Mechanisms of Invasion in Head and Neck Cancer.
THE CLINICAL RELEVANCE OF INVASION
Head and neck squamous cell carcinoma (HNSCC) is an epithelial cancer, arising from the mucosa of the upper aerodigestive tract. Therefore, an invasive cancer, by definition, must first invade the basement membrane of the native epithelium. This property is achieved during the process of carcinogenesis when multiple genetic insults are accumulated, and in turn, the acquisition of invasive capabilities allows invasion through the basement membrane. This primary event of invasion differentiates carcinoma in situ from invasive carcinoma. In oral tongue cancer, when this layer of connective tissue is breached, the invading tumor encounters the underlying tongue musculature. Depth of invasion for these tumors is directly associated with patient outcome, and when the depth of invasion approaches 2 to 4 mm, the rate of lymph node metastasis increases greatly, and prognosis worsens. (2-4) Therefore, elective neck dissection is recommended for oral tongue cancers with a depth of invasion of more than 2 to 4 mm, even when no clinical evidence of lymph node spread exists. A modified staging system has been developed that integrates depth of invasion into the T category of the currently accepted American Joint Committee on Cancer TNM staging. (3,5) Depth of invasion was significantly associated with disease-specific survival in oral squamous cell carcinoma (OSCC). (3)
In addition to depth of invasion, analysis of histologic parameters in resected tumors has indicated that invasion of tumor cells into the normal host tissue can predict patient outcome. The invasive front of an invading tumor is an important indicator of patient prognosis. (2,6-10) Bryne et al (10) developed an invasive-front grading system for tumors, which incorporates 4 parameters: (1) the pattern of invasion, (2) the degree of keratinization, (3) nuclear polymorphism, and (4) host response (inflammatory cell infiltration). (9) The scores for each parameter were combined to yield a total malignancy score for the whole tumor. The worst part of the tumor (most invasive) was used for the assessment. A higher malignancy score meant a poorer prognosis. Sandu et al (2) used the invasive-front grading system described by Bryne et al (10) and found that patients with a higher invasive-front grading system score had significantly lower diseasefree survival. Brandwein-Gensler et al (6) found the histologic parameter--the pattern of invasion--to predict patient outcome in patients with OSCC as part of their risk assessment model. (8) The worst pattern of invasion was significantly associated with overall survival, as well as local recurrence. (6,8) Li et al (7) demonstrated that this risk model predicted local recurrence and disease-specific survival in an independent study for patients with low-stage OSCC.
Bone and cartilage are ensheathed in a dense layer of connective tissue. Therefore, the mandible has a natural barrier to tumor invasion. If a mandible cancer is able to breach the mandibular periosteum and invade the bony cortex, the relatively uninhibited spread of tumor in the bone marrow space can lead to extensive involvement and ill-defined tumor margins with poor prognosis. (11-14) The presence of mandibular invasion places a tumor in an advanced primary stage (T4a) and an overall stage (IV) according to the American Joint Committee on Cancer staging system. (11) Similar to invasion of OSCCs into surrounding normal tissue, the histologic pattern of invasion by OSCC into bone can also be assessed and correlated with outcome. Invasion into bone displaying a broad pushing front and a sharp interface between tumor and bone is categorized as an erosive pattern. Projections of tumor cells along an irregular front with residual bone islands within the tumor are categorized as an infiltrative pattern. (11-14) A retrospective study of patients with mandibular invasion by OSCC found that patients with an infiltrative pattern of invasion into the bone had a 4-fold increased risk of death with disease compared with patients who displayed an erosive pattern of invasion. (13) The infiltrative pattern of invasion into bone tissue is associated with more-aggressive tumor behavior, increased likelihood of positive margins, recurrence, death with disease, and shorter disease-free survival. (11-13) Tumors with mandibular spread are generally treated aggressively, most often with segmental mandible resection, followed by postoperative radiation or chemoradiation. Similarly, the laryngeal cartilage poses a barrier to tumor spread. A cancer of the larynx that exhibits frank cartilage destruction is deemed a T4/stage IV cancer by American Joint Committee on Cancer criteria. If extensive cartilage invasion is present, often an organ-sparing approach with chemoradiation is not feasible, and laryngectomy with postoperative adjuvant therapy is the treatment of choice.
Another feature that HNSCC can exhibit is a propensity for invasion into neural structures. The head and neck region is populated by a rich network of innervation. The hypoglossal nerve extensively innervates the fine musculature of the tongue, whereas the lingual nerve provides a network for tongue sensation and carries distal fibers from the chorda tympani to provide special sensory taste fibers. The mandible carries the V3 branch of the trigeminal nerve, which innervates the teeth and transits to the mental foramen and provides sensation to the buccal mucosa of the lower lip and the skin of the chin. Similarly, the pharynx and larynx are innervated by vagus nerve fibers and branches of the glossopharyngeal nerve. When tumor cells invade the perineurium and nerve sheaths, the cranial nerves can essentially provide a direct route of spread toward the base of the skull and intracranial extension. This is particularly evident for adenoid cystic carcinoma of the salivary gland, which has a very high affinity for perineural spread. In addition, HNSCC commonly exhibits perineural invasion, and this feature has been cited as a harbinger for an increased rate of locoregional recurrence and poor outcome.
