BIOMOLECULES OF METASTASIS

❖ Cellular & Tissue changes of Metastasis: Occur in 5 steps

Mediated by biomolecules
❖ Pattern of Invasion: 3 types- EMT,
Partial EMT,
CCM
❖ Depth of Invasion: Endophytic ,
Exophytic,
Ulcerative
❖ Clinical criteria of diagnosis of Metastasis : TNM Classification,
Imaging techniques
SLN Concept
❖ Histopathological Investigations
❖ Gentic changes
❖ Molecular changes
❖ Small RNA changes
❖ Additional biomolecules of metastasis
 Cellular & Tissue changes of Metastasis

• Metastasis is one of the main causes of oral cancer patients’ death.

• The process of oral cancer metastasis is a series of sequential and interdependent events involving :
a) detachment of cells from tumor tissue,
b) increased cell motility and local invasion,
c) angiogenesis,
d) intravasation of invading cells into the vasculature or lymphatic systems
e) ,extravasation & cross-talk with components of the new microenvironment, including parenchymal ,
stromal and inflammatory cells followed by
f) deposition & proliferation at a second site.
• It is clear that only a minority of malignant cells undertake the metastatic route, due to an interplay
between host factors and intrinsic characteristics of cancer cells. Thus metastasis may represent an
escape of these cells from the hostile environment they themselves created, such as shortage of
oxygen and nutrients, inflammation and immune system attacks.
 • This process occurs due to –
1. loss of E-cadherin leading to reduced intercellular adhesion &
2. loss of cell polarity of tumor cells leading to morphological transition,
called epithelialmesenchymal transition (EMT) as, the tissue cells progress
to malignancy because of molecular alterations infering to the change of
behavior of these cells along with Basement Membrane
alterations.(Step1)
• The major determinant of the prognosis of oral carcinoma is the risk of cervical
metastasis , particularly, in the absence of clinically detectable neck disease.
• Other main hallmarks of cancer progression are the “pattern of invasion” &
“depth of invasion” which are an important indicator of patient's prognosis.
 Reduced Homophilic Cell-Cell Adhesions(Step1A)
• Step-1 is as a direct consequences of
a net increase in glucose consumption , due to abnormal lactic acid release and a more
acidic extracellular pH (pHe) .
• Hypoxia, low pHe, and glucose utilization are established features of many solid tumors
and concur to EMT and cancer dissemination.
• Thus, for treatment purpose,interfering with the glycolytic pathway could impair EMT
and subsequent tumor progression and could be a potential anticancer strategy.
• Several components of the molecular pathways driving EMT have a clear impact on cell
metabolism and vice versa, resulting in a metabolic rewiring which sustains the EMT
transcriptional program.
• So, EMT is regulated by
1. ECM components,
2. hypoxic conditions,
3. exosomes
4. soluble factors such as hepatocyte growth factor (HGF), fibroblast
growth factor (FGF) and members of transforming growth factor-βs
(TGF-βs)
EMT is accompanied by the modulation of well-known markers, like, the loss of epithelial
marker E-cadherin, induced by the upregulation of its transcriptional repressors (i.e.,
Snail1/2, Twist, ZEB1/2), as a priming event &
The concomitant acquisition of mesenchymal markers that sustains and stabilizes the
newly acquired phenotype.
FUNCTIONALSIGNIFICANCE OF EMT
• EMT is the coordinated de-stabilisation of cell–cell contacts and
acquisition of a more migratory and invasive mesenchymal
phenotype, together with respective changes in gene expression
patterns, and is regarded as a potentially important event in the
metastatic spread of tumour cells 
• It is a process to acquire migration and invasion abilities, alongwith
a more complex rewiring of signaling, metabolic, genetic, and
epigenetic networks that allow differentiated epithelial cancer cells
to revert into a more undifferentiated state and stem cell functions
in order to gain a plethora of strategies in order to survive in
different environmental conditions and/or in presence of anticancer
drugs.
• This plasticity reinforces the conclusion that “it is not the fittest of
the species (in this case of the tumor cell bulk) that survives but the
most adaptable.”
• The challenges we now face are (i) the identification of
