CANCER BIOLOGY ASSIGNMENT

UNIT – III: PRINCIPLES OF CANCER METASTASIS

B.TECH BIOTECHNOLOGY VIII-SEMESTER

SR.NO. TOPIC ROLL NO. NAME

1 CLINICAL SIGNIFICANCES OF INVASION 251503029 AJAY

2 HETEROGENEITY OF METASTATIC 251503027 SIMRAN PHUTELA

PHENOTYPE
3 METASTATIC CASCADE 251503026 DIKSHA GAUTAM

4 BASEMENT MEMBRANE DISRUPTION 251503023 ANSH

5 THREE-STEP THEORY OF INVASION 251503022 GOURAV

6 PROTEINASES AND TUMOUR CELL 251503021 MONAMI

7 THE BIOLOGY OF ANGIOGENESIS 251503020 MONIKA

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 THE CLINICAL SIGNIFICANCE OF INVASION
(251503029 – AJAY)
Invasion

Invasion refers to the direct extension and penetration by cancer cells into neighbouring tissues. The
proliferation of transformed cells and the progressive increase in tumour size eventually leads to a breach
in the barriers between tissues, leading to tumour extension into adjacent tissue. Local invasion is also the
first stage in the process that leads to the development of secondary tumors.

Moleculer aspects of invasion and metastasis

Genomics

Molecular alterations implicated in invasion and metastasis have been described at the genomic (genetic
and epigenetic), transcriptional, translational, and post-translational level. Cancer is a genomic disease,
implicating inactivation of tumor suppressor genes and activation of promoter genes. Some of these genes
are more specifically involvedin invasion and metastasis .Mechanisms of gene inactivation in human
cancer comprise loss of heterozygosity, deletion, mutation, chromatin modification and promoter
methylation, the latter being reversible through reactivation of silenced genes .. Mechanisms of gene
activation comprise mutation, multiplication,translocation, and promoter acetylation.
Proteomics

The number of proteins involved in invasion and metastasis is much larger than the number of promoter
and suppressor genes ,many of which encode transcriptional and translational regulators. The
transcription of CDH1 encoding the invasion-suppressor E-cadherin is regulated by Snail, Slug, ZEBs
(ZEB1=δEF1; ZEB2=SIP1), and bHLH, receiving signals from more than 20 autocrine and paracrine
ligands that reach them via multiple signal transduction pathways.These transcription factors are
themselves regulated post transcriptionally through phosphorylation and sumoylation; their transcriptional
repression is mediated throughinteraction with corepressors like HDAC. Adding to the complexity is the
fact that CDH1 transcription factors also modulated of genes.

The clinical significance of Invasion and metastasis

Invasion and metastasis, the most insidious aspect of cancer, is the major cause of treatment failure for
cancer patients. Approximately 30% of patients with newly diagnosed solid tumors (excluding skin
cancers other than melanoma) already have clinically detectable metastases. Of those 70% of cancer
patients who are clinically free of metastasis, approximately half can be cured by local tumor therapy
alone (1). The remaining patients have clinically occult micrometastases which, if untreated, will grow
and become manifest. Thus, 60% of patients have microscopic or clinically evidence metastases at the
time of primary tumor treatent. The size and age variation in metastases, their dispersed anatomic
location, and their heterogeneous composition hinder surgical removal and limit the effective
concentration of anticancer drugs that can be delivered to the metastatic colonies. The patient with
metastatic disease succumbs to the direct anatomic compromise caused by the metastasis or to
complications associated with antimetastatic therapy.

Individual patients’ tumors vary widely in aggressiveness. One patient’s tumor may disseminate at a very
early stage of growth, while anoQer patient’s tumor of similar histologic appearance may grow to a large
size and fail to metastasize. Clearly, there is a great need to
accomplish the following goals: (a) accurately predict the aggressiveness of a patient’s individual tumor;
(b) detect clinically occult micrometastasis, and (c) eradicate established metastasis. Strategies toward

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these goals will emerge from a fundamental understanding of biochemical and genetic mechanisms
necessary for cancer invasion and metastasis.

