has occurred. Whether through promoter methylation, transcriptional repression, or protein phosphorylation followed by protein degradation, E-cadherin expression has been shown to be decreased in multiple human and mouse studies of HCC [25–30]. Loss of E-cadherin expression has also been linked to a more metastatic phenotype in a variety of other cancers [31–34].

In addition to the downregulation of epithelial markers, EMT is also characterized by simultaneous upregulation of mesenchymal markers. These mesenchymal markers include N-cadherin, vimentin, fi  CD44, and integrin B6 [21]. During EMT, N-cadherin expression typically increases as E-cadherin expression decreases, thereby leading to a change that both disturb cell adhesion and promote cell migration and invasion [35–37]. Another signifi molecular change that typically occurs as epithelial cell transition to a mesenchymal phenotype involves the transition from cytokeratin to more vimentin intermediate fi  proteins. Increased vimentin expression results in greater contractibility and stability of cells in response to mechanical activity, thereby supporting greater migration [38–40]. Similarly, fi (an extracellular matrix glycoprotein) [41–43], CD44 [44, 45] (a transmembrane glycoprotein), and integrins (transmembrane receptors controlling cell-cell and cell-matrix adhesion) [46] are all upregulated in cells that have undergone EMT, and they are associated with poor prognosis [7].

Транскрипционные факторы, оркестрирующие EMT: центральные регуляторы

Another set of molecules critically important for EMT are the key regulating transcription factors. These include molecules such as Snail zinc finger family, Zeb homeobox family, and the basic helix-loop-helix (bHLH) family of transcription factors. Each of these EMT-regulating transcription factors has well-established roles in migration, invasion, and proliferation [47, 48].

The Snail transcription factors, for example, include proteins such as Snail and Slug. Both Snail and Slug have been noted to be upregulated in a variety of metastatic cancers. Snail and Slug are also known to have increased expression in cells that have been treated with EMT-inducing agents [49, 50]. Snail and Slug are also associated with the disassembly of cell-cell and cell-extracellular matrix adhesions, such as desmosomes, tight junctions, and gap junctions. They are also negative regulators of epithelial molecular markers like E-cadherin and accomplish their repression of E-cadherin and other markers of the epithelial phenotype by binding to the enhancer box DNA sequences of these genes to inhibit their transcription [51–54]. In addition, Snail and Slug transcription factors promote the transcription of genes contributing to the mesenchymal phenotype [18, 55, 56].

Similarly, the ZEB transcription factors repress epithelial gene transcription such as transcription of the E-cadherin gene. The ZEB transcription factors also activate gene transcription of mesenchymal markers by binding to the regulatory enhancer box sequences of mesenchymal marker genes. They can be induced by tumor-promoting TGF-β signaling as well as growth factors activating the RAS-MAPK pathway. In addition, they have been shown to be activated by the EMT-regulating Snail 1 transcription factor, and they also inhibit the expression of claudins and ZO-1 which are both important for cell junction adhesion [20, 55, 56].

The basic helix-loop-helix (bHLH) transcription factor family, which includes Twist 1, Twist 2, and E12/47, can induce EMT by acting alone or cooperatively with one another or other molecules. Their activities include inhibition of E-cadherin, induction of N-cadherin, and activation of molecular signaling pathways promoting invasion, among many other tumor-promoting activities. In addition, these transcription factors function as either homodimers or heterodimers to regulate gene expression [20, 57–59].

Внеклеточные факторы, индуцирующие клетки подвергнуться EMT: индукторы/активаторы

In addition to the molecules that characterize mesenchymal and epithelial cellular identity, and the transcription factors that execute the EMT program, the agents that induce EMT to occur are also of critical importance. These are the molecules that serve as “on” and “off” switches and regulate EMT’s occurrence during both normal development and tumorigenesis. For example, different major signaling pathways, such as Notch, Wnt, and growth factor signaling cascades (transforming growth factor beta, TGFB; fibroblast growth factor, FGF; hepatocyte growth factor, HGF; epidermal growth factor, EGF; insulin-like growth factor 1, IGF1; and platelet-derived growth factor, PDGF), have all been implicated in EMT induction. These regulatory signals tend to be tissuespecific and involve multiple signaling pathways in order to promote a more mesenchymal cell phenotype, favoring invasion and migration [20]. Table 8.1 summarizes the different signaling pathways that will be discussed in Sects. 8.2.3.1–8.2.3.3, with particular interest in those demonstrated to have a role in HCC. Other EMT inducers will also be discussed, including hypoxia, inflammation, and microRNAs.

