|Year : 2016 | Volume
| Issue : 4 | Page : 235-241
Cellular and molecular aspects at invasive tumor front in oral squamous cell carcinoma (Part-II)
Rekha Bhaskar Chaudhari
Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Nagpur, Maharashtra, India
|Date of Web Publication||13-Dec-2016|
Rekha Bhaskar Chaudhari
Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Nagpur - 444 003, Maharashtra
Oral squamous cell carcinoma (OSCC) is the most prevalent malignant cancer in the oral cavity. Despite considerable advances in diagnostic and therapeutic field, its prognosis remains poor. Grading by histopathological features does not influence prognosis until deeply invasive margins are evaluated by pathologists. The most invasive part of malignant tumor reflects disturbance in molecular mechanism, alteration in gene expression that controls cell differentiation, growth, death (apoptosis) as well as interactions between cancer cells and stromal components that may lead to the formation of oncofetal extracellular matrix, enabling tumor cell phenotype transition and migration. In recent years, analysis of cell proliferation, DNA content, and epithelial-mesenchymal transition (EMT) phenomenon has been used in an attempt to identify new prognostic indicators. Great interest is also shown in matrix metalloproteinase which plays a key role in invasion and metastasis. This review focuses on altered pathways in OSCC at gene expression and protein level, role of proteinases and EMT phenomenon in tumor progression, invasion, and metastasis with respect to invasive tumor front.
Keywords: Genetic markers, matrix metalloproteinases, molecular factors, oral squamous cell carcinoma, tumor budding and epithelial-mesenchymal transition
|How to cite this article:|
Chaudhari RB. Cellular and molecular aspects at invasive tumor front in oral squamous cell carcinoma (Part-II). SRM J Res Dent Sci 2016;7:235-41
|How to cite this URL:|
Chaudhari RB. Cellular and molecular aspects at invasive tumor front in oral squamous cell carcinoma (Part-II). SRM J Res Dent Sci [serial online] 2016 [cited 2019 Aug 18];7:235-41. Available from: http://www.srmjrds.in/text.asp?2016/7/4/235/195635
| Introduction|| |
Oral squamous cell carcinoma (OSCC) is one of the most common malignancies. Despite diagnostic and therapeutic progress in oncology, during the past decade, it has fairly onerous prognosis. Its morphological and functional characteristics indicate the biological aggressiveness, reflected by alteration and mutation that alter cell-matrix interaction and cell signal pathways.  Progress of OSCC primarily depends on clinicopathological factors that however has shown limited predictive value. Prognostic information is essential for evaluation and optimal treatment of patients.
Oral carcinogenesis is a multistep process which involves precancerous lesions, invasion and metastasis. Degradation of cell cycle, proliferation of malignant cells results in loss of control mechanism that ensures normal function of tissue. Transformed neoplastic cells determine biologic behavior of the tumor.  The activated tissue microenvironment at the advancing tumor front seems to provide appropriate stimuli for triggering molecular alterations. Invasive tumor front (ITF) is considered as a "biologic horizon" of the tumor with high prognostic significance. It also represents the area where cancer cells are bathed in the host milieu of oxygen and other nutrients from angiogenesis, enabling them to grow actively.  Tumor cells have the property to invade and spread through surrounding tissues. Cellular and molecular mechanism that illustrates this complex biology includes - overexpression of extracellular matrix (ECM) degrading enzymes, cytoskeleton protein interplay, abnormal DNA contents, proliferation, and epithelial-mesenchymal transition (EMT).