Conclusions and perspectives

Malignant, i.e. invasive and metastatic, tumors involve communicating ecosystems, namely: a primary
tumor,lymph node metastases, distant metastases, a reactive bone marrow, and the circulation. Each of
these ecosystemscontains cancer cells and tumor-associated host cells that are in continuous molecular
conversation with each other.Multiple autocrine and paracrine ligands signaling through multiple and
branching pathways, together with activation of promoter genes and inactivation of suppressor
genes,modulate programs of cellular activities, comprising cell–cell adhesion, cell–matrix adhesion,
breakdown of extracellular matrix,migration, survival, and growth. These programs take tumor cells
through the multistep process of metastasis, comprising: invasion into foreign tissues, intravasation,
survival in the circulation, arrest at distant organs, extravasation, homing, survival, and growth. It is our
hope that future therapeutic strategies will take into account the dynamic molecular complexity of
invasion and metastasis implicating both cancer cells and tumor-associated host cells.

References

1. Tarin D (2006–2007) New insights into the pathogenesis ofbreast cancer metastasis. Breast Dis
26:13–25
2. Stafford LJ, Vaidya KS, Welch DR (2008) Metastasis suppressors genes in cancer. Int J Biochem
Cell Biol 40:874–891
3. Oliveira MJ (2004) The effect of bacteria on colon cancer cell invasion: molecular mechanisms
associated. Faculty of Medicine and Health Sciences. Ghent University. PhD thesis
4. Madani I, De Neve W, Mareel M (2008) Does ionizing radiation stimulate cancer invasion and
metastasis? Bull Cancer 95:292–300
5. De Wever O, Mareel M (2003) Role of tissue stroma in cancer cell invasion. J Pathol 200:429–447
6. Bracke ME, Vanhoecke BW, Derycke L et al (2008) Plant polyphenolics as anti invasive cancer
agents. Anticancer Agents
Med Chem 8:171–185
7. Ottewell PD, Coleman RE, Holen I (2006) From genetic abnormality to metastases
8. Langley RR, Fidler IJ (2007) Tumor cell-organ microenvironment interactions in the pathogenesis of
cancer metastasis. Endocr Rev 28:297–32
9. Kuperwasser C, Chavarria T, Wu M et al (2004) Reconstruction of functionally normal and
malignant human breast tissues in mice. Proc Natl Acad Sci U S A 101:4966–4971
10. Hoffman RM (2006) Real-time subcellular imaging in live animals: new visible targets for cancer
drug discovery. IDrugs 9:632–635
11. Fomchenko EI, Holland EC (2006) Mouse models of brain tumors and their applications in
preclinical trials. Clin Cancer Res 12:5288–5297
12. Gingrich JR, Barrios RJ, Morton RA et al (1996) Metastatic prostate cancer in a transgenic mouse.
Cancer Res 56:4096–4102

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 HETEROGENEITY OF METASTATIC PHENOTYPE
(251503027 – SIMRAN PHUTELA)
Introduction

In oncology, molecular, cellular and architectural variability are often referred to “heterogeneity”, a
concept that increases the complexity of the pathogenesis of malignant tumours. In terms of cell
phenotype, cell density or cell location, cell heterogeneity can be observed between tumours that occur in
the same organ and/or between patients. Tumour heterogeneity has several key clinical impacts: (i) it has
been associated with acquired drug resistance; and (ii) it limits the precision of histological diagnoses and
consequently reduces the value of a biopsy.( Michor, F.; Polyak, K. The origins and implications of
intratumor heterogeneity. Cancer Prev. Res. (Phila.)2010, 3, 1361–1364)(Fisher, R.; Pusztai, L.; Swanton,
C. Cancer heterogeneity: Implications for targeted therapeutics. Br. J. Cancer 2013, 108, 479–485)
Circulating tumour cells (CTCs) are a potential surrogate for tissue-based cancer diagnostic and may thus
provide the opportunity for monitoring serial changes in tumour biology. Recent progress has made
possible accurate and reliable quantification and molecular characterization of CTCs.( Alix-Panabieres,
C.; Pantel, K. The circulating tumor cells: Liquid biopsy of cancer. Klin. Lab. Diagn. 2014, 4, 60–64.)