Таблица 8.1. Некоторые сигнальные пути, регулирующие EMT в HCC

TGF-β
Fransvea, E et al. (2008) Blocking transforming growth factor beta upregulates E-cadherin and reduces migration and invasion of hepatocellular carcinoma cells [74]
Fransvea, E. et al. (2009) Targeting transforming growth factor (TGF)-betaRI inhibits activation of beta 1 integrin and blocks vascular invasion in hepatocellular carcinoma [75]
Reichl, P. et al. (2012) TGFβ in epithelial-to-mesenchymal transition and metastasis of hepatocellular carcinoma [76]
Dituri, F et al. (2013) Differential inhibition of TGFβ signaling pathway in HCC cells using the small molecule inhibitor LY2157299 and the D10 monoclonal antibody against TGFβ receptor type II [77]
Steinway, SN et al. (2014) Network modeling of TGFβ signaling in hepatocellular carcinoma epithelial-to-mesenchymal transition reveals joint sonic hedgehog and Wnt pathway activation [78]
Qin, G. et al. (2016) Reciprocal activation between MMP-8 and TGFβ1 stimulates EMT and malignant progression of hepatocellular carcinoma [79]
HGF
Nagai, T. et al. (2011) Sorafenib inhibits the hepatocyte growth factor-mediated epithelial-to- mesenchymal transition in hepatocellular carcinoma [80]
Ogunwobi, O and Liu C (2011) Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-to-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways [81]
Ogunwobi, O et al. (2013) Epigenetic upregulation of HGF and c-Met drives metastasis in hepatocellular carcinoma [82]
PI3K
Wang, H et al. (2014) Activation of phosphatidylinositol 3-kinase/Akt signaling mediates sorafenib-induced invasion and metastasis in hepatocellular carcinoma [83]
Zhang, PF, et al. (2016) Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling [84]
WNT
Zhang, Q, et al. (2013) Wnt/B-catenin signaling enhances hypoxia-induced epithelial- mesenchymal transition in hepatocellular carcinoma via cross talk with HIF-1alpha signaling [85]
Yang, M. et al. (2013) A double-negative feedback loop between Wnt/β-catenin signaling and HNF4a regulates epithelial-mesenchymal transition in hepatocellular carcinoma [86]
Jiang, Lei et al. (2014) CLDN3 inhibits cancer aggressiveness via Wnt-EMT signaling and is a potential prognostic biomarker for hepatocellular carcinoma [87]
Notch
Wan, X et al. (2016) CD24 promotes HCC progression via triggering Notch-related EMT and modulation of tumor microenvironment [88]
Jia, Meng et al. (2016) LincRNA-p21 inhibits invasion and metastasis of hepatocellular carcinoma through Notch signaling-induced epithelial-to-mesenchymal transition [89]
Xiao, S. et al. (2016) Actin-like 6A predicts poor prognosis of hepatocellular carcinoma and promotes metastasis and epithelial-to-mesenchymal transition [90]

Трансформирующий фактор роста β (TGFβ)

Of the growth factor signaling cascades, TGF-β is considered to be one of the most potent inducers of EMT. It is a key signaling pathway very important in development that also presents serious consequences when dysregulated. It has been commonly implicated in a variety of cancers. It consists of several family members including the TGF-βs, BMPs, and activins. Ligand binding to TGF-β type II receptors will activate TGF-β type I receptors, which will in turn lead to the activation of a cascade of SMAD molecules depending on which particular TGF-β pathway is activated (SMAD2 and/or SMAD3, followed by SMAD4 if the TGF-β/activin mechanism has been activated, SMAD1/5/9 followed by SMAD4 if the BMP mechanism has been activated). These cytoplasmic SMAD complexes will then travel to the nucleus and combine with other transcription factors and co-activators/corepressors to regulate gene expression [60]. Some of the genes induced by the SMAD complexes are genes promoting different aspects of tumorigenesis including suppression of the immune response as well as promotion of cell proliferation, angiogenesis, cancer cell stemness, metastasis, and EMT—the focus of this chapter [60, 61]. Genes induced by TGF-β that contribute to EMT include mesenchymal markers (fi vimentin, and collagen I) as well as EMT transcriptional regulators (Snail1/2 and ZEB1). SMAD activation has also been shown to repress E-cadherin and occludin gene expression, ultimately reducing cell-cell adhesion [62, 63].