distinct metabolic hallmarks exclusive to
cancer-associated EMT and (ii) the identification of
therapeutic window that allows EMT targeting in cancer
patients.
 Partial EMT
• When cancer cells express epithelial and mesenchymal markers
(epithelial/mesenchymal (E/M) phenotype) concurrently, it is what is
termed as partial EMT .
• Benefits of Partial EMT
i. CTCs that survive in blood exhibit E/M phenotypes, become resistant
to anoikis, and exit the bloodstream more efficiently.
ii. Effective metastasis is caused by such cancer cells without complete
loss of epithelial morphology and complete acquisition of
mesenchymal morphology.
iii. The partial EMT cancer cells with E/M phenotypes can undergo
collective cell migration through their remaining epithelial character
and enhance attachment to the ECM by achieving mesenchymal
character.
• As a result of the ability of cancer cells to undergo partial EMT (E/M
phenotype), rather than complete EMT,their presence poses a higher
metastatic risk.
TUMOR BUDDING USUALLY SHOWS PARTIAL
EMT
 E-Cadherin(Glycoprotein of Step1A)
• E-cad is a single-pass transmembrane glycoprotein & is a key
molecule that plays a critical role in cell–cell adhesion either as a
monomer or as cadherin ‘clusters’ via homotypic
calcium-dependent interactions & its association with cytoplasmic
proteins, termed “catenin” (α-, β-, and γ-catenins) that link E-cad to
the actin cytoskeleton .So, E-cad , Catenin & actin cytoskeleton
together maintain dynamic Adherens cell junctions with help of
other modulators . For cleavage of E-CADs to occur:
• TGF-β activates FGFreceptors & makes cells more sensitive to
EGF-2 leading to formation of carcinoma activated fibroblasts(CAF)
• CAFs then -
a) secrete MMP2 & MMP9 which cleave E-cads,
b) aid in release of oxygen reactive species via certain pathwys
c) enhances EMT of cancer cells so, making epi.cells ANOIKIS
resistent
d) CAFs also secrete paracrine signaling factors including IL-6, IL-8,
NFκB, IFN-γ, HGF, CTGF, CCL5, and PGE2,
e) CAFs secrete ECM molecules such as collagenases, MMPs, tenascin
C, and periostin
• *ANOIKIS: Programmed cell death of anchorage dependent cells on detachment
 E-Cadherin
• A low level of E-cadherin thus,
1. facilitates the crucial process of Epithelial-mesenchymal transition
(EMT)that is responsible for poor prognosis &
2. lead to the phenotypic changes of increased motility and invasiveness of a
cancer cell.
• Loss of E-cadherin, claudins and ZO-1, and a subsequent increase in
mesenchymal markers such as N-cadherin, vimentin and fibronectin and
cytoskeletal reorganization are the hallmark of EMT.
• During EMT cancer cells lose their cell-to-cell attachment by decreasing
E-cadherin expression, due to hypermethylation of the promoter region or
transcriptional repression caused by Zinc finger E-box-binding homeobox 1
(ZEB1), ZEB2, Snail, Slug (also known as SNAI2), and TWIST.
• In EMT-caused cells, hypermethylation of the promoter region occurs due
to the downregulation of miR-200 family that induces expression of
ZEB1/ZEB2, resulting in E-cadherin suppression.
• A recent paper showed that cells expressing the partial EMT program,
spatially localized to the leading edge of primary tumors in head and neck
squamous cell carcinoma .This is important because, partial EMT is an
independent & distinct predictor of nodal metastasis in head and neck
squamous cell carcinoma.
• The levels of soluble E-cadherin in the circulation reflect the progression of
cancer and can be used as a diagnostic marker .
illustration of the tumor microenvironment showing representative
cell types, tissues, and signaling factors involved
 Schematic representation of E-cadherin-mediated cell–cell
adherens junctions and cell–ECM integrin-mediated adhesions.
 Collective Cell Migration
Research has shown, that tumour cells can invade
even with fully intact and functional cell–cell
adhesions as, collective groups of cells, and that a
loosening of cell–cell contacts is sufficient to permit
this collective migration and invasion.
• This requires coordination of cues from the
surrounding tumour environment, to regulate both
cell–cell and cell–ECM interactions.
• It also involves interplay between E-cadherin and
cell–ECM interactions that are mediated by integrin
matrix receptors.
 Step 1B