Brandwein-Gensler et al (6) found that perineural invasion (PNI) of small and large nerves was associated with reduced overall survival, and PNI of large nerves was specifically associated with local recurrence. To independently assess another set of OSCC cases, de Matos et al (15) applied the same criteria (6,8) and found a significant, positive correlation between PNI and the pattern of invasion, as well as between the pattern of invasion and tumor thickness, or depth of invasion. (3) High levels of nerve growth factor have been correlated with PNI and worse survival. (16) When perineural spread is identified after surgical resection, this feature can influence the decision to administer postoperative adjuvant treatment. Clinicians have evaluated PNI as an indicator in patients with early stage (I and II) OSCC tumors to determine treatment strategies, but PNI and lymphovascular invasion were not significant risk factors for disease-free or overall survival. (17)
The clinical significance of invasive properties is not limited to local disease. In regional lymph node metastases, invasion outside of the lymph node capsule is associated with poor outcome. (18,19) Two large, randomized clinical trials demonstrated that extracapsular spread of lymph node disease, identified after surgical resection, is an indication for maximized treatment with postoperative radiation and concurrent cisplatin. (20)
The strategies that cancer cells use to achieve all the invasive properties highlighted above likely require genetic and molecular alterations that are highly complex. The details of how these cancers acquire the ability to invade normal, organized, anatomic structures are being revealed. An improved understanding of the molecular events that lead to these invasive phenotypes may provide insight that could improve both prognostication and treatment. The following sections summarize
what is known about the mechanisms of invasion of the head and neck by cancer cells.
CELL SURFACE RECEPTORS INFLUENCE INVASIVE BEHAVIOR IN HNSCC
The best-characterized receptor for stimulation of HNSCC invasion is the epidermal growth factor receptor (EGFR; Figure 1). (21-26) Epidermal growth factor (EGF) is present in saliva at concentrations of about 1 ng/mL. (27-29) These concentrations (30,31) or higher (32-39) are able to stimulate migration and invasion in oral carcinoma lines in vitro, as well as in vivo. (40) Other EGFR ligands besides EGF have been shown to stimulate invasion, including transforming growth factor [alpha] (TGF[alpha]), betacellulin, heparin-binding EGF-like growth factor (HB-EGF), and amphiregulin (AREG). (32) Conversely, inhibition of the EGFR blocks invasion in response to EGF. (32,37,41-43) Intriguingly, EGFR also mediates invasion induced by some other receptors, notably G protein-coupled receptors (GPCRs) (44) for lysophosphatidic acid (LPA), (45,46) bradykinin, prostaglandin E2, (47) and gastrin-releasing peptide. (48) In some cases, the mechanism of crossactivation of EGFR by GPCRs has been identified as the release of EGFR ligands. The EGFR ligands are initially produced as cell-surface transmembrane proteins, (49) which can then be released from their transmembrane tethers by members of a disintegrin and metalloproteinase (ADAM) family of proteases. (50) The ADAM17 mediates the stimulated release of TGF[alpha] by bradykinin and prostaglandin E2. (47) Gastrin-releasing peptide stimulates the release of both AREG and TGF[alpha]. (48) In the case of gastrin-releasing peptide,
EGFR ligand release involves a pathway in which Src activates phosphatidylinositol 3-kinase (PI3K), leading to phosphoinositide-dependent kinase 1 activation and phosphorylation of ADAM17. (51) Estrogen-receptor activation can also trigger EGFR ligand release. (30) In addition, autocrine release of AREG, HB-EGF, betacellulin, and TGF[alpha] occur during serum starvation, (48,52-55) indicating that autocrine stimulation of EGFR can contribute to basal invasion capability.
A number of other receptors that can regulate HNSCC invasion have been identified. Other GPCRs that stimulate invasion with extracellular signal-regulated kinase (ERK) and matrix metalloproteinase 9 (MMP-9) activation include chemokine (C-X-C motif) receptors CXCR1 and CXCR2 (56,57) and CXCR4. (58,59) Interleukin (IL) 6 receptors can activate the ERK and c-JUN N-terminal kinase pathways. (60,61) Toll-like receptors have also been shown to stimulate invasion with activation of the nuclear factor of k light chain polypeptide gene enhancer in B cells (NF-[kappa]B) pathway. (62,63) The NF-[kappa]B signaling can synergize with the ERK/activator protein 1 (AP-1) pathways to stimulate invasion. (64,65) Wingless-type MMTV integration site family member 5B (WNT5B) knockdown suppresses MMP-10 production and invasion indicating that the WNT signaling pathways can also contribute to invasion. (66,67)
Transforming growth factor [beta] (TGF[beta]) can be produced by tumor-associated fibroblasts (68) and induce NF-[kappa]B activation (69) and SMAD-dependent MMP production, (70) as well as epithelial to mesenchymal transition (EMT) factors, such as Snail and Slug. (71-76) Invasion is stimulated by hypoxia in a Notch-dependent fashion. (77) Inhibition of Notch can also suppress MMP production. (78)
SIGNALING PATHWAYS INVOLVED IN HNSCC INVASION