Types of Heterogeneity

 Intra-Tumour Heterogeneity

Intra-tumour heterogeneity was shown by Fidler and Hart more than 30 years ago in murine models,
which refers to the existence of distinct subpopulations of cancer cells within tumours, within various
metastatic sites, and between metastatic sites and primary foci. Furthermore, intra-tumour heterogeneity
applies not only to tumour cells, but also to the components of their microenvironment. The cancer cell
populations detected usually differ in terms of tumorigenicity, activation of signalling pathways, evasion
from antitumour immunity, induction of senescence, production of secreted factors, migration, metastasis,
angiogenic capacity, genetic make-up, response to anticancer agents and activation of metabolic
pathways. Intra-tumour diversity is also thought to develop due to either genetic (epigenetic) disorder/s in
tumour cells themselves, or under the influence of the tumour microenvironment, or in the background of
interactions between these factors.

Figure 1. Two models for tumour heterogeneity and propagation: (A) In the cancer stem cell (CSC)
model, only the CSCs can generate a tumour based on their self-renewal properties and enormous
proliferative potential. The tumour heterogeneity is associated with the capacity of differentiation of these
CSCs and series of mutations and/or epigenetic events. The other cancer cells (CS) are non tumorigenic in

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immunodeficient mice for instance; (B) in the clonal evolution model, all undifferentiated cells (CSC)
have initially similar tumorigenic capacity. However, CSCs acquire a series of mutations resulting in
dominant clones; and (C) Both tumour maintenance models may underlie tumorigenesis. Initially, tumour
growth is driven by a specific CSC (CSC1). With tumour progression, another distinct CSC (CSC2) may
arise as a result of clonal evolution in CSC1. This may be a result of the acquisition of an additional
mutation or epigenetic modification. CSC2 is more aggressive and becomes dominant, driving tumour
formation. (Visvader, J.E.; Lindeman, G.J. Nat. Rev. Cancer 2008, 8, 755–768. Copyright 2008 Nature
Reviews Cancer.).

 Inter-Tumour Heterogeneity

Inter-tumour heterogeneity defines differences between tumours of the same origin in different patients.
These tumour subtypes have specific individual molecular signatures, different biological behaviours and,
as a result, have a differential impact on clinical outcomes. Two main mechanisms have been used to
explain inter-tumour heterogeneity: (i) genetic mutations or/and epigenetic modifications occurring within
th e same target cell and resulting in different tumour phenotypes (Figure 2A); and (ii) different tumour
subtypes arising from distinct cells within the tissue that serve as the cell of origin (Figure 2B). In
addition, extrinsic mechanisms may generate tumour variability, such as stromal heterogeneity (i.e., the
existence of different populations of cancer-associated fibroblasts), the complexity of immune system
infiltration into the tumour bulk, or dysregulation of the extracellular matrix. All these mechanisms are
crucial for determining malignant growth.

Figure 2. Two models of inter-tumour heterogeneity: (A) In the genetic and/or epigenetic mutation
model, mutations/modifications primarily determine the phenotype of the tumour. For this reason,
different mutations/modifications result in different tumour subtypes; and (B) In the cell-of-origin model,
different cell populations in the lineage hierarchy are used as the cells of origin for the different cancer
subtypes.( Cassidy, J.W.; Caldas, C.; Bruna, A. Maintaining Tumor Heterogeneity in Patient-Derived
Tumor Xenografts. Cancer Res. 2015, 75, 2963–2968)( Tlsty, T.D.; Coussens, L.M. Tumor stroma and
regulation of cancer development. Annu. Rev. Pathol. 2006, 1, 119–150.)
3. Sources of Heterogeneity

However, it has since been proved that genetic differences in cells within the tumour, between different
metastases diagnosed simultaneously, and even within the same excised primary or metastatic tumour,
can occur in the absence of any intervening treatment. Phenotypic differences among cells within a
tumour may reflect the genetic differences between them. However, tumour cell diversification is not only
due to genetic factors, but also to different non-genetic causes such as epigenetic processes, the
microenvironment or stochastic mechanisms, all of which can result in this cell heterogeneity.

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Figure 3. Development factors for tumour heterogeneity. This diagram shows the genetic and non-genetic
mechanisms that occur in tumour cells enhancing genome instability and leading to both increased clonal
diversity, and the development of genetic, phenotypic and epigenetic heterogeneity. Solid arrows indicate
strict regularities and dotted arrows indicate possible relations.( Turner, N.C.; Reis-Filho, J.S. Genetic
heterogeneity and cancer drug resistance. Lancet Oncol. 2012, 13, e178–e185.)