• Carcinoma cells attach to Basement membrane via

fibronectin , laminin ,Integrin , Type-IV collagen etc.
 Integrins
• Integrins are cell membrane adhesion molecules, which can
interact with ECM and have role in cell proliferation, growth,
differentiation, and migration.
• Integrins are composed of α and β chains.
• Most of them are down-regulated in OSCC but those
involved in migratory cell phenotype are retained &/or
upregulated eg.αv-family which besides giving a more
malignant phenotype also regulates survival of newly formed
bloodvessels & increases expression of MMP2 .
• In OSCC expression of α6β4 is retained allowing epitheloid
colony formation & metastasis to regional lymph
nodes.Thus,it can be used as an indicator of propensity of
OSCC to metastasize.
• Additionally, metastatic cell colonies show increased
expression of α2, α3, α4 & α6 but not the primary tumors
 adhesions. Integrin engagement triggers several
signalling cascades including those that are mediated
by ILK, FAK and Src and Rho GTPases, such as
Rac1, RhoA and CDC42. These signaling pathways
are not independent but linked.

The net input from these signalling pathways can,

therefore, shift the balance between stabilization or
remodelling of AJs, which ultimately governs the
migratory capacity of epithelial cells.

• Integrins transmit signals from the outside to the inside

of the cell through the assembly of multi-protein
complexes that link integrins to the actin cytoskeleton.
• These are comprised of structural, adaptor and signalling
proteins, and this complex network of protein
interactions has been termed the adhesome, in which
180 protein–protein interactions have been identified to
date.
 INTEGRIN
• Integrin engagement leads to rapid changes in lipid
kinase activity and the activation of the focal
adhesion-linked protein tyrosine kinases ,Src and FAK
causing adhesion changes at the cell membrane &
promoting dynamic actin asssociated migratory
activities.
• The crosstalk between epithelial cell–cell adhesion and
cell–matrix adhesion signalling, and the dynamic
interplay between the two, contribute to the plasticity
within tumour cells that allows them to respond to
external cues, which in turn drives effective migration
and invasion.
 Crosstalk by E-cadherin
• The crosstalk between E-cadherin-mediated cell–cell
adhesions and integrin-mediated cell–matrix contacts is
regulated by signalling mechanisms that are still being
uncovered.
• Recent findings point towards reactive oxygen species as
emerging candidates for modulating the crosstalk between
integrin adhesions and AJs, as well as the Hippo signalling
pathway .
• It is important to more fully understand how the molecular
events downstream of adhesion dynamics and crosstalk
orchestrate tumour progression and survival, as well as the
response to therapy, so that new therapeutic strategies can
be devised to target metastatic disease.
 Collective Cell Migration
• To migrate in group, the tumor epithelial cells need
to retain and remodel their cell–cell junctions but it
involves reorganization of the actin cytoskeleton
(AC) and concomitant formation of F-actin-rich
membrane protrusions.
• This essentiates,coordination of cues from the
surrounding tumor environment & involves interplay
between E-cadherin and cell–ECM interactions
mediated by Integrins.
• The F-actin protrusion consists of noninvasive and
invasive protrusions.
• These protrusions at the leading edge of mobile cells
are in the form of lamellipodia, filopodia,
podosomes, or invadopodia.
 Invadopodia
• Invadopodia contributes to the formation of an actin
filament-rich core and a multimeric protein complex
enclosing the actin core with protein complex
consisting of integrin-associated proteins (vinculin,
talin, and paxillin).
• They express matrix metalloproteinases (MMPs) for
ECM degradation.
Invadopodia formation and function involve
a) initiation caused by growth factors,