However, TGF-β also promotes EMT regulatory gene expression through other signaling pathways independent of the SMAD protein complexes. These alternative pathways include the phosphatidylinositol 3-kinase (PI3K) and MAPK pathways as well as signaling through the Rho, CDC42, and Rac GTPases [64, 65]. Induction of the PI3K pathway leads to the activation of AKT, followed by activation of the mTORC1 and mTORC2 complexes, leading to stabilization of the Snail 1 transcription factor, the inhibition of E-cadherin, and ultimately the acquisition of more invasive/motile characteristics, all characteristics of EMT [66]. The ERK, p38, and JNK MAPK pathways are distinct MAPK pathways, which have been shown to be necessary for TGF-β-induced transcription of genes promoting EMT [67]. Finally, TGF-β activation of the Rho, Cdc42, and Rac GTPases leads to the reorganization of actin and the formation of filopodia and lamellipodia, favoring mesenchymal and motile activity [64].

Другие факторы роста

In addition to TGF-β, a number of other growth factors serve as inducers of the EMT process: FGF, HGF, EGF, IGF1, and PDGF. Similar to TGF-β, they promote EMT via ligand binding of specific receptors and subsequent induction of signaling cascades [20]. Most of these growth factors, however, work by induction of transmembrane receptor tyrosine kinases (RTKs), which often activate the PI3K/AKT pathway or the ERK or p38 or JNK MAPK pathways for EMT induction [68]. Although most of these growth factors have roles in inducing EMT during normal development, these growth factors can also lead to the promotion of a more mesenchymal phenotype in the context of pathological processes, leading to greater invasiveness, migration, as well as tumorigenesis [69, 70]. This is most often achieved by the transcription and stabilization of mesenchymal markers (such as snail, twist, N-cadherin, and vimentin) as well as repression of markers important for cell adhesion, such as E-cadherin [20].

Дополнительные сигнальные пути

Other signaling pathways that regulate EMT besides the TGF-β/SMAD, PI3K/AKT, ERK, p38, and JNK MAPK pathways include the Wnt, Hedgehog, and Notch signaling pathways [20].

Usually, when the Wnt signaling pathway is off, GSK3β kinase is active, and together with axin and adenomatous polyposis coli (APC), it phosphorylates β-catenin to keep it in the cytoplasm where it will get tagged and eventually get degraded. However, when Wnt ligand binds to its Frizzled receptor, this receptor inhibits GSK3β kinase from phosphorylating β-catenin. Instead of being ubiquitinylated and labeled for degradation, β-catenin is now free to regulate gene expression to induce EMT by promoting the stability of snail, increasing fibronectin, and lowering levels of E-cadherin [71].

For the hedgehog signaling pathway, when the sonic hedgehog (SHH) ligand is not there, patched (PTC) receptor inhibits the cytoplasmic protein smoothened (SMO) from activating GLI transcription factors. However, the binding of SHH to PTC receptor relieves the inhibition of SMO, which then goes on to activate the GLI transcription factors which travel into the nucleus. Once in the nucleus, these GLI transcription factors increase SNAIL1 transcription, reduce E-cadherin expression, and result in increased cell motility [72].

Another signaling pathway involved in regulating EMT is the notch signaling pathway. The binding of the notch ligand (Delta-like or Jagged) to the notch receptor on another cell will lead to the proteolytic cleavage of the intracellular domain of the notch receptor, which will then enter the nucleus and begin regulating notch target gene expression to promote EMT, for example, SNAIL2 induction and E-cadherin repression [73].