4. Clinical Implications of Tumour Heterogeneity

The main reason why cancer treatments fail is that treatment programmes are not designed to address
tumour heterogeneity. Most of the methods used to characterise tumours lead to a global overview of the
characteristics of the cancer tissue, corresponding to an average picture of all the tumour clones and their
microenvironment. The intra- and inter-tumour variations of biomarkers may affect the success of
biomarker-driven clinical trials if the biomarker used to both predict the therapeutic response and stratify
the patients into subgroups shows spatial variability.

Figure 4. Effects of tumour heterogeneity on the predictive value of biomarkers. Cancer diagnosis is
commonly based on a biopsy (1) that contains only a small fraction of tumour and may thus not be
representative of all the subclones (cells in different colours). The first line treatment can be successful in
eliminating dominant clones (2), but resistant clones are selected and drive disease progression (3).
Metastases can develop from primary tumour cells, or from clones that survive the initial therapy.
Therefore, the clonal composition of metastatic lesions may be completely different from that of the
primary tumour sample, and treatments based on the initial diagnostic sample may be suboptimal for the
treatment of metastatic disease (4). New diagnosis after a relapse must be made before applying a second
line treatment (dashed-red lines) (5) from (Almendro, V.; Marusyk, A.; Polyak, K. Cellular heterogeneity
and molecular evolution in cancer. Annu. Rev. Pathol. 2013, 8, 277–302)

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5. Methods for Studying Tumour Heterogeneity

(Tellez-Gabriel, M., Ory, B., Lamoureux, F., Heymann, M. F., & Heymann, D. (2016, December 20).
Tumour heterogeneity: The key advantages of single-cell analysis. International Journal of Molecular
Sciences. MDPI AG. https://doi.org/10.3390/ijms17122142)

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 METASTATIC CASCADE
(251503026 – DIKSHA GAUTAM)
A tumor cell that leaves the first cancer site goes through a journey called the metastatic cascade . Many
steps of the metastatic cascade and the different targets within each step. One of the early steps happens
when a tumor cell leaves the first tumor site and enters the vasculature system. Here, targets can include
inhibiting tumor cells from getting into the bloodstream and inhibiting the blood vessel cells from
allowing cells to pass through. Another step happens when a tumor cell stops at another organ site,
although research has not yet shown how a tumor cell either decides to or is forced to metastasize to
certain organs. Here, we need to answer if it is possible to target all the tumor cells while they are in
circulation before they stop at another target, or if it would be better to target the cell in the new
microenvironment of the metastasis site. In the final steps of the metastatic cascade, the tumor must
contend with the new microenvironment. In a new microenvironment, targets for therapy can include
targets also utilized for primary tumors, including forcing tumor cell death or inhibiting tumor cell
growth.(https://www.metavivor.org/research/research-news/the-metastatic-cascade-a-cells-journey-from-
one-organ-to-another/)

Steps of Metastatis

 Invasion

Cell invasion is related to cell migration, and defines the ability of cells to become motile and to
navigate through the extracellular matrix within a tissue or to infiltrate neighbouring tissues. Cancer
cells that become invasive may disseminate to secondary sites and form metastases.
(https://www.google.com/search?q=Metastatic+cascade&source=lnms&tbm=isch&sa=X&ved=0ahU
KEwj717_ehP3gAhUK8HMBHX2ZBqcQ_AUIDigB&biw=1346&bih=598#imgrc=EMzBMY_6c2As
ZM:)

 Intravasation

Entry of tumor cell into blood or lymphotic vessel . It require 3 steps:

o Cell must attach to stromal phase of vessels

o It should degrade basement membrane using MMP.
o It must pass between endothelial cell into blood stream.

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 Transport

One way traffecking , cells can travel signally or a as clumps with plateplets called Emboli in direction
of blood flow.Emboli protect cells from shear forces inside blood stream.Specific cancers have
favoured site of metastatisis & explained by first-pass organ that lies down stream from primary site.

 Extravasation

Cell must attach endothelial cell &site. It pass through endohtelial cell & basement membrane .It
migrate into surrounding tissue.Membrane of selecting family of adhesion molecule e.g E-selection i.e
specific expressed on endothelial cell. These E-selection calcium dependent that are important for
attachment of cancer cell through endothelial cell.