Several signaling pathways have been shown to be important for invasion of HNSCC. Inhibition of ERK results in reduced invasion. (37,41,79) An important ERK target is AP-1, (80) which can increase transcription of MMPs, such as MMP-9. (79,81,82) The PI3K activity is also important for invasion. (37,79,83,84) Inhibition of PI3K also results in reduced MMP-9 expression. (37,79) Additional contributions of PI3K to invasion can occur through AKT stimulating Slug and Snail expression to enhance EMT (85,86) or through phosphorylation of ezrin (87) and yes-associated protein. (88) Phospholipase C [gamma]1 is important in EGFR-induced invasion in some cases, (79,89) potentially in a complex with c-Src. (35) The Src family members can contribute to invasion either through enhanced release of receptor ligands as described above or through stimulation of invadopod formation, which is described in more detail below. (48,55,90-94) A number of HNSCC cell lines have constitutively high levels of Rac1 activity, and inhibition of Rac activity results in reduced invasion capability. (95) In most cases, the increased Rac1 activity correlated with increased tyrosine phosphorylation of the Rac GEF Vav2 induced by EGFR, but an alternative pathway used Ras activation. The role of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in HNSCC invasion is complex. STAT3 is important for ligand-independent invasion induced by the EGFR vIII isoform, (96) but JAK2-STAT5a signaling is important for erythropoietin (EPO)-induced invasion but not EGF-induced invasion. (96) STAT3 activity may generally enhance invasion capability through suppression of phosphatase and tensin homolog (PTEN) (97) and E-cadherin. (98)
PROTEASES IMPLICATED IN HNSCC INVASION
A key distinction between migration and invasion is the ability to degrade extracellular matrix (ECM) barriers in the latter activity. Thus, expression of proteases, especially MMPs, is important for HNSCC invasion in many cases. (99) The most commonly identified MMP has been MMP-9. Its activity is increased by EGFR (32,37,42,54,100-102) and integrins, (75) and its inhibition reduces invasion. (81,103,104) In addition, MMP-9 can degrade type IV collagen, (105-107) a key constituent of the basement membrane, and thus has the potential for an important role in enabling HNSCC cells to become frankly invasive. Analysis of OSCC cases by immunohistochemistry revealed that expression of MMPs corresponded to areas with loss of type IV collagen [alpha] chain. The enzymatic activity of MMPs was assessed by zymography and was enhanced in higher-grade OSCC and along the advancing tumor front. (108) MMP-9 is used as a marker of invasive OSCC in many studies, and the extent of its expression is related to the infiltration pattern of squamous cell carcinoma at the invasive front. (109) In vitro invasion assays that use Matrigel, which is derived from basement membranes, often show a dependence on MMP-9 activity. However, MMP-9 can cleave other proteins exposed to the extracellular milieu as well, activating ligands such as TGF[beta] and chemokines as well as cleaving cell surface receptors. (110) In addition, MMP-9 may be involved in cleavage of E-cadherin, resulting in reduced cell-cell adhesion and increased invasion. (37)
MMP-2 is another MMP that has been implicated in HNSCC invasion. (103,104) Although MMP-2 and MMP-9 are secreted MMPs, the transmembrane protease membranetype 1 MMP (MT1-MMP) also has a significant role. MT1-MMP, together with the tissue inhibitor of metalloproteinases 2, (111,112) is important in the activation of secreted proteases, such as MMP-2, (113) and suppression of MT1-MMP reduces overall matrix degradation activity (114) and invasion. (43) Another protein that enhances protease activity and is important for invasion is ECM metalloprotease inducer (EMMPRIN). (100,115-117) Although the mechanism is unclear, EMMPRIN homophilic binding induces the expression of a number of proteases, including MMPs, cathepsin B, and urokinase plasminogen activator receptor (uPAR). (100,115,117,118) Other proteases that have been shown to contribute to HNSCC invasion include MMP-10, (66) MMP-13, (80) matriptase, (119) fibroblast activation protein, (120) and uPA/ uPAR, (117,121-124) as well as the glycosidase heparanase. (125)
THE ROLES OF ADHESION IN HNSCC INVASION
Cell-cell adhesion mediated by E-cadherin generally inhibits invasion. (26,126) Induction of cell-cell junctions can inhibit invasion. (127) Reduction in E-cadherin expression can occur through methylation of the promoter, and demethylating agents, which reverse this, can enhance invasion. (128) Inhibition of the E-cadherin function also enhances invasion. (129) However, the inhibitory effect of E-cadherin may be overcome by expression of other invasion-inducing proteins. (130,131) Desmosomes are another cell-cell adhesion structure, which can suppress invasion. (132) However, over-expression of certain adhesion proteins, such as claudin 1 (133) and desmoglein 3, (134) can enhance invasion.
Although degradation of the ECM, especially the basement membrane, is a key property of invasive tumors, interaction with the ECM is also important for invasion and prognosis. (135-138) Knockdown of the integrin [beta]1 using short interfering RNA (siRNA) can reduce MMP-2 activity and invasive capability, (139) mimicking the ability of miR-124, which has been shown to target integrin [beta]1 and reduce invasion. (140) The integrin [[alpha].sub.v][beta]6 is upregulated in wound healing, and inhibition of integrin [[alpha].sub.v][beta]6 also inhibits invasion. (141-143) Integrin [[alpha].sub.v][beta]6 is important to inducing protease expression. (142,144) Conversely, expression of specific ECM proteins, such as collagen I, (145) collagen XVI, (146,147) or laminin, (148) can enhance invasion. Binding of integrins to these matrix molecules triggers a signaling cascade, including integrin-linked kinase (ILK), (149) talin, (92,150) focal adhesion kinase (FAK), (151-153) and Src family members, (90,154) which then link to the MAP kinase and PI3 kinase pathways for stimulation of MMPs. (82,145,149,154) It is unclear why both growth factor signaling and adhesion signaling are needed for invasion--the connection between adhesion and growth factor signaling in invadopod formation is an active area of investigation. (155-159)
MicroRNAs THAT REGULATE HNSCC INVASION
MicroRNAs (miRNAs) are a class of gene-expression regulators that often have altered expression in various human cancers, including HNSCC. (160-165) The MiRNAs have emerged as having key roles in diverse cellular processes, including cancer cell proliferation, migration, invasion, and metastasis. (160,161,166,167) Dicer, a key enzyme involved in siRNA and miRNA function, was reduced in expression in tongue squamous cell carcinoma lines, and reduction of Dicer led to increased cell proliferation and invasion. (168) Consistent with these observations, most miRNAs that affect invasion in HNSCC are inhibitory (see Table 1).