Гипоксия

Besides growth factors and the signaling pathways we have discussed so far, there are additional factors in the tumor microenvironment that can induce EMT. One such factor is hypoxia, a condition of low oxygen tension at tissue level. The hypoxic state is often prevalent in growing tumors and serves to turn on HIF1a transcription factor expression, which promotes EMT by activating twist and SNAIL1 expression [91, 92]. In a study using hepatocellular carcinoma cell lines, it was found that the Wnt/β-catenin pathway may further enhance HIF1a’s induction of EMT, demonstrating the intricate cross talk that occurs among the different EMT-inducing signaling pathways in the tumor microenvironment [20, 85].

Воспалительные цитокины

Another essential component of the tumor microenvironment contributing to EMT is the inflammatory state. Numerous studies have shown that there is a link between chronic inflammation and the progression of cancer [93]. For example, patients who have chronic hepatitis B or C virus infection (both conditions well characterized by chronic inflammation in the liver) are significantly much more likely to develop hepatocellular carcinoma than patients without chronic viral hepatitis [2]. Some of the cells responsible for releasing inflammatory cytokines are immune cells recruited to the cancer microenvironment, cancer-associated fibroblasts (CAFs), and endothelial cells in the surrounding area. Among the cytokines that are often released by such cells and have been implicated in EMT include interleukin-6 (IL-6) [94]. In breast cancer cell lines, for example, this cytokine has been associated with lower E-cadherin expression as well as increased expression of N-cadherin, vimentin, and EMT-promoting transcription factors: twist and SNAIL1 [95]. Similarly, another inflammatory cytokine often released by immune cells such as tumor-associated macrophages (TAMs), which contributes to carcinogenesis, is TNFa. This cytokine has been shown to induce snail expression as well as upregulate TGF-β expression, which is one of the most potent inducers of EMT, as discussed above [20, 96, 97].

MicroRNA

EMT can also be regulated and induced by microRNAs. MicroRNAs are small noncoding strands of RNA that are highly conserved and control gene expression by either targeting specifi mRNA sequences for degradation or inhibiting their translation into proteins. Interestingly, these 19–22-nucleotide-long molecules can function as tumor suppressors or oncogenes, and their dysregulated expression has been noted in several human cancers [98]. Specifi in hepatocellular carcinoma, the following microRNAs (miRNAs) have been observed to have tumor-suppressive properties: the miR-200 family, miR-205 (which inhibits EMT by decreasing vimentin and increasing E-cadherin expression), miR-449a (which suppresses EMT via multiple targets), miR-26a (which suppresses EMT by decreasing EZH2 and increasing E-cadherin), and miR-124 (which inhibits EMT by decreasing cytoskeletal changes brought about by ROCK2 and by inhibiting EZH2). Likewise, there are specifi miRNAs that have been observed to be oncogenic and promote EMT in hepatocellular carcinoma, for example, miR-520g (which induces EMT by targeting SMAD7), miR-155 (which induces invasion by targeting RhoA and facilitating TGF-βinduced EMT), and miR-124 (which supports metastasis and EMT via oncogenic RAS signaling) [20, 99, 100].

In summary, multiple factors in the tumor microenvironment likely play a role in the induction or suppression of EMT, including growth factors, hypoxic conditions, multiple signaling pathways, inflammatory cytokines, as well as microRNAs, as indicated in Fig. 8.2. All of these, as well as the effectors and transcription factors needed for EMT to occur, work in a complex and intricate manner to be able to promote tumorigenicity.

Фиг. 8.2. Различные внеклеточные факторы, которые могут направить клетки подвергнуться EMT

Фиг. 8.3. Вовлечение EMT в различные стадии метастазирования

EMT и метастазирование рака

Metastasis is a reason for which patients with HCC succumb to the disease. This is because by the time most people present with noticeable symptoms, the disease has already progressed beyond a focal lesion in the liver. As such, identifying the roles EMT plays in HCC progression is very useful. The importance of this lies in possibly using EMT markers as prognostic biomarkers in HCC or as potential novel therapeutic targets.

Although originally thought to occur during later stages of tumorigenesis, studies have implicated EMT in earlier stages of tumorigenesis (as shown in Fig. 8.3), as early as malignant cell conversion [55, 101–103].