 Metastatic colonization:

Metastasis is the main cause of death from cancer. To colonize distant organs, circulating cancer cells
must overcome many obstacles through mechanisms that we are starting to understand. Infiltrating
distant tissue, evading immune defences, adapting to supportive niches, surviving as latent tumour-
initiating seeds, and eventually breaking out to replace the host tissue, are key steps for metastatic
colonization. These obstacles make metastasis a highly inefficient process, but once metastases are
established current treatments frequently fail to provide durable responses. A better understanding of
the mechanistic determinants of metastatic colonization is needed to better prevent and treat metastatic
cancer.(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029466/).

 Angiogenisis:

The role of angiogenesis in tumor growth has been studied continuously for over 45 years. It is now
appreciated that angiogenesis is also essential for the dissemination and establishment of tumor
metastases. In this review, we focus on the role of angiogenesis as a necessity for the escape of tumor
cells into the bloodstream and for the establishment of metastatic colonies in secondary sites. We also
discuss the role of tumor lymphangiogenesis as a means of dissemination of lymphatic metastases.
Appropriate combination therapies may be used in the future to both prevent and treat metastatic
disease through the rational use of anti-angiogenic and anti-lymphangiogenic therapies in ways that
are informed by the current and future work in the field. In vitro, tumor cells grown in spheroids have
an upper size limit based on the distance that nutrients can diffuse in the media into the core of the
spheroid. Similarly, tumors grown in organs ex vivo can only expand to ~1–2 mm. In vivo, tumors
cannot expand beyond the diffusion limit of nutrients from the nearest capillary, which is
approximately 100–500 microns, unless new blood vessels grow toward the tumor. The process by
which these tumor-associated neovessels sprout from existing blood vessels was first referred to as
“tumor-angiogenesis”.(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670555/)

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 BASEMENT MEMBRANE DISRUPTION
(251503023 – ANSH)
Basement Membrane

The basement membrane is a thin, fibrous, extreacellular matrix of tissue that separates the lining of an
internal or external body surface from underlying connective tissue in metazoans. This surface may
be epithelium (skin, respiratory tract, gastrointestinal tract, etc.), mesothelium (pleural cavity, peritoneal
cavity, pericardial cavity, etc.) and endothelium (blood vessels, lymph vessels, etc.)

Fig 1: Depicting extracellular matrix in relation to

epithelium, endothelium and connective tissue. Fig 2: Structure of basement membrane

Structure

The basement membrane is composed of two layers, the basal lamina and the underlying layer of reticular
connective tissue. The underlying connective tissue attaches to the basal lamina with collagen
VII anchoring fibrils and fibrillin microfibrils. The two layers together are collectively referred to as the
basement membrane.

The basal lamina layer can further be divided into two layers. The clear layer closer to the epithelium is
called the lamina lucida, while the dense layer closer to the connective tissue is called the lamina densa.
The electron-dense lamina densa membrane is about 30–70 nanometers thick, and consists of an
underlying network of reticular collagen IV fibrils which average 30 nanometers in diameter and 0.1–
2 micrometers in thickness. In addition to collagen, this supportive matrix contains intrinsic
macromolecular components. The lamina densa, whose collagen IV fibers are coated with the heparan
sulfate-rich proteoglycan perlecan, and the lamina lucida (made up of laminin, integrins, entactins,
and dystroglycans) together make up the basal lamina.

Integrins are not part of the basal lamina, they are part of desmosomes which are in the basement
membrane but not the basal lamina.

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Function

The primary function of the basement membrane is to anchor down the epithelium to its loose connective
tissue (the dermis) underneath. This is achieved by cell-matrix adhesions through substrate adhesion
molecules (SAMs).The basement membrane acts as a mechanical barrier, preventing malignant cells from
invading the deeper tissues. Early stages of malignancy that are thus limited to the epithelial layer by the
basement membrane are called carcinoma in situ.The basement membrane is also essential
for angiogenesis (development of new blood vessels). Basement membrane proteins have been found to
accelerate differentiation of endothelial cells.

The most notable examples of basement membranes is the glomerular basement membrane of the kidney,
by the fusion of the basal lamina from the endothelium of glomerular capillaries and the podocyte basal
lamina and between lung alveoli and pulmonary capillaries, by the fusion of the basal lamina of the lung
alveoli and of the basal lamina of the lung capillaries, which is where oxygen and CO2 diffusion happens.