The targets of the miRNAs identified thus far can be categorized according to the various functions involved in invasion. Many miRNAs target proteins associated with signal transduction, including ligands, receptors, and intracellular regulators. MiR-126 down-regulates protein levels of ligands epidermal growth factorlike domain 7 (EGFL7), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF2). (169) Dickkopf 2 (DKK2), a target of miR-21, promotes cell invasion by antagonizing Wnt/[beta]-catenin signaling. (170) MiR-99a is a metastasis suppressor in OSCC by regulating expression of the insulin-like growth factor 1 receptor (IGF1R). (171) The regulation of neurofibromin 1 (NF1) by miR-193b impaired cell migration and invasion via inhibiting ERK1/2 phosphorylation. (172) MiR-155 promotes the migration and invasion of laryngeal squamous cell carcinoma. Knockdown of miR-155 in laryngeal squamous cell carcinoma cells suppressed invasion by negatively regulating suppressor of cytokine signaling 1 (SOCS1), which allowed increased STAT3 signaling. (173) HNSCC cells overexpressing miR-107 had reduced cell invasion in vitro and tumor growth in vivo resulting from the targeting of protein kinase C epsilon (PRKCE). (174)
Many adhesion proteins are also targets of miRNAs. Laminin, beta 3 (LAMB3), a component of laminin-332 in the ECM, is targeted by miR-218. (175) The miR-29s (miR-29a, miR-29b, and miR-29c) target another component of laminin-322, laminin, gamma 2 (LAMC2). (148) The miR-29s also target adhesion molecule integrin, alpha 6 (ITGA6). (148) In addition, miR-29a was found to upregulate the ECM protease MMP2. (176) ADAM10, another protease, is a target of miR-140-5p. (177) There is also a significant group of miRNAs whose targets are associated with cell protrusions and the cytoskeleton, including transgelin 2 (TAGLN2), targeted by miR-1, (178) moesin (MSN), and actin-related protein 2/3 complex, subunit 5 (ARPC5), targeted by miR-133a, (179,180) and podoplanin (PDPN) targeted by miR-363. (181) The cytoskeleton proteins ras homology family member C (RHOC) and Rho-associated, coiled-coil containing protein kinase (ROCK2) are targeted by miR-138, which also targets the EMT-associated protein vimentin (VIM). (184,185) Other miRNA targets associated with EMT for general gene expression are twist basic helix-loop-helix transcription factor 1 (TWIST1) and forkhead box C1 (FOXC1), targeted by miR-181a and miR-639. (186,187)
Transcription factors that may regulate genes associated with motility and invasion are also targets of miRNAs. MiR-29b suppresses specificity protein 1 (SP1) expression, which impairs OSCC invasion via reduced Akt activation. (188) MiR-375 targets metadherin (MTDH) (189-193) and miR-504 targets forkhead box P1 (FOXPl), (194) also important for invasion. Finally, there is a small group of targets whose direct function in invasion are unclear, such as programmed cell death 4 (PDCD4), (195) targeted by miR-21, BCL2 binding component 3 (BBC3), (196) and mitochondrial superoxide dismutase 2 (SOD2), (197) targeted by miR-222.
INVADOPODIA AS MEDIATORS OF HNSCC INVASION
Invadopodia (Figure 2) are specialized, actin-rich structures that mediate ECM proteolysis and are thought to have a key role in cell invasion. (198-201) An important component and marker of invadopodia is cortactin. The HNSCC cells that have band 11q13 amplification and subsequently overexpress cortactin have elevated binding and activity of Arp2/3 complex together with increased HNSCC cell motility and invasion. (202) The EGFR inhibitor, gefitinib, impairs motility in HNSCC cells with the degree of inhibition positively correlated with the level of cortactin in the cells. (202) High levels of cortactin reduce EGFR down-regulation and extend ERK activation. (203) Cortactin is important for both invadopodium assembly and ECM degradation. (204) Cortactin was shown to promote MMP-2 and MMP-9 secretion, as well as the cell surface levels of MT1-MMP in invadopodia of HNSCC cells. (204) Figure 3 shows an example of an in vitro assay to assess invadopodial matrix degradation, with cortactin and tyrosine kinase substrate with 5 SH3 domains (Tks5) as markers for invadopodia.