b) assembly, and
c) maturation.
 Motility of CCM
• In addition to protrusions at the leading edge of mobile
cells,some kind of biomechanical dynamics occurs within the
sheet of collective cells which involves:
• the organization and structural change of the substrate, either
in the form of gel or fibrillar.
• This has an impact on the mobility of cells.
• Collective tumor cells migrate from one location to another in
search of oxygen, nutrients, and space.
• Each,CCM is different in cells per unit and route of metastasis
as compared with single cell movement.
• N-cadherin is thought to regulate the CCM in cancer cells. Its
extracellular domain interacts with the FGF and intracellular
domain acts independently to promote the promigratory role
of cancer cells by regulating AC.(Actin cytoskeleton)
 Collective Cell Migration/Invasion
• CCM (Collective Cell Migration)exhibits two patterns which
were demonstrated by Friedl et al.
• In the first pattern, the invading cells maintain a contact with
its primary site. This type of pattern is more common in
OSCC and basal cell carcinoma.
• In another pattern, “cell files” detached from their parent site
and migrate along the path of least resistance.Cells move in
the chain where the cell contacts with each other by “tip-like”
junction. The single cell then “lines up” in a stromal fiber,
forming “Indian file” appearance. This pattern dictates high
metastatic potential and poor prognosis and mostly seen in
melanomas.
• Podoplanin (Pod) has been suggested to play an essential
role in CCM and its expression in the invasive front of
tumors, may be associated with this type of invasion
 Benefits of CCM over EMT in defining Pattern of Invasion
• In CCM, higher levels of promigratory elements and matrix
proteases exist at the invasive front, because they are secreted by
multiple cells that form the groups or sheets, characteristic of CCM.
• Important FACTS about CCM
1) The central cells in the collective group are protected against the
destructive effects of the host immune response by the outer cells.
2) Since the migrating cells constituting the functional units are
heterogeneous, we basically have a group of cells with various
biological capabilities and different clonal origins which can work
together to induce the diverse set of characteristics required for
invasion.
3) A group of cells which have metastasized to the vasculature can
resist elimination better than single cells and therefore produce
metastasis with less difficulty.
4) Tumor stromas that are more organized have been seen to support
collective migration.
5) Tissues with higher muscle fiber content and thick collagen bundles
tend to promote CCI, while fatty tissues and those with
less-organized short collagen fibers are in favor of single-cell
migration.
6) CCM facilitates greater plasticity & hence better invasion ability.
Single cancer cell migration, also known as mesenchymal migration, is characterized by fibroblast-like morphology
(upper panel).
Collective cancer cell migration is characterized by multicellular clusters analogous to a bunch of grapes, and
includes fibroblast-led collective cancer cell migration/invasion, promotion of invasion by non-EMT cancer cells
through crosstalk between EMT and non-EMT cancer cells , and putative EMT cell-mediated collective cancer cell
migration
 Plasticity of cancer cell
invasion. Tumour cell invasion can
occur as single cells or as collective
groups of cells moving together.
Cancer cells display plasticity in the
chosen mode of migration, and this is
dependent on diverse signalling inputs
from the surrounding tumour
microenvironment that control the
interplay between adhesive structures
Both types of invasion are dependent
on integrin-mediated adhesion to the
ECM, whereas collective invasion also
requires the generation and
maintenance of dynamic cell–cell
adhesions, with loosening of cell
junctions being sufficient to permit
movement