The aim of the present article is to review the current knowledge on prognostic value of cellular and genetic/molecular expressions at ITF. Published scientific data were collected and scanned. Articles pertaining to studies undertaken to find genetic alterations, abnormal DNA contents, overexpression of ECM-degrading enzymes, cytoskeleton protein interplay, angiogenesis, and cell motility/migration (EMT and tumor budding) with respect to ITF were identified and selected.
| Matrix Metalloproteinases|| |
Invasion and metastasis of cancer cells are a complex multistep processes involving cooperation of multiple proteolytic enzymes, secreted by tumor or host cells.  Matrix metalloproteinases (MMPs) are the most important ECM-degrading enzymes. Activation of MMP cascade is accompanied by altered expression of many adhesion molecules by transformed cells. Trafficking of neoplastic cells through ECM is necessary for invasive activity.  Surrounding tumor stroma is an integral player in this process. Tumor cells are capable of utilizing MMPs, produced by stromal cells, indicating the active role of stroma in tumor invasion.  Because ECM shows complex structure combined action of several MMPs is crucial for degradation of ECM components which acts as a barrier.  Tumor cells frequently overexpress MMP, and this has been shown to be correlated with invasive capacity, metastatic potential, and poor prognosis. Marked expression of MMP-1 (collagenase), MMP-2 (gelatinase), MMP-8, and MMP-9 at ITF has been demonstrated to be related with cell motility and recurrence and poor survival, ,,,, whereas MT1-MMP (MMP-14) expression was shown to be inversely associated with MMP-2 and MMP-9, suggesting a sequential action of these proteases in different moments of invasive process.  In addition, localization of MMP-7 (matrilysin), exclusively observed in cancer cells, at invasive front has been shown. MMP-7 mediated Fas-L cleavage provides a mechanism to evade apoptosis and thereby facilitating cell survival and progression.  Thus, MMPs are key mediators of cancer invasion.  Expression of MMP-2 and -9 (gelatinases) has not been shown to be correlated with any clinicopathological or prognostic variables by Mäkinen et al.  On the contrary, Kurahara et al.  showed that expressions of MMP-2 and -9 correlated significantly with nodal status in OSCC. A correlation of nuclear immunoexpression of MMP-13 with invasive depth, tumor size, and disease-specific survival in oral tongue squamous cell carcinoma (OTSCC) has been detected by Mäkinen et al.  They suggested that MMP-13 may be regarded as a prognostic biomarker in OTSCC. MMP-7 combined with other markers may be used to predict the metastatic potential of OSCC,  whereas MMP-2 is a marker of poor survival. 
| Tissue Inhibitor of Metalloproteinases|| |
Activity of MMPs is regulated by tissue inhibitor of metalloproteinases (TIMPs). It seems that concerted action of MMPs and TIMPS is involved in ECM remodeling in OSCC.  Overproduction of MMP appears to be important for metastasis than production of TIMPs in vivo.  However, many authors have demonstrated high levels of TIMPs, correlating with local invasion and metastasis. ,,, Overexpression of TIMPs is characteristic in head and neck squamous cell carcinoma (HNSCC). O-Charoenrat et al.  explained elevated expressions of TIMP-1 in HNSCC by the growth promoting activity of TIMPs on a variety of cell types or the induction of TIMPs by secreted MMPs, from tumor-host interaction in the extracellular milieu. Among the best known and widely studied are TIMP-1, -2, -3 and have long been associated with metastasis. ,,, A significant association of MT1-MMP with TIMP2 has been shown by de Vicente et al.  and Katayama et al.  Role of these factors for activation of pro-MMP-2 is explained by the mechanism of cell-mediated MMP-2 activation in which pro-MMP-2 binds to TIMP-2 in combination with MT1-MMP on cell surface forming a tertiary complex. The pro-MMP-2 in this complex is activated by adjacent MT1-MMP and activated MMP-2 degrades ECM components that correlate well with OSCC progression.  Singh et al.  showed correlation between MMP-2, TIMP-1, and TIMP-2 levels with lymph node involvement, stage, and differentiation. In addition, Ondruschka et al.  detected marked TIMP-2 expression at invasive front. Further Kurahara et al.  reported that marked TIMP-2 expression might be considered as the most valuable indicator in identifying patient's risk for poor prognosis. However, reports on in vivo involvement of MMPs and TIMPs in oral cancer are still contradictory. Evidence suggests that MMPs and their physiological tissue inhibitor TIMPs play a causal role in oral cancer progression. 