Src is an important regulator of invadopodium formation and activity. Saracatinib, an Src inhibitor, inhibited Src activation and phosphorylation of FAK, p130 Crk-associated substrate and cortactin in HNSCC cells along with reduction of invadopodia formation, ECM degradation, and MMP-9 secretion. (91) Intriguingly, although constitutively active Src induces invadopodium formation, wild-type Src expression is also needed in HNSCC cells for invadopodium-associated matrix degradation. (205) Abelson (Abl) kinase activity diminishes invadopodial ECM degradation in HNSCC cells: The inhibition of Abl and Arg by imatinib, an Abl family inhibitor, led to EGFR activation through increased HB-EGF production and shedding, and because of increased EGFR signaling, Src and ERK activity induced tyrosine and serine phosphorylation of cortactin. (55) These findings differ from a proposed model of EGFR-invadopodia signaling in breast cancer where elevated EGFR signaling induces Src activation, which subsequently leads to activation of Abl/Arg and tyrosine phosphorylation of cortactin by Abl/Arg. (206) In addition to the EGFR, (207) TGF[beta] receptor 1 can induce invadopodium formation through stimulation of insulinlike growth factor II messenger RNA (mRNA) binding protein 3 and podoplanin, both of which are important for invadopodium formation, ECM degradation, and MMP-9 activity. (73,208)
PI3K activity also regulates the formation and activity of invadopodia. (209,210) In breast cancer cell lines, PI3K signaling induces formation of invadopodia, and specifically, the p100[alpha] catalytic subunit of PI3K was responsible for the effect. (210) In HNSCC cells, the mechanism of PI3K signaling differs from the breast cancer model. (209) LY294002, a PI3K inhibitor, suppressed invadopodia numbers and ECM degradation, as well as focal adhesion numbers and size. Stimulation of invadopodia by PI3K may be due in part to production of PI(3,4)[P.sub.2] (phosphatidylinositol [3,4]-bisphosphate) from PIP3 (phosphatidylinositol [3,4,5]-trisphosphate) through the action of SH2-containing inositol 5'-phosphatase. (209) Expression levels of formin homology domain protein 1 (FHOD1), an actin nucleating protein, are dependent on PI3K. (211) Silencing FHOD1 reduced cell migration, invasion, invadopodium formation, and invadopodium-mediated ECM degradation. (211) Protein kinase C [alpha] (PKC-[alpha]) can provide feedback negative regulation of cells with mutant PI3K, although in cells with wild-type PI3K, PKC-[alpha] appears to stimulate invadopodium formation. (209)
Adhesion signaling is also important for invadopodium formation and maturation. Adhesion rings form around invadopodia and are recruited after invadopodium formation. (114) Integrin activity and ILK are needed for adhesion ring formation, MT1-MMP accumulation in invadopodia, and invadopodia-associated ECM degradation. (114) Although MT1-MMP is necessary for ECM degradation, it is not necessary for adhesion ring formation. Invadopodia are key docking sites for multivesicular endosomes containing smaller, secreted vesicles, termed exosomes. Invadopodia are also secretion sites for exosomes, (212) which promote invadopodia formation, enable the exocytosis of MT1-MMP at invadopodia, and stimulate invadopodia-associated matrix degradation. (212)
EPITHELIAL TO MESENCHYMAL TRANSITION AND CANCER STEM CELLS IN HNSCC INVASION
Epithelial to mesenchymal transition is a highly regulated process in which epithelial cells acquire a mesenchymal morphology through coordinated changes in gene and protein expression that lead to decreased cell adhesion and cell polarity, resulting in a more-invasive phenotype. (26,213-219) Common protein indicators of EMT are decreased expression of E-cadherin, and increased expression of N-cadherin and vimentin. The expression of these genes is controlled by transcription factors, including Snail, the zinc finger family of transcription factors (ZEB), and Twist. There are also changes in cellular proteins, such as integrins and [alpha]-smooth muscle actin, as well as ECM proteins, such as collagen, laminin, and fibronectin. (149,213-216,220-223) The HNSCC cells that undergo EMT have been shown to be more invasive.* The loss of E-cadherin associated with EMT is an indicator of poor prognosis in HNSCC. (26)
Epithelial to mesenchymal transition can induce the generation of a subpopulation of cells, called cancer stem cells, which have the potential for tumor initiation. (225,226) Cancer stem cells exhibit the properties of self-renewal, potential to behave as tumor progenitor cells, and the ability to differentiate. (223,225-227) Cancer stem cells are slow-dividing and therefore often more resistant to chemotherapy. (225-227) Cancer stem cells are found in invasive fronts, and in many tumors are near the blood vessels. (217,223,225-227) Epithelial to mesenchymal transition can generate cells within tumors that have stemlike properties. (216,219,223,228) In head and neck cancer, Twist, a regulator of EMT, was found to induce expression of Bmi-1, a regulator of sternness. (219,228) Twist and Bmi-1 can act together to repress expression of E-cadherin and p16INK4a. Higher expression of Twist and Bmi-1 has been correlated with lower E-cadherin and p16INK4a and is associated with poor prognosis. (219)
There are many common molecules and pathways involved in invasion, EMT, and stemness. TGF[beta] regulates transcription of EMT transcription factors via SMADs. (229) CD44, a cell surface glycoprotein that binds hyaluronic acid, is involved in cell-cell crosstalk, cell adhesion, and migration and interacts with c-Met and EGFR. (227,230) Epidermal growth factor can induce EMT in HNSCC cell lines through PI3K/ Akt signaling. (223) Expression of Bmi-1, aldehyde dehydrogenase, and CD44 also increases with EGF treatment, indicating an acquisition of stemlike properties. Notch signaling can induce EMT under hypoxic conditions, with increased expression of Notch pathway molecules and Snail, decreased expression of E-cadherin, and an increase in invasiveness. (77) In addition to inducing EMT and increased invasiveness, Snail has also been shown to induce stem-like properties in OSCC. (224) Adhesion-related signaling molecules that are important for EMT and invasion include ILK (149) and FAK. (152) For proteases, MT1-MMP has been shown to induce EMT and stemlike properties in OSCC, as well as invasion, in parallel with increased levels of Twist and ZEB. (221) However, there may be subtypes of cancer stem cells, which differ in their invasive ability. (231)