• Exclusively in head and neck squamous cell carcinoma, transforming growth

factor-beta 1 (TGF-b1) stimulates CAF(carcinoma associated fibroblasts) to release
hepatocyte growth factor (HGF) which later on results in the invasiveness of tumor
cells by regulating distribution & expression of CLAUDIN in cell-specific manner
probably due to promoter hypermethylation .
• Recently, recognized molecules are Src family kinase and p130 Crk-associated
substrate.
Molecules responsible for collective cell
migration
 Therapeutic approach for CCM
• It will be beneficial if the therapeutic implications will be targeted
towards inhibiting the motility of tumor cells.
• Various approaches could be implicated in arresting the distant
metastasis,like :
• First, suppression of action of the molecules which are responsible for
modulating AC. Disorganization of AC does not allow the cell to gain
its shape for motility which is called as an antimotility approach.
• Second, one can stop forming “tip guiding” cells which, will help in
stopping the navigation of the tumor island to the distant site. Thus,
targeting specific components of ECM provides achievable cessation
which results in metastasis and invasion.
• Another therapeutic approach is focusing on cessation of cell–cell
interaction or by stopping the formation of swarms of cells.
• Also,Genes responsible for signal transduction can be ablated. One
of such novel drugs which is responsible for Src gene protein inhibitor
is Saracatinib.
 Schematic illustrations of EMT regulated by
transcriptional and post-transcriptional mechanisms
 Step-2
• Cancer cells secrete degradative,hydrolytic enzymes
like Type IV collagenase & Plasminogen together
with(+) new basement membrane components
secreted at the invasive front resulting in -
connective tissue matrix (ECM) lysis locally.
 Step-3
• Matrix lysis in turn facilitates infiltration & locomotion
of cancer cells into stroma across Basement
membrane into ECM modified by proteolysis.
However, it occurs in a highly localised region close
to the tumor cell surface only.
• Continued invasion of matrix may occur by cyclic
repetition of these three steps.
 Step 4
• In connective tissue, cancer cells stimulate
Fibroblasts to increase no. & activity of
CAFs/Myofibroblasts. This is called DESMOPLASIA.
• The reorganization of the stromal network is
primarily mediated by stromal cells, most
prominently fibroblasts.
• Cancer-associated fibroblasts (CAFs)
,Myofibroblasts & Tumor-associated macrophages
reorganize the stroma by secreting ECM and
enzymes that covalently cross-link collagens fibers
and by physically pulling on the collagen network
,they behave as ‘tumor-promoting’ drivers.
• As a result, the stromal network becomes stiffer.
 Step-5
• There is recruitment of ED-B FN(Oncofetal
FN) messenger RNA positive cells to
further change the stromal behaviour in
favour of tumor cells.
• ED-B FN(Oncofetal FN) messenger RNA
positive or synthesising cells are confined
to tumor stroma & inflammatory cells at
INVASIVE FRONT only.
 CLINICAL CRITERIA OF METASTASIS ASSESSMENT
• Oral cancer tends to spread primarily to the regional lymph nodes of the
neck first before it spreads to remote sites. Lymph node metastasis is
called locoregional metastasis
• Lung (or other organs) metastasis is called distant metastasis .
• Early diagnosis of lymph node metastasis is important for improving
clinical outcomes of oral cancer patients . The clinical diagnosis of lymph
node metastasis of oral cancer is currently based on imaging techniques
and sentinel lymph node biopsy (SLNB).
• Current imaging tests have been proven to be unreliable, especially in the
detection of early nodal diseases. Localization of SLNB in patients with
certain cancers such as floor-of-mouth carcinoma is difficult. The
difficulties of detecting micrometastasis in frozen sections also limit SLNB
as an adequate guide to clinical decision-making.
• Better understanding of the lymphatic metastasis of oral cancer and
developing new diagnostic strategies to predict the clinical behavior of the
disease are desired.
• According to published data, the incidence of occult metastases to the neck
can range from 15% to 60% depending on different prognostic factors
Representative schema of tumour depth and
measurement of depth of invasion.
 SLN Concept
• SLN biopsy has become of interest in oral squamous
cell carcinoma as it has ability to identify metastases
and unpredictable lymphatic drainage patterns.
• SLN utilizes lymphatic mapping to locate and
harvest the small group of lymph nodes most likely
to harbor metastases, minimizing the invasiveness of
the procedure .
• The SLN concept states that tumor will spread from
the primary site to a single node or group of nodes,
termed the sentinel nodes, before progressing to the
remainder of the lymph node basin .
Histopathological evaluation of SLNs allows for
accurate prediction of the disease status of the rest
of the basin as positive or negative.
 INVESTIGATIVE CRITERIA
• Although,there is no agreement on a reliable predictor of
prognosis,various histopathological parameterslike:
a) keratinization,
b) mode of invasion, and
c) lymphocyte infiltration have been described as being predictors of
lymph node metastasis .
• Tumor thickness is an important prognostic factor in carcinomas of
the oral cavity. The treatment of tumors smaller than 3 mm might
need to be less aggressive than if the tumor is larger than 5 mm .
• Therefore, the use of biomarkers could help to avoid the
unnecessary surgical treatment of metastasis free patients .
• Notable is the fact that, although the TMN staging system is used
routinely, the technique accurately determines only the size and
location of tumor and does not predict their metastatic potential.
• Clinical examination can only identify regional metastasis with an
accuracy of 70%.
• Hence, several proteins and genes are candidates for use as
predictors of metastasis due to the heterogeinty of the cells.
GENETIC SIGNATURES INDICATIVE OF ONCOGENESIS