Конверсия злокачественных клеток и локализованная инвазия

This first step of tumorigenesis is characterized by the conversion of a normal healthy cell into a malignant one as well as factors in the tumor microenvironment supporting progression of the disease. EMT transcription factors have been shown to be involved in this early stage by promoting transformation. Apart from its welldefined role of serving as a transcription factor to lower E-cadherin expression, increase N-cadherin expression, and promote invasion, Twist 1 has also been shown to override cellular senescence and apoptosis as well as cooperate with other molecules to promote malignant transformation. In addition, EMT makes cancer cells lose adhesion to one another through downregulation of E-cadherin and other cell junction proteins and upregulation of mesenchymal markers and makes them migrate to and invade into local tissues around the site of primary cancer origin. Some of the EMT transcription factors such as SNAIL1 have also been shown to upregulate enzymes that degrade the extracellular matrix, such as matric metalloproteinases (MMPs). And still other EMT transcription factors such as Twist 1 have been shown to induce invadopodia formation, which leads to the recruitment of various proteases that will degrade the ECM and help facilitate invasion by cancer cells [102–104].

Интравазация раковых клеток в циркуляцию

This step in the metastatic process occurs after local invasion by cancer cells and is otherwise known as intravasation. This is the process by which cancer cells are able to cross the endothelium to enter the bloodstream and lymphatic system for dissemination into other parts of the body [101]. Studies have shown that EMT plays a role in modulating cancer cell migratory properties such that entry of cancer cells into the vasculature will be facilitated. For example, using the transendothelial migration assay, it was found that the EMT transcription factor, Zeb1, is needed for greater ability of cells to pass through the endothelial cell barrier [105]. It was also found in a breast cancer cell model that overexpression of the EMT transcription factor Snail1 was needed for greater intravasation via the activation of certain membrane-bound matrix metalloproteinases (MMPs) [106].

Системный транспорт в кровотоке

When cancer cells have breached the endothelial barrier and enter the bloodstream, the next step is transportation through the circulatory system. At this point, tumor cells are known as circulating tumor cells (CTCs). CTCs have been particularly difficult to study because there are only a few cancer cells that are able to make it and survive the harsh conditions of being outside their normal environment and traveling through the bloodstream [107]. Studies on CTCs have revealed that many CTCs have mesenchymal features indicating that EMT may have taken place [108–111]. Indeed, it has also been observed that expression of the EMT transcription factors Twist1 and Snail1 significantly increased CTC numbers and promoted microtubule membrane protrusions, which are thought to help CTCs aggregate and attach to the endothelial wall to aid in their survival and in the next step of the metastatic cascade. The fact that several studies also found that mesenchymal CTCs were associated with platelets, which are major secretors of TGF-β (a major inducer of EMT), supports the fact that EMT may be a characteristic feature of CTCs in the bloodstream and may contribute to the promotion of metastasis.

Экстравазация раковых клеток из кровотка в отдаленные ткани

As CTCs travel through the bloodstream and aggregate and attach to the endothelial wall of blood vessels, the cancer cells will cross the endothelial barrier yet again, this time to leave the bloodstream and spread into the surrounding tissue [101]. Extravasation assays using zebra fish showed that the EMT transcription factor Twist 1 was able to form large membrane protrusions in cancer cells ready to cross the endothelial blood vessel barrier and enhance cancer cell extravasation into the surrounding tissue [112]. Others showed that the protrusions formed in tumor cells depended on the mesenchymal state of the cancer cells and could also be induced by the EMT transcription factors Twist1 and Snail1 [113]. Thus, in this way, EMT may facilitate extravasation of cancer cells into the surrounding parenchyma.

Колонизация раковых клеток вторичных областей

There is evidence to suggest that when CTCs get to secondary sites, colonization of the secondary site by cancer cells involves gradual transition of cancer cells from a predominantly mesenchymal cell phenotype to a more epithelial state [19, 101, 114–116]. Whereas cancer cells in the primary tumor and CTCs exhibit EMT, there is evidence suggesting that cancer cells colonizing distant tissues display mesenchymal-to-epithelial transition (MET), this being possible since EMT is a readily reversible biological process [19].