| Cytogenetic Changes|| |
Biologic behavior of oral cancer is considered to reflect genetic alterations in cancer cells. These alterations include reduced expression or overexpression of proteins. Genetic instability is the intrinsic characteristic of malignant tumor. The genes that are altered include p53, cyclin D1 (CCD1), epithelial growth factor receptor (EGFR), signal transduction, vascular endothelial growth factor (VEGF) as well as other molecules. These alterations affect cell cycle regulation, cellular differentiation, proliferation, death, DNA repair, and cellular immunity.  Among these, p53 is the most notable. ,, Inactivation of tumor suppressor gene (TSG) can occur via epigenetic or genetic mechanism.  The interactions between activation of oncogenes and loss of function of TSGs drive uncontrolled growth and invasion. Tumorigenic alterations involve activation of oncogene (genetic instability due to impairment of DNA repair) and inactivation of TSGs, exemplified by the loss of heterozygosity (LOH), deletion, and epigenetically through DNA methylation or chromatin remodeling. 
Kato et al.  and Kato et al.  measured p53 labeling index (LI) at ITF in OSCC and reported a significant correlation of overexpression of p53 LI with clinical outcome. High p53 LI at ITF was associated with worse prognosis. However, Piffkò et al.  could not demonstrate biological significance of p53 alteration at ITF. In OSCC, p53 is mutated, resulting in loss of p53 control of cell cycle progression and apoptosis. Thus, aberrant cellular expression of p53 is a good prognostic biomarker of stage and malignant vigor of cancer and predictor of tumorigenesis. Verma et al.  demonstrated a linear increase in p53 expression at ITF through varying grades in OSCC, suggesting it to be independent of histological grade.
p21 protein (WAF1/CIP1) is a cyclin-dependent kinase inhibitor which is one target of the transcriptionally active p53 and mediates cell cycle arrest after DNA damage. Horta et al.  observed overexpressions of p53 and p21 WAF1/CIP1 at the invasive front of lip SCC with no correlation between their expressions at ITF or at whole tumor and reinforced that ITF is the most important area for tumor behavior and further supports that p53-independent mechanisms are involved in the expression of p21 (WAF1/CIP1). Further, overexpression of p53 as well as decreased intensity of p63 immunostaining has been shown to be associated with metastases and correlated with poor outcome. 
Noguchi et al.  examined prognostic significance of cancer cells with abnormal DNA content at ITF in OSCC and observed that high DNA content of tumor cells implies bad prognosis for clinical outcome or survival and considered it as powerful independent prognostic factor, influencing survival. LOH and microsatellite instability (MSI) have been documented as important events in OSCC and were studied by Wang et al.  in invasive front/center, superficial part, and stromal cells. Statistically high frequency of LOH and MSI at chromosomal loci TP53 and RP56 at invasive front than at other region was observed. It has been postulated that relationship between histological grading and genotypes of OSCC exists. Chromosomal aberrations such as deletion, amplification, and structural rearrangement are hallmark of malignancy. There is allelic loss/imbalance during progression from premalignant to malignant phenotype. Yamamoto et al.  demonstrated that allelic loss in 2q, 3p, and 21q region was associated with OSCC progression and correlated it with worse prognosis, particularly regarding 2q. Partridge et al.  observed a correlation between AI/LOH and high recurrence rate in OSCC patients and stated that it could be a better prognostic factor than tumor-node-metastasis (TNM) staging system.
| Cyclin D1|| |
It is a key regulator of cell proliferation and exerts its function in late G1 phase. It is frequently expressed in OSCC.  A positive CCD1 expression has been shown in high-grade OSCC by Lam et al. CCD1 expression appears to have a predictive value for the prognosis of the patients with lower TNM stage oral carcinoma. 