Diseases due to disruption of Basement Membrane :

 Basement membrane assembly in tubulogenesis

Basement membrane assembly is required not only for tubulogenesis, but also for maintenance of
epithelial tubular structures containing polarized cells. Basement membranes are thin sheet-like structures
assembled through complex interactions among the major components, laminins, collagen IV, perlecans,
and nidogens. Several groups have shown that the lack of laminins disrupt formation of basement
membranes in vitro and in vivo. However, although a laminin self-assembly model involved in formation
of basement membrane has been hypothesized, the details remain to be defined.

Static cell adhesion to basement membrane is essential to maintain epithelial tubular structure. Therefore,
the imbalance of cell adhesion to basement membrane in epithelial tubes could be involved in pathologic
features, such as tumor invasion.

Fig 3: Basement membrane assembly in tubulogenesis

 Mechanisms of human prostate cancer invasion:

Basement membrane degradation and basal cell layer disruption

The epithelium of normal and pre-invasive human prostate tissue is physically separated from the stroma
by both the basement membrane and the basal cell layer. Physical or functional disruptions of these two
layers are pre-requisite for tumor progression from in situ to invasive tumor. The basement membrane

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layer may be degraded by matrix metalloproteinases (MMPs) and other proteases. MMP-
26/endometase/matrilysin-2 is specifically expressed in human prostate cancer carcinoma and prostatitis
tissues, it promotes the activation of pro-gelatinase B/MMP-9 and cancer cell invasion through type IV
collagen and fibronectin. A putative mechanism for basal cell layer disruptions and human prostate tumor
invasion has been investigated. To assess the potential correlation between focal basal cell layer
disruptions and leukocyte infiltration, serial sections of normal (n=5) and tumor (n=50) prostate tissues
were double immunostained for cytokeratin 34 betaE12, leukocyte common antigen, and Ki-67. Among
2,047 acini and ducts examined, 201 (9.8%) were found to contain focal basal cell layer disruptions. Of
those, 183 (91%) were with leukocyte infiltration, compared to 67 (33.3%) in 201 morphologically similar
counterparts with intact basal cell layers. Leukocytes were generally located at or near the disruptions.
Focal prostate basal cell layer disruptions and leukocyte infiltration are correlated events. Basal cell layers
surrounded by or subjacent to leukocytes were generally attenuated or fragmented. Cells overlying focal
basal cell layer disruptions often showed distinct changes in the cell size, nuclear shape, cell density, and
polarity, compared to those away from disruptions. A vast majority of the proliferating cells were located
at or near basal cell layer disruptions. These findings suggest that leukocytes and inflammation might
contribute to basal cell layer disruptions, basement membrane degradation, and tumor cell proliferation
and invasion.

References

1. Kierszenbaum, Abraham; Tres, Laura (2012). Histology and Cell Biology, An Introduction to
Pathology (3rd ed.). Elsevier. ISBN 978-0-323-07842-9.
2. Paulsson M (1992). "Basement membrane proteins: structure, assembly, and cellular
interactions". Crit.Rev.Biochem.Mol.Biol. 27 (1):93127. doi:10.3109/10409239209082560. PMID 13
09319. Archived from the original on 2007-10-13.
3. Noonan DM, Fulle A, Valente P, et al. (December 1991). "The complete sequence of perlecan, a
basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain,
low density lipoprotein-receptor, and the neural cell adhesion molecule". J. Biol. Chem. 266 (34):
22939–47. PMID 1744087.
4. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S (March 1980). "Metastatic potential
correlates with enzymatic degradation of basement membrane collagen". Nature. 284 (5751): 67–
8. doi:10.1038/284067a0. PMID 6243750.
5. Kubota Y, Kleinman HK, Martin GR, Lawley TJ (October 1988). "Role of laminin and basement
membrane in the morphological differentiation of human endothelial cells into capillary-like
structures". J.CellBiol. 107 (4): 1589–98. doi:10.1083/jcb.107.4.1589. PMC 2115245. PMID 3049626.
6. "Sect. 7, Ch. 4: Basement Membrane". 1 April 2008. Archived from the original on 1 April 2008.
Retrieved 7 May 2018.
7. Pozzi, Ambra; Yurchenco, Peter D.; Iozzo, Renato V. (January 2017). "The nature and biology of
basementmembranes". MatrixBiology.5758:111. doi:10.1016/j.matbio.2016.12.009. PMC 5387862. P
MID 28040522.
8. Henig, Robin Marantz (February 22, 2009). "What's Wrong With Summer Stiers?". New York
Times. Archived from the original on November 9, 2016.
9. Janeway, Charles; Janeway, Charles A. (2001). Immunobiology (5th ed.). Garland. ISBN 978-0-8153-
3642-6.
10. Mechanisms of human prostate cancer invasion: Basement membrane degradation and basal cell layer
disruption. Qing-Xiang Amy Sang, Yun-Ge Zhao and Yan-Gao Man DOI: Published May 2005