One aspect of invasion that needs more examination in HNSCC is collective invasion, in which cell-cell contacts may be maintained during invasion. Three-dimensional reconstructions of the invasive front of 14 oral tongue SCCs revealed that most invasive tumor cells are in multicellular structures of variable size, (232) suggesting the presence of cell-cell contacts. There is an ongoing debate regarding the relative importance of single cell and collective invasion for patient prognosis. One possibility is that a partial EMT could maintain cell-cell contacts but stimulate invasive properties sufficient to generate a more aggressive tumor that results in a worse prognosis. (233)
TUMOR MICROENVIRONMENT AND INVASION
Cancer-associated fibroblasts are the major stromal cells present in the microenvironment of HNSCC, and they tend to be myofibroblasts showing increased expression of [alpha]-smooth muscle actin. (234-237) Myofibroblasts and cancer-associated fibroblasts have been shown to enhance HNSCC invasion in vitro in a variety of assays. (238-247) Treatments that can enhance the ability of fibroblasts to stimulate tumor cell invasion include irradiation (182) and reactive oxygen species, (183,248,249) as well as lifestyle-correlated factors, such as cigarette smoke (250) and areca nut extract. (251) Fibroblasts have been shown to stimulate tumor cell invasion through secretion of a number of factors, including chemokine (CC motif) ligand 2 (CCL2), (183,252) CCL7, (253) stromal cell-derived factor-1 (SDF-1), (254) TGF[beta], (73,248,249,255,256) IL-33, (257) MMP-2, (145,249-251,258,259) EGFR ligands, (260) and hepatocyte growth factor (HGF). (254,261-265) Cancer-associated fibroblasts can also produce ECM molecules that enhance invasion, such as thrombospondin 1266 and fibronectin. (267) The mechanism of stimulation of tumor cell invasion by cancer-associated fibroblasts can include induction of EMT. (245,268)
In several studies, a paracrine interaction between tumor cells and fibroblasts has been shown to stimulate invasion. For example, CCL2 from fibroblasts can stimulate invasion and reactive oxygen species production by tumor cells, which, in turn, stimulates fibroblast senescence and CCL2 production from the fibroblasts. (183) Galectin production by tumor cells can also stimulate CCL2 production from fibroblasts. (252) Interleukin 1 from OSCC stimulates CCL7, HGF, and TGF[beta] production by fibroblasts, (73,253,265) whereas endothelin stimulates ADAM17-mediated release of EGFR ligands. (246,260) Interestingly, there is a report of TGF[beta] from tumor cells inducing HGF production by fibroblasts as well. (264)
Other features of the tumor microenvironment have also been identified as stimulating HNSCC invasion, although not as thoroughly as cancer-associated fibroblasts. The perivascular niche has been associated with cancer stem cells and invasion. (227) Endothelial cells can secrete EGF to induce EMT and invasion. (223) Tumor-associated endothelial cells have been shown to stimulate invasion of HNSCC cells in vitro through the secretion of IL-8269 and CXCL1. (270) These chemokines are produced and secreted upon stimulation of endothelial cells by VEGF via BCL2 upregulation. (269,271) Macrophages have been shown to stimulate Axl-mediated invasion (272) and perineural invasion through production of glial cell-derived neurotrophic factor (GDNF). (273) However, macrophages did not enhance invasion in response to EGF in an in vivo invasion assay. (40) Hypoxia in the tumor microenvironment can induce Notch and EMT factors, such as Slug and Snail, to increase invasion. (77,274) In addition, MMP-9 can be induced by hypoxia to enhance invasion in a [Na.sup.+]/[H.sup.+] exchanger 1-dependent fashion. (275) Hypoxia-inducible factor 2a leads to EGFR activation, (276) whereas hypoxia-inducible factor 1a increases integrin [alpha]5 and fibronectin (277) in invasion. Of concern for treatments using EPO to mitigate chemotherapy side effects, hypoxia can induce the EPO receptor in tumor cells, enabling EPO to stimulate tumor cell invasion. (278)
GENOMIC CHANGES IN INVASION-ASSOCIATED GENES
DNA changes, such as mutations and copy number variations (CNVs) at the genomic level have been implicated in cancer progression. (279) Higher numbers of CNVs in the genome are associated with the development of cancer, and more CNVs are accumulated with tumor progression. (279) In HNSCC, disease-specific survival and recurrence can be predicted by CNVs. (280) We used HNSCC TCGA (The Cancer Genome Atlas, National Cancer Institute Center for Bioinformatics, Bethesda, Maryland) data downloaded from the UCSC Cancer Genomics Browser (University of California, Santa Cruz) to evaluate the genes discussed in this review. (281) The 25 genes with the greatest CNVs or number of mutations in HNSCC discussed here are presented in Table 2. PIK3CA is highest in copy number increase and second in mutations, with EGFR and RACI also being high in both mutations and copy number increases, emphasizing the potential importance of these genes in HNSCC.
Studies of invasion have identified a number of possible applications in the treatment of HNSCC. The most straightforward approach is to develop specific inhibitors targeted to molecules that are identified as being important in invasion. In particular, proteases, such as MMP-2, MMP9, and MT1-MMP, are commonly identified as being important for invasion and, thus, could be targeted for therapeutic development. However, there has been extensive development of broad-spectrum MMP inhibitors with very limited success. (282) Inhibition of the beneficial roles of MMPs resulted in unacceptable side effects, (283) and thus, there is now caution in considering therapeutics targeting MMPs, with the assumption that such treatments likely need to be brief to avoid side effects caused by longer-term treatments. (284) A possible use for brief treatments could be in counteracting the effects of radiation. Radiation has been found to induce migration and invasion, (285) and treatment with invasion inhibitors during radiation therapy could potentially enhance its efficacy. (286,287) Inhibitors targeting EGFR, (288) [Na.sup.+]/[H.sup.+] exchanger 1,275 Src, (91) and microtubules (289) can inhibit invasion at concentrations that are not toxic to cells and could be useful in counteracting radiation-induced spread.