•Identifying reliable gene signatures or molecular biomarkers for oral cancer lymph node
metastasis is highly valuable to use as a potential diagnostic or prognostic tool for routine
clinical practice of oral cancer.
•Comparing the gene expression profiles of primary tumors from non-metastatic and metastatic
tumor generated a list of genes whose expression was significantly different in these two groups.
The list of genes is called “metastasis signatures”. Several “metastasis signatures” have been
generated in oral cancer aimed at predicting lymph node metastasis.
• “Metastasis signatures” from different studies are highly variable, & therefore it hinders their
translation to clinical applications.
•So far, none of the identified signatures or molecular biomarkers has been successfully
implemented as a diagnostic or prognostic tool applicable to routine clinical practice of oral
cancer.
The higher expression level of ECM-degrading enzyme genes in OSCC tissues suggests that
activated ECM degradation plays a fundamental role in the progression of OSCC.
Studies also show that there is a colocalization of five ECM-degrading enzymes in inflammatory
mononuclear cells of the neoplastic stroma. These findings alternatively support the existence of
coregulated and synergistic functions among ECM-degrading enzymes in tumor tissues & also
indicate that the same are associated with lymph node metastasis
GENETIC SIGNATURES INDICATIVE OF ONCOGENESIS

According to a review by H K Williams (2000), Chromosome

breakpoints are frequently seen in centromeric regions of
chromosomes 1, 3, 8, 14, 15, 1p22, 11q13, and 19p13. Because genes
bcl-1, int-2, and hst-1 have been mapped to 11q13 and n-ras to 11q13, it
has been suggested that activation of these oncogenes is the result of
these cytogenic alterations.

Approximately two thirds of all head and neck cancers contain a deleted
region in chromosome 9p21–22. The cyclin dependent kinase inhibitor
2/multiple tumour suppressor gene 1 (CDKN2/MTSI) has been mapped
to this chromosome region, and inactivation of its protein product
p16INK4 by mutation and deletion has been found in 10% and 33% of
head and neck squamous carcinomas, respectively, along with frequent
inactivation of p16 in oral premalignant lesions.
This suggests an important role for this gene in the early stages of oral
carcinogenesis.
 ROLE OF PROTEINS IN CARCINOGENESIS &
METASTASIS LEADING TO GENETIC ABNORMALITIES:
• Cyclins, cyclin dependant kinases (CDKs), and cyclin dependent kinase
inhibitors regulate progress through key transitions in the cell cycle.
p16INK4 binds to and inhibits phosphorylation of pRb by the cyclin
dependent kinases CDK4 and CDK6.
• Other proteins that regulate crucial checkpoints in the cell cycle, and
which are important contributors to increased cell proliferation, include
cyclin D, E, and A, which regulate the G1 to S phase transition, and
cyclin B, which regulates the G2 to M transition. The cyclin D1 gene is
frequently overexpressed in oral cancers as a result of amplification of
the 11q13 region. Overexpression of cyclin A has been reported in oral
carcinomas, with the increase in expression being associated with tumour
grade.
• This suggests that frequent abnormalities in cyclin B/p34 kinetics in oral
carcinomas lead to deregulation of the G2 to M transition.
GENETIC SIGNATURES INDICATIVE OF METASTASES OF OSCC