This progressive understanding may help us determine which steps during carcinogenesis that EMT detection and targeting may be useful for potential clinical applications [117]. For example, it is conceivable that our knowledge of EMT may have diagnostic applications in HCC and other cancers given that EMT plays an early role in the malignant conversion of a normal cell into a cancerous cell. Thus EMT markers may be useful biomarkers for earlier detection of HCC, if imageguided biopsy is performed early. It is also conceivable that “liquid biopsies” to obtain CTCs from the blood can be used to detect EMT markers in CTCs, thus diagnosing progression of HCC noninvasively. Moreover, the knowledge that increased expression of mesenchymal markers is observed and may contribute to several of the steps of the metastatic cascade opens up the possibility of targeting them to lower their expression, EMT, and possibly to inhibit or eliminate metastasis.

Таблица 8.2. Эпителиальные и мезенхимальные маркеры, которые вовлечены в специфические случаи гепатоцеллюлярной карциномы

E-Cadherin Epithelial Hashiguchi, M, et al. (2013) [26]; Wei Y et al. (2002) [29]; Matsumura, T et al. (2001) [27]; Wang XQ et al. (2012) [28]; Cho SB et al. (2008) [25]
N-Cadherin Mesenchymal Cho SB et al. (2008) [25]; Gwak, Geum-Youn et al. (2006) [37]
Vimentin Mesenchymal Hu L et al. (2004) [40]
Fibronectin Mesenchymal Gupta, N et al. (2006) [118]; Torbenson, M et al. (2002) [119]
Snail Mesenchymal Sugimachi, K et al. (2003) [54]; Yang, M et al. (2009) [58]; Wang XQ et al. (2012) [28]
Slug Mesenchymal Sun Y et al. (2014) [120]
ZEB Mesenchymal Hashiguchi, M, et al. (2013) [26]
Twist Mesenchymal Lee TK, et al. (2006) [59]; Yang, M et al. (2009) [58]
Goosecoid Mesenchymal Xue TC, et al. (2014) [121]
MMP9 Mesenchymal Nart D et al. (2010) [122]

Потенциальные биомаркеры для наиболее ранней диагностики HCC

There are several molecular features that have been associated with epithelial-tomesenchymal transition (EMT) that can serve as biomarkers of HCC. During EMT, for example, cell-to-cell adhesion is lost, and molecules that usually connect cells together such as some cadherins, catenins, and claudins are downregulated. In addition, transcription factors such as Snail, Slug, Twist, Zeb1, and Goosecoid are also activated. And genes associated with the mesenchymal phenotype, such as vimentin, fibronectin, CD44, integrin β6, and matrix metalloproteinases (MMPs), are also commonly upregulated [55, 101–104]. Table 8.2 shows the different epithelial and mesenchymal markers as detected in HCC cell lines or biopsies from patients with HCC.

Таргетинг EMT как потенциальная лечебная стратегия HCC

As discussed above, it is important to be able to diagnose HCC earlier to optimize treatment and be more successful at removing this disease. The diffi in treating HCC lies in the fact that by the time patients present with defi ve symptoms, the cancer has likely advanced, and curative treatment options are either very limited or not available. As mentioned previously, EMT markers have a potential role in earlier HCC diagnosis and in the noninvasive monitoring of HCC progression.

However, EMT markers may also have potential application in HCC as therapeutic targets for inhibiting or eliminating progression and metastasis. There are several ways EMT can be targeted: through its effectors, transcription factors, or inducers. Each of these will be discussed below [123–125].

EMT эффекторы как возможные мишени для HCC

EMT effectors are molecules that define the epithelial and mesenchymal state, for example, those mentioned before: E-cadherin, N-cadherin, vimentin, fibronectin, etc. While there are as yet no clinical applications of these EMT effectors as therapeutic targets, in vitro and preclinical studies have shown promising results [123]. For example, peptide ADH1 and quercetin both lower N-cadherin expression and thus prevent migration and tumor progression [126, 127]. Similarly, agents that lower vimentin levels such as withaferin A, silibinin, flavonolignan, and salinomycin exhibit antitumorigenic effects [128–131]. However, despite these observations, many EMT effectors have complex, time-dependent, and context-specific roles. Consequently, targeting them may prove challenging [123].