For longer-term treatments, potentially lower-toxicity compounds for inhibiting invasion have been identified whose targets are unknown. These include extracts from herbs, green tea, and other natural sources. (41,101,290-300) Such compounds could constrain further tumor spread and extend patient survival without directly killing tumor cells. Limiting tumor spread could develop additional benefits because of the consequences of keeping tumor cells clustered together. As noted above, there is an intimate relationship between invasion, EMT, and stem cell properties. It is possible that by inhibiting invasion of HNSCC cells, there is partial reversion of EMT and loss of stemness, which could result in greater sensitivity to cytotoxic treatments. Compounds have been identified that, on their own, inhibit invasion and EMT without affecting viability, (292) but then sensitize HNSCC cells to cisplatin. (292,301) Identification of invasion inhibitors that can enhance sensitivity to chemoradiation while suppressing the spread of tumor cells would provide a valuable addition to the therapy options for patients with head and neck cancer.
Please Note: Illustration(s) are not available due to copyright restrictions.
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Lizandra Jimenez, MS; Sangeeta K. Jayakar, MS; Thomas J. Ow, MD, MS; Jeffrey E. Segall, PhD
Accepted for publication January 14, 201 5.
Published as an Early Online Release June 5, 201 5.
From the Departments of Pathology (Mss Jimenez and Jayakar, and Drs Ow and Segall) and Anatomy and Structural Biology (Mss Jimenez and Jayakar, and Dr Segall), Albert Einstein College of Medicine, Bronx, New York.
Mss Jimenez and Jayakar are co-first authors on this article.
The authors have no relevant financial interest in the products or companies described in this article.
Corresponding author: Lizandra Jimenez, MS, Department of Pathology, Albert Einstein College of Medicine, 1301 Morris Park Ave, Price Center Room 207, Bronx, NY 10461 (e-mail: lizandra. firstname.lastname@example.org).
Reprints: Jeffrey E. Segall, PhD, Department of Pathology, Albert Einstein College of Medicine, 1301 Morris Park Avenue, Price Center Room 201 Bronx, NY 10461 (e-mail: email@example.com).
* References 57, 77, 152, 215, 217, 221-224.
Caption: Figure 1. Signaling pathways in head and neck squamous cell carcinoma invasion involving the epidermal growth factor receptor (EGFR). The EGFR, activated by EGFR ligands, can activate extracellular signal-regulated kinase (ERK)/activator protein 1 (AP-1), phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt), or Src activity, which all lead to increased invasion in head and neck cancer. The EGFR can be cross-activated by G-protein coupled receptors (GPCRs), whose downstream signaling leads to the release of EGFR ligands from their transmembrane precursors through activity by the members of a disintegrin and metalloproteinase (ADAM) family of proteases.
Caption: Figure 2. Proteins that contribute to invadopodium structure and function. Invadopodia are actin-rich structures that specialize in mediating the degradation of the extracellular matrix. Their formation can be induced by epidermal growth factor receptor (EGFR) signaling. The key components of the core structure are cortactin, tyrosine kinase substrate with 5 SH3 domains (Tks5), neural Wiskott-Aldrich syndrome protein (N-WASP), actinrelated protein 2/3 complex (Arp2/3), noncatalytic region of tyrosine kinase adaptor protein 1 and 2 (Nck-1,2), and cofilin. Adhesion rings, made up of adhesion proteins, such as integrins and integrin-linked kinases (ILKs), are important for the formation and stabilization of invadopodia. Proteases, such as matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9), are secreted at invadopodia. Membrane type matrix metalloproteinase (MT1-MMP) can be transported by exosomes, which are secreted from multi-vesicular endosomes. Abbreviations: IQGAP, IQ motif containing GTPase activating protein; MVE, multivesicular endosome.
Caption: Figure 3. Invadopodial matrix degradation assay. UMSCC1 cells, a human oral cavity squamous cell carcinoma cell line, were plated onto an Alexa Fluor-405-labeled gelatin matrix (Life Technologies, Norwalk, Connecticut). After 4 hours, the cells were fixed and stained for cortactin and tyrosine kinase substrate with 5 SH3 domains (Tks5), 2 invadopodia markers. Representative images of the degraded fluorescent matrix (A), Tks5 (B), and cortactin (C), and the merged (cortactin, red; Tks5, green; matrix, blue) staining (D). The merged image shows colocalization of Tks5- and cortactinrich structures associated with degradation holes in the Alexa Fluor-405 matrix (original magnification X380 [A through D]).