Zhanget al in 2018 identified a new oral cancer metastatic gene signature: CCND1, JUN
and SPP1. Their gene knockdown experiment on the oral cancer cell line and clinical data
correlation analysis indicated strong association of this gene signature expression with
oral cancer invasion and metastasis.
c-Jun is a subunit of transcription factor AP-1. Activated AP-1 increases the transcription of
target genes and plays roles in cell division, proliferation, differentiation, apoptosis and so on.
Although overexpression of c-Jun promotes invasion and metastasis of various tumors.
c-Jun plays a role during the initiation and progression of OSCC. High expression of c-Jun is
associated with poor prognosis of OSCC . Through a gene knockdown experiment in oral cancer
cell line and clinical data analysis, their study confirmed that it is also required for oral cancer
invasion and metastasis.
SPP1 (secreted phosphoprotein 1) encodes osteopontin (OPN). Osteopontin is a secreted
non-collagenous, sialic acid rich, chemokine-like, matricellular phosphoglycoprotein that facilitates
cell–matrix interactions and promotes tumor progression . Osteopontin is a multifunctional
cytokine regulating cell proliferation, survival, drug resistance, invasion, and stem-like behavior.
Its aberrant expression and/or splicing is functionally responsible for many disease pathologies
including cancer . The expression of OPN was elevated in 95% of OSCC and can be used as a
diagnostic marker for oral cancer. It is also a poor prognostic factor in OSCC treated with
cisplatin-based IC followed by CCRT .
However, The gene knockdown experiment in an oral cancer cell line and clinical data analysis
showed that SPP1 is important for oral cancer invasion and metastasis in this study.
So,besides aberrant expression, the pattern of isoform expression (gene splicing) and
post-translational modification are other SPP1 regulation methods. This regulation is
cell-type specific and may influence the potential role of OPN in malignancy as a cancer
biomarker.
CCND1 (cyclin D1) : A recent study showed that CCND1 (cyclin D1)
together with its binding partner CDK4 does not only simply act as a
transcriptional regulator to control cell proliferation, but also controls
cell adhesion, migration and metastasis under normal and pathological
conditions.
The focal adhesion component paxillin is a cytoplasmic substrate of
CCND1·CDK4.
This complex phosphorylates a fraction of paxillin specifically associated
with the cell membrane, and promotes Rac1 activation, thereby triggering
membrane ruffling and cell invasion in both normal fibroblasts and tumor
cells .
CCND1 is one of the DEGS with elevated expression in both metastatic
lymph nodes and metastatic primary tumor of oral cancer in author’s
analysis.
The results suggested the important function of CCND1 in promoting oral
cancer lymph node metastasis although the detailed mechanism still
needs to be explored.
Molecular mechanisms underlying OSCC progression
According to a study by Liu et al (2018)
•Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that
controls the expression of various antioxidant and cytoprotective genes regulating the cellular
response to oxidative and electrophilic stresses .
•Under conditions of oxidative stress, Keap1 is modified and allows Nrf2 to be released and
translocates into the nucleus where it recognizes an enhancer sequence known as the
antioxidant-response element, resulting in the transcription of antioxidants, phase II detoxification
enzymes, and drug transporters.
•Nrf2 is a primary cellular defense protein against the cytotoxic effects of oxidative stress but,
mounting evidence reveals the potential oncogenic roles of Nrf2 and its transcriptional target
genes, such as heme oxygenase 1 (HO1) , one of the main effectors of Nrf2-dependent cell
responses which plays crucial roles in the malignant transformation of cancer cells .
Nrf2 is highly expressed in many types of tumors, including OSCC .
Nrf2/HO-1 signaling activation promotes oncogenesis, metastasis, angiogenesis, and
chemotherapy and radiotherapy resistance of cancer cells and was inversely associated with
differentiation and prognosis of OSCC patients.
•In this study, Nrf2 was highly expressed in OSCC tissues and cell lines & as mentioned was
positively associated with lymphatic and distant metastases, high histological grades, and
advanced stages, but negatively associated with tumor differentiation and the prognosis of OSCC
patients.
•Fan et al . also showed that Nrf2 overexpression increases proliferation, migration, and invasion of
OSCC cells by regulating Notch signaling.
Enhancement of Nrf2 expression can thus be, considered a poor prognostic factor.
HO-1
•Increased levels of HO-1 in patients with ovarian cancer
and non-small-cell lung carcinoma were positively
associated with a high metastatic state and poor
prognosis.
• Similarly, high expression of HO-1 in OSCC specimens was
also associated with lymph node metastasis .
•It has been reported that HO-1 increased the expression of
metastasis-associated protein MMP-9 in many types of
tumors.
•Angiogenesis is essential not only for tumor growth but
also for metastasis . The pro-angiogenic roles of HO-1 are
directly evidenced in human pancreatic cancer as well as
in experimental models of many tumors.
•So,HO-1 has impact of being oncogenic,
pro-angiogenetic & causing high rate of metastasis .
•Aberrant elevations of HO-1 and Nrf2 have been found in
many different types of human malignancies, suggesting
their oncogenic roles.
Small RNAs in Metastatic & Non-metastatic OSCC
In the study by Saverino et al(2015) the authors, report the identification of small
RNAs linked to the metastatic status of a group of OSCC, both in tissue and plasma
samples.
Six miRNAs were found to be up regulated in metastatic OSCC with statistic
significance (p < 0.05).
The most expressed molecule in N+ samples was, miR-181, which has been
previously associated with metastasis and, in fact, it has been considered as a marker
for lymph-node metastasis in OSCC.
Another miRNA involved in metastatic processes is miR-296, possibly through targeting
ICAM-1 . Corroborating these sequencing results, in the additional set of 15 tumor
samples miR-296 was over-expressed in N+ samples.. Besides,two metastasis
inhibitors studied , miR-31 and miR-130b, showed expression only in N0 samples.
For known Small RNAs other than miRNAs, the research with help of BLAST of the
remaining data,showed , 68 reads that were associated with specific ncRNA categories,
in particular: piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), snRNA
(small nuclear ribonucleic acid), Y RNA, easRNA (exon-associated small RNA), rasRNA
(repeat-associated small RNA) or pasRNA (promoter-associated small RNA).
Their work shows that despite homogeneity in their global expression, small RNAs
other than miRNAs are expressed and could exert a regulatory function associated with
the cancer phenotype.
Other Biomolecules of Metastasis
•Tenascin (Tn) is an ECM glycoprotein that modulates adhesion of cells.
•Mesenchymal cells are its major source.However, carcinoma cells are also capable of
synthesizing Tn-C at ITF
•Evidence of correlation between Tn-C1-Ln-5/γ2 colocalization in reestablished BM
structures and malignancy grade in OSCC is available.
•Colocalization of these molecules, is exhibiting different patterns & is shown to be
biologically meaningful and hence, reflects sequential modulation and reorganization of
ECM, at tumor-stroma interface during different stages of OSCC.