| Epithelial Growth Factor Receptor|| |
EGFR is a transmembrane glycoprotein and tyrosine kinase receptor, expressed in HNSCC. Mutation can result in abnormal activation of EGFR that promotes cell proliferation and inhibits apoptosis.  A statistically significant correlation between increased EGFR and tumor front score, and with advanced lymph node involvement in OSCC has been shown by Bánkfalvi et al.  and Gröbe et al.,  respectively. This stresses the importance of ITF in the molecular cross-talk between tumor and host. This molecular change is accompanied by invasive growth of the tumor and poor survival of the patients. Increased expression of EGFR has been linked to advanced OSCC and to poor prognosis and suggested as supplementary tool for prognostic assessment and predicting metastasis  and locoregional control in OSCC patients. 
| Proliferative Activity of Cancer Cells at Invasive Tumor Front|| |
OSCC is a disease resulting from genetic damage leading to uncontrolled cell proliferation of damaged cells.  Cell proliferations are defined as increase in a number of cells resulting from completion of cell cycle.  Tumor cell proliferative activity is believed to indicate the degree of aggressiveness of the tumor.  Control of this important biologic process is believed to be lost in cancer. ITF might have putative biological significance because of accumulation of abnormal cells.  The two most common IHC markers used to study cell proliferation are - Ki-67 and proliferative cell nuclear antigen (PCNA).
| KI-67|| |
It is a nonhistone nuclear protein. It is expressed in all active phases of cell cycle (G1, S, G2, and mitosis), but it is absent from resting cells (G0) and reaches its peak in G2 and M phases.  Various studies have shown positive correlation between Ki-67 expression at ITF and OSCC grading. ,,, A significant positive relationship between cell proliferation (using Ki-67 LI) at ITF and number of prognostic (clinical stage and tumor thickness) and risk factors (smoking, alcohol consumption) in human OSCC has been shown by Tumuluri et al.  Whereas a strong relationship of Ki-67 LI with histological grading in OSCC was reported by other investigators. ,, Further p53 expression was shown to be significantly correlated with that of Ki-67 in OSCC, suggesting that altered p53 overexpression may promote increased cell proliferation.  High Ki-67 labeling values may indicate biologic malignancy with high proliferative activity.  Kurokawa et al.  further suggested that high values of Ki-67 and p53 positive status may be related in turn to the progression and metastatic spread in OSCC. All these results emphasize the potential of Ki-67 as prognostic marker for OSCC. 
| Proliferative Cell Nuclear Antigen|| |
It is the most representative proliferative marker that plays an important role in DNA replication, chromatin remodeling, and DNA repair. Alteration in PCNA activity is common genetic event in various cancers including OSCC. Kato et al.  recorded high expression of PCNA at ITF and correlated it with the abnormal proliferative activity. It can be used as an independent predictor of histologic grade, recurrence rate, and prognosis. 
| Apoptosis Regulated Proteins|| |
Kato et al.  evaluated prognostic value of interrelation of opposing apoptotic proteins ( Bcl-2 and Bax) expression at invasive front of OSCC and concluded that Bax expression was found to be the strongest independent prognostic parameter. The Bax (Pro-apoptotic protein) binds to antiapoptotic Bcl-2, leading to inactivation of anti apoptotic function of Bcl-2. Patients with negative Bcl-2 expression and + Bax expression had a significant better prognosis. Results of their study suggested that Bax is a better prognostic marker than Bcl-2 in OSCC.
| Phosphatase and Tensin Homolog|| |
Phosphatase and tensin homolog (PTEN) is a TSG that regulates cell physiology such as division, differentiation, apoptosis, migration, and adhesion. Immunohistochemical study of aggressive OSCC with high score malignancy has not demonstrated PTEN expression at invasive edge. At ITF − ve immunoreactivity for PTEN suggested relation of PTEN to histologic pattern and biological behavior. Inactivation or absence of PTEN results in increased accumulation of phosphatidylinositol triphosphate which increases phosphatidylinositol-3 kinase (PI3K) activity, contributing to cell survival and antiapoptotic stimuli. 