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 THREE-STEP THEORY OF INVASION

(251503022 – GOURAV)
Matrix metalloproteinases in cancer

Multicellular organisms consist of cells and a complex network of extracellular macromolecules such as
collagens, proteoglycans, fibronectin, lamins and many other glycoproteins. This network, referred to as
the extracellular matrix (ECM), holds cells together in an organized assembly, guides cell migration and
creates correct cellular environments.

The extracellular matrix (ECM) holds cells together and maintains the threedimensional structure of the
body. It also plays critical roles in cell growth, differentiation, survival and motility. Tumour cell to
metastasize from the primary tumour to other organs, it have to locally degrade ECM components that are
the physical barriers for cell migration. The key enzymes responsible for ECM breakdown are matrix
metalloproteinases (MMPs).

To date, 23 MMP genes have been identified in humans and many are implicated in cancer. ECM
degradation by MMPs not only enhances tumour invasion, but also affects tumour cell behaviour and
leads to cancer progression. This review highlights recent developments with regard to the cellular and
molecular mechanisms of MMPs that influence tumour cell growth, invasion and metastasis.

Roles Of ECM

 Complex network of extracellular macromolecules such as collagens, proteoglycans, fibronectin,

lamins and many other glycoproteins.
 Holds cells together in an organized assembly.
 Acts as a reservoir of growth factors and provides signals to the cell through ECM receptors on
thecell surface.
 Essential roles in many biological processes, e.g. embryonic development, morphogenesis, tissue
resorption and repair, cell differentiation, migration, growth and apoptosis.
 Degradation of the ECM modifies not only the structure of tissue but also cellular function and
behaviour.
The activities of ECM-degrading proteinases must be precisely regulated. Although many proteinases are
implicated in ECM degradation, a group of metalloproteinases called matrix metalloproteinases (MMPs),
or matrixins, is considered to play a major role.

ECM turnover associated with uncontrolled matrixin activities is involved in diseases such as arthritis,
atherosclerosis, fibrosis and cancer. In addition to the involvement in metastasis, recent studies indicate
that MMPs are also involved in vascularization and initial tumour development. In this review we discuss
the current understanding of the role of MMPs in cancer metastasis and tumour progression.

MMPs

MMPs are structurally related zinc metalloproteinases (proteinases that contain a zinc atom at the
catalytic site that is essential for hydrolysis of a peptide bond. They are secreted from the cell (soluble
MMPs) or bound to the cell surface (membrane-type MMPs; TMT-MMPs) and degrade ECM and other
proteins. At present, 23 mammalian MMPs have been identified and they are classified according to their
substrate specificity and structural similarity. All MMPs share common domain structures including a
signal sequence, a propeptide, a catalytic domain, and a hemopexin-like (Hpx) domain. Propeptides
contain a unique sequence signature called the ‘cysteine switch’ with a PRCGXPD motif, whose cysteine
residue interacts with the catalytic zinc in the catalytic domain as a fourth ligand, thereby keeping the

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precursor zymogen (the inactive precursor form of an enzyme) proMMP inactive. Catalytic domains have
a zinc-binding motif, in which the three histidine residues are ligands of the catalytic zinc atom.

MMPs and tumour cell invasion

Metastasis is the spread of cancer cells from the primary tumour to distant sites in the body. It is the
leading cause of death in cancer patients.