Table 1. MicroRNAs (miRNAs) That Affect Invasion in Head and Neck Squamous Cell Carcinoma miRNA Effect Target on Invasion hsa-miR-1 Reduction TAGLN2 hsa-miR-107 Reduction PRKCE hsa-miR-29a,b,c Reduction LAMC2, ITGA6, MMP-2, SP1 hsa-miR-99a Reduction IGF1R hsa-miR-126 Reduction EGFL7, VEGF, FGF2 hsa-miR-133a Reduction CAV1, MSN, ARPC5 hsa-miR-138 Reduction RHOC, ROCK2, VIM, ZEB2 hsa-miR-140-5p Reduction ADAM10 hsa-miR-181a Reduction TWIST1 hsa-miR-218 Reduction LAMB3 hsa-miR-363 Reduction PDPN hsa-miR-375 Reduction MTDH hsa-miR-491-5p Reduction GIT1 hsa-miR-639 Reduction FOXC1 hsa-miR-21 Increase DKK2, PDCD4 hsa-miR-134 Increase WWOX hsa-miR-155 Increase SOCS1 hsa-miR-193b Increase NF1 hsa-miR-504 Increase FOXP1 hsa-miR-222 Mixed BBC3, SOD2 miRNA Function hsa-miR-1 Cytoskeleton hsa-miR-107 Signaling hsa-miR-29a,b,c Adhesion, proteolysis, gene expression hsa-miR-99a Signaling hsa-miR-126 Signaling hsa-miR-133a Cytoskeleton hsa-miR-138 Cytoskeleton, EMT hsa-miR-140-5p Proteolysis hsa-miR-181a EMT hsa-miR-218 Adhesion hsa-miR-363 Cytoskeleton hsa-miR-375 Gene expression hsa-miR-491-5p Adhesion hsa-miR-639 Gene expression, EMT hsa-miR-21 Signaling, protein synthesis hsa-miR-134 Cell death hsa-miR-155 Signaling hsa-miR-193b Signaling hsa-miR-504 Gene expression hsa-miR-222 Cell death/ROS miRNA Source, y hsa-miR-1 Nohata et al, (178) 2011 hsa-miR-107 Datta et al, (174) 2012 hsa-miR-29a,b,c Kinoshita et al, (148) 2013; Lu et al, (176) 2014; Jia et al, (188) 2014 hsa-miR-99a Yen et al, (171) 2014 hsa-miR-126 Yang et al, (169) 2014 hsa-miR-133a Kinoshita et al, (179) 2011; Kinoshita et al, (180) 2012; Nohata et al, (302) 2011 hsa-miR-138 Islam et al, (184) 2014; Liu et al, (185) 2011 hsa-miR-140-5p Kai et al, (177) 2014 hsa-miR-181a Liu et al, (187) 2013 hsa-miR-218 Kinoshita et al, (175) 2012 hsa-miR-363 Sun et al, (181) 2013 hsa-miR-375 Hui et al, (189) 2011; Hui et al, (190) 2010; Nohata et al, (191) 2011; Harris et al, (192) 2011; Chen et al, (193) 2012 hsa-miR-491-5p Huang et al, (303) 2014 hsa-miR-639 Lin et al, (186) 2014 hsa-miR-21 Zhou et al, (97) 2014; Kawakita et al, (170) 2013; Reis et al, (195) 2010 hsa-miR-134 Liu et al, (304) 2014 hsa-miR-155 Zhao et al, (173) 2013 hsa-miR-193b Lenarduzzi et al, (172) 2013 hsa-miR-504 Yang et al, (194) 2012 hsa-miR-222 Jiang et al, (196) 2014; Liu et al, (197) 2009 Abbreviations: EMT, epithelial to mesenchymal transition; ROS, reactive oxygen species. Table 2. Ranking of the 25 Genes With the Greatest Number of Mutations or Copy Number Variation (CNV) in Head and Neck Squamous Cell Carcinoma Rank Mutations Gene CNV Mutations 1 CDKN2A -0.77299 66 2 PIK3CA 0.919765 64 3 NOTCH1 0.166341 59 4 EGFR 0.485323 14 5 NOTCH3 -0.04892 14 6 NOTCH2 -0.03327 13 7 FN1 -0.20744 13 8 TLN1 0.009785 12 9 ITGB1 -0.18395 10 10 RAC1 0.373777 9 11 NF1 0.060665 9 12 ZEB2 0.050881 9 13 PIK3CG 0.23092 8 14 HGF 0.203523 8 15 ZEB1 -0.18591 8 16 TLR4 0.242661 7 17 ABL2 0.195695 7 18 IGF1R 0.023483 7 19 COL1A1 0.107632 6 20 ROCK2 0.099804 6 21 COL16A1 -0.03523 6 22 AXL -0.04501 6 23 THBS1 -0.08023 6 24 EGF -0.20548 6 25 PIK3R1 -0.34442 6 Rank CNV Gene CNV Mutations 1 PIK3CA 0.919765 64 2 CLDN1 0.896282 0 3 PTK2 0.827789 4 4 MTDH 0.704501 2 5 PIK3CB 0.696673 5 6 SNAI2 0.614481 1 7 CTTN 0.571429 4 8 EGFR 0.485323 14 9 GDNF 0.401174 1 10 RAC1 0.373777 9 11 SRC 0.362035 1 12 TWIST1 0.358121 1 13 IGF2BP3 0.34638 1 14 SNAI1 0.320939 0 15 MMP-9 0.318982 0 16 PLCG1 0.313112 5 17 EPO 0.293542 2 18 WNT5B 0.25636 1 19 LPAR5 0.25636 0 20 TLR4 0.242661 7 21 ABL1 0.238748 0 22 LPAR1 0.236791 2 23 FOSL1 0.236791 0 24 PIK3CG 0.23092 8 25 VAV2 0.23092 1
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|Author:||Jimenez, Lizandra; Jayakar, Sangeeta K.; Ow, Thomas J.; Segall, Jeffrey E.|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Nov 1, 2015|
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