| Hepatocyte Growth Factor|| |
Hepatocyte growth factor (HGF), also known a scatter factor, is a pleiotropic growth factor with many diverse biological effects such as cell proliferation, migration, tumor angiogenesis, invasion, and metastasis. These are mediated via interaction with a transmembrane tyrosine kinase receptor - c-met protein. A significant correlation of high expression of HGF LI and c-met LI in ITF with clinical parameters (large tumor size and regional lymph node metastasis) has been shown by Chen et al.  HGF can activate extracellular signal-related kinases (ERK), the PI3K/AKT or nuclear factor-κβ signaling pathway, thus inhibiting apoptosis and thereby inducing tumor growth and promote cancer metastasis, through production of MMPs. Thus, HGF LI in ITF and c-met can be a good biomarker for prognosis of OSCC. 
| Vascular Endothelial Growth Factor|| |
Angiogenesis and lymphangiogenesis are well known to be essential for tumor growth and metastasis. VEGF secreted by cancer cells and stromal cells proved to play a major role in tumor angiogenesis.  VEGF family members include -A, -B, -C, and -D. Among these, VEGF-A promotes angiogenesis, whereas VEGF-C is a potent inducer of lymphangiogenesis, leading to tumor cell dissemination.  Overexpression of VEGF-A and -C has been shown to be related to lymph node metastasis by Yanase et al.  In contrast, downregulation of VEGF-C in deep area near invasive front has been shown by Ohno et al.,  indicating poor lymphangiogenesis. VEGF-C may be considered as predictor factor of OSCC outcome. 
| Prognostic Value of Signaling Molecules - Phosphorylated Extracellular Signal-Regulated Kinases (P-Extracellular Signal Related Kinase1/2) and Cyclo-Oxygenase-2|| |
Interaction between tumor cells and neighboring stromal components at ITF is of crucial importance for tumor behavior. Søland et al.  investigated prognostic value of P-ERK1/2 and cyclooxygenase (COX)-2 at ITF. Patients with tumor showing P-ERK1/2 staining and upregulation or absence of COX-2 reported to be unfavorable for clinical outcome and poor prognosis. Activation of intracellular signaling cascade involving Ras, ERK1/2 can promote cell proliferation, invasion, and prognosis in HNSCC. Overexpression of COX-2 and resultant synthesis of prostaglandins such as PGE2 can affect several processes such as angiogenesis, invasion, and proliferation during tumorigenesis. Because of diverse expressions, COX-2 was not considered of prognostic significance at ITF or in other tumor area. These molecules are of established importance in cell proliferation and tumor development. 
| Basic Fibroblast Growth Factor|| |
Basic fibroblast growth factor (bFGF) is a cytokine, closely associated with activated fibroblasts and mediates the interaction between cancer cells and host tissue stroma. Increased prevalence of bFGF expression, FGF receptor-1 expression at invasive front has been demonstrated to be linked to mode of invasion and prognosis in OSCC. bFGF promotes production of cancer cells proteinases and enhances their invasive ability. It is reported that bFGF exerts influences upon fibroblasts to produce HGF, transforming growth factor (TGF)-β, and MMP-2 which are associated with tumor invasion. Furthermore, bFGF also initiates angiogenesis actively by interacting with various endothelial cell receptors. Proliferative activity of tumor has been correlated with the expression of bFGF in OSCC. 
| Aldehyde Dehydrogenase-1|| |
Aldehyde dehydrogenase (ALDH)-1 is considered as putative marker of highly tumorigenic cells (i.e., cancer stem-like cells) in HNSCC. Hildebrand et al.  evaluated pattern of ALDH expression in the center and ITF region of the tumor. ALDH-1 immunostaining indicated more aggressive behavior and was positively related with clinical parameter (tumor size and regional metastasis) and histopathological grading and tumor progression in HNSCC. ALDH-1 positive neoplastic cells have proliferative ability similar to that observed in stem cells. The lack of similar association in tumor cells may be due to cell hierarchy with less aggressive biological behavior. 