•Syndecans are family of cell surface HS-PG, which participate in cell-matrix adhesion,
modulating epithelial-mesenchymal interactions, cell migration, and proliferation.
• These biologic effects are mediated through its binding with growth factors, for
example, FGF, basic FGF, vascular endothelial growth factor, and HGF.
•S-1, also known as CD138, is the prototype member of syndecan family, which
maintains normal architecture of epithelium and is the most widely investigated
member.
• Reduced expression of S-1 seems to be valuable marker of malignancy at ITF and
useful prognostic factor.
•Loss of S-1 in tumor cells leads to decreased intercellular adhesion and increased
potential for uncontrolled proliferation, disturbed differentiation, and tumor invasion.
•Statistically significant correlation between downregulation of S-1 expression and
prognosis, differentiation, and pattern of invasion has also been noted at ITF.
• Fascin:
• Usually overexpressed in OSCC & is involved in the
formation of cell protrusions by branched actinrich
structures
• Hence,it contributes special motility to cells &
facilitates invasion.
• Cortactin:
a) It regulates actin dynamics along with Podoplanin.
b) In aggresive lesions with high TNM classification
score, it is overexpressed & stimulates cell motility,
invasion & metastasis.
c) It causes Invadopodia formation
d) It promotes MMP secretion
 • Galectin:
• Shows increased expression at early stage of OSCC at
Invasive tumor front (ITF) & leads to –
a) Increased MMP2 & MMP9 expression
b) Reorganisation of actin cytoskeleton by Cdc42
c) early metastasis & poorer prognosis
CD44(Growth factor):
a) Ubiquitous,multifunctional cell surface adhesion molecule
b) It interactes with Hyaluronan & causes tumor progression
c) Often overexpressed at stromal surface at ITF of tumor
indicating a role in Epi-Con tissue interaction of Metastatic
cascade
d) Variant changes causing CD44s,v3,v9 accumulate at ITF &
poor prognosis CD44v4,v7,v5,v8 reduce at ITF
Role of galectin-1, fascin, cortactin, and podoplanin (molecular pathways)
in formation of cell protrusions, extracellular matrix degradation, and invasion
GOOD –DAY & THANKS Sonalee