EMT транскрипционные факторы как возможные мишени для HCC

EMT transcription factors are the molecules that carry out EMT (such as Snail1, basic HLH family such as Twist1, and Zeb1). While preclinical studies have revealed promising results suggesting that EMT transcription factors are good therapeutic targets [132–134], these transcription factors have proven challenging to target in a clinical context [123]. Nevertheless, agents shown to decrease Twist1 expression (such as sulforaphane and moscatilin) all show a disruption of EMT as well as inhibition of tumorigenicity [135, 136]. Similarly, fucoidan, which decreases Twist1, Snail1, and Slug expression via various signaling pathways and microRNAs, also inhibits EMT [137–139].

EMT индукторы как возможные терапевтические мишени для HCC

In addition to EMT effectors and transcription factors being possible therapeutic targets, we may also be able to target the factors that induce EMT for potential therapeutic applications [123]. With the recent clinical use of sorafenib for treatment of advanced HCC, high-throughput screening and an ongoing search are underway for other agents that could possibly have antitumorigenic benefits and extend lifespan while demonstrating limited toxicity [140, 141]. Interestingly, most of the relevant clinical trials currently ongoing target EMT inducers rather than target EMT effectors or transcription factors [123]. Indeed, sorafenib, while possessing anti-angiogenic effects, also inhibits tumor progression by suppressing EMT in HCC cases where percutaneous ablation is not an option. Sorafenib also inhibits migration and invasion by suppressing MMPs and HGF-induced EMT via the c-Met and ERKMAPK pathways. And sorafenib’s efficacy in inhibiting TGF-β has been demonstrated in mouse hepatocytes [140–144]. Several other agents inhibiting TGF-β (e.g., LY2157299, a selective inhibitor of TGF-βR1) and c-Met (e.g., tivantinib) have also been investigated, with seemingly promising results in HCC [123].

Other agents being examined in clinical trials target other EMT inducers such as EGF, FGF, PI3K/AKT/mTOR, MEK, and IGF signaling and perhaps target microRNAs that modulate EMT status [123].

Заключение

Unfortunately, because HCC patients are frequently diagnosed when the cancer has already advanced, their prognosis is often dismal. To date, there is a lack of effective therapeutics for the treatment of advanced HCC [1]. Currently, sorafenib is the only FDA-approved drug for the treatment of advanced HCC, and it prolongs survival of patients with advanced HCC by only a few months [5]. Consequently, we urgently need discovery of molecular mechanisms that can be exploited for early diagnosis of HCC, monitoring of HCC progression, effective treatment of advanced HCC, and monitoring of response to drug treatment of HCC.

Although clinical applications of EMT markers may take a few more years to actualize, current research suggests that EMT markers may hold promise as potential biomarkers for early diagnosis and development of novel targeted therapeutic strategies for HCC (see Table 8.2). Because EMT is a phenomenon observed as early as the malignant conversion of a healthy cell into a cancer cell and present through multiple stages of tumorigenesis, it is likely to have potential applications for early detection and curative treatments of early cancers. Indeed, as mentioned above, there are a number of preclinical and clinical trials that are currently ongoing to determine potential therapeutic applications from targeting specific EMT markers [123]. While dysregulated expression of EMT effectors and transcription factors may one day prove useful as biomarkers for early diagnosis of HCC, it appears that some clinical trials are showing promising results from targeting EMT inducers [145–147]. It is possible that combining sorafenib with one or more of these novel EMT marker-based therapeutics that may yet come through may provide additional beneficial outcomes for patients with advanced HCC [148].

Of course, therapeutic targeting of EMT markers may be associated with currently unknown side effects. These may be related to issues such as intratumor heterogeneous expression of EMT markers. Also, EMT appears to be contextand time-dependent and transient. These issues may create side effects from targeting EMT markers. However, with increasingly greater knowledge being gained from large cancer sequencing projects, it may be that it is possible to combine other novel targeted therapies with therapeutic targeting of EMT markers in such a way as to minimize side effects [145–147]. Moreover, better understanding of the most appropriate timing of when EMT inhibition would be most effective at combating HCC and other cancers could contribute to developing effective new drug treatments. Our current understanding of the role of EMT in HCC certainly suggests that EMT is important in HCC development and progression and that further research into the role of EMT in HCC is required to create beneficial clinical applications in HCC.

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