| Transforming growth factor-β|| |
TGF-β1 is thought to be involved in cell attachment to ECM; thereby influencing invasion and lymph node metastasis of cancer cells. TGF-β1 transforms OSCC cell lines into mesenchymal phenotype. It has been demonstrated that TGF-β signaling induces EMT by forming activated transcription complexes that directly inhibits E-cad gene activity. Subsequently, loss of E-cad and cell-cell adhesion allows oral cancer cells to become motile and invasive. It is also considered as potent inducer of oral cancer progression. 
| Tumor Budding and Epithelial-Mesenchymal Transition|| |
Recently, a number of unique features of cancer cells located at ITF have been recognized including tumor budding and EMT that are associated with increased cell motility and invasiveness. Tumor budding is a morphological marker of tumor invasion  and is defined as the presence of either isolated single cell or small cluster (up to 4) scattered in stroma at ITF, and high intensity tumor budding has been demonstrated as adverse prognostic factor in patients with tongue SCC.  Budding represents two malignant features - discohesion and active invasion.  Association of Ln-5 expression with high intensity of tumor budding and with high density of stromal MF has been shown to suggests permissive environment for invasion in OSCC.  Wang et al.  observed reduction in E-cadherin in ITF and budding cells which is accompanied by increase in vimentin expression. Microscopically, tumor cells in ITF and tumor buds exhibit distinct switch in morphological features including dedifferentiation and loss of cell-cell adhesion. This fibroblast-like morphological appearance is characteristic of the cell undergoing EMT. 
EMT is defined as a process during which epithelial cells downregulate specific adhesive proteins (e.g., E-cadherins), induce mesenchymal proteins (e.g., vimentin), and acquire migrating properties.  Differential expression of E-cadherin/β catenin  and demonstration of vimentin-positive cells at ITF, expressing Laminin-5/γ2chain mRNA,  implicated EMT origin  for cells in ITF. The PI3K/AKT pathway is directly linked to EMT-like phenotype in human OSCC cell lines. AKT activation induces the decrease of proteins-E-cadherin/β-catenin and triggers the expression of mesenchymal marker such as vimentin. It also changes cellular morphology, leads to loss of cellular polarization and decrease in cell-ECM adhesion and production of MMPs. Thus, EMT causes an increase in cell motility, invasion and is associated with more aggressive behavior of cancer cells.  EMT is considered to be crucial for the development of an invasive and metastatic phenotype potential. However, carcinoma cell phenotype conversion at OSCC invasive front is poorly understood.  Furthermore, Yang et al.  demonstrated EMT positive immunoreactions with tumor satellite formation, located at ITF, indicating a link between EMT and tumor invasive front and suggesting a cross-talk between tumor cells and tumor microenvironment. Thus, EMT may be considered a new signal from invasive front.
| Conclusion and Perspectives|| |
Several cellular and molecular characteristic at ITF have an impact on progress of OSCC and may be used as valuable tool for predicting OSCC patient outcome. Genetic expression profile may offer greater accuracy in diagnosis and prognosis. MMPs are important group of proteolytic enzymes used by cancer cells to degrade ECM. Molecular factors modulate cell behavior and enhance progression by regulating angiogenesis, migration, proliferation, and invasion. Advances in cancer research have provided new knowledge about cellular/molecular mechanism in the complex biology of tumor progression and invasion. Several molecular factors have been strongly correlated with outcome showing promising pathways for future development of more effective prognostic system and anticancer treatment therapies. Diagnostic and prognostic value of genetic and other molecular factors has been emphasized. Understanding of cellular and molecular events at ITF is of paramount importance with respect to improving prognosis.
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Conflicts of interest
There are no conflicts of interest.
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