For a tumour cell to metastasize it must accomplish the following four events:

1. Detachment from the primary tumour and subsequent invasion into the connective tissue
stroma.
2. Entrance to the blood vessel or lymphatic system (intravasation) to traverse to distant sites in
the body.
3. Edation from the circulation (extavasation).
4. Formation of metastatic colonies for migratory cells to achieve these tasks, ECM
components are major physical barriers.

First, the tumour cells attach to the matrix macromolecules of the basement membrane or stroma via
specific cell-surface receptors. This is followed by local degradation of the ECM.

At this point, the anchored tumour cell needs to utilize proteolytic enzymes, which may arise from the
tumour cell itself or come from the surrounding stroma.

Finally, the cell migrates towards the degraded ECM, which dictates the directionality of cell
locomotion.

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3 Step Theory of Invasion

When cells invade a tissue, it is not necessary to degrade a large area of ECM, but a more focal
pericellular area (the area immediately surrounding cell) in the direction of the migration. Thus,
proteinases that are bound to the plasma membrane are likely to be more suitable for this purpose than
soluble enzymes. Despite the fact that the expression of many soluble MMPs is upregulated at the
invasive sites of tumours, these studies emphasize that pericellular proteolysis is closely associated with
the invasive phenotype and malignancy of cancer cells. More recently, it has been shown that MT1-MMP
sheds CD44 from the cell surface, an activity that is essential for migration of the MIA PaCa-2 pancreatic
cancer cell line. CD44 is a major hyaluronan receptor and is expressed in many malignant tumour cells. It
is therefore proposed that these invasive tumour cells use the CD44/MT1-MMP system for migration in
vivo.

MMPs and tumour neovascularization

In order for tumours to grow to the size of more than a few millimetres in diameter, neovascularization or
angiogenesis (the formation of new blood vessels from existing ones) must take place to supply oxygen
and nutrients. Vascularization of the tumour also facilitates the invasion of the blood stream by tumour
cells. To achieve neovascularization, it is generally thought that tumourcells produce angiogenic factors
such as vascular endothelial cell growth factor (VEGF) or fibroblast growth factor (FGF) to stimulate
local neovascularization. VEGF and FGF-1 (acidic FGF) are present in both the normal tissue of control
mice and in the tumour tissue of a transgenic mouse model. Neurovascularization is only observed in the
tumour tissue. Furthermore, in this tumour model there were no differences in the expression of two
VEGF receptors (Flk-1 and Flt-1) before and after angiogenesis took place, although VEGF signalling
was still essential for angiogenesis.

VEGF increases vascular permeability, causing the leakage of blood proteins from the vascular bed. This
is accompanied by activation of the blood clotting cascade and the formation of cross-linked fibrins. The
deposited fibrin network then serves as a scaffold for angiogenic endothelial cells, but at the same time
the highly cross-linked fibrin structure is also a major barrier for endothelial cell migration.

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MMPs and Tumour development

The role of MMP-7 in the development of intestinal tumours was investigated using a mouse strain which
spontaneously develops intestinal tumours. This mouse model closely mimics the hereditary human colon
cancer syndrome, familial adenomatous polyposis. MMP-7 production is elevated in the human intestinal
tumour. As expected, MMP-7 was also expressed in epithelial-derived tumour cells in these mice, but not
in normal intestinal mucosa. When these mice were crossed with MMP-7-deficient mice, the generated
MMP-7-null mice developed 58% less tumours and the size of the tumours was 20% smaller than those of
the wild-type.

Reference:

1. Werb, Z. (1997) ECM and cell surface proteolysis: regulating cellular ecology. Cell 91, 439–442
2. Nagase, H. & Woessner, Jr, J.F. (1999) Matrix metalloproteinases. J. Biol. Chem. 274, 21491–
21494
3. Seiki, M. (1999) Membrane-type matrix metalloproteinases. APMIS 107, 137–143
4. Itoh, Y., Kajita, M., Kinoh, H., Mori, H., Okada, A. & Seiki, M. (1999) Membrane type 4 matrix
metalloproteinase (MT4-MMP, MMP-17) is a glycosylphosphatidylinositol-anchored proteinase.
J. Biol. Chem. 274, 34260–34266
5. Brew, K., Dinakarpandian, D. & Nagase, H. (2000) Tissue inhibitors of metalloproteinases:
evolution, structure and function. Biochim. Biophys. Acta 1477, 267–283
6. Liotta, L.A., Thorgeirsson, U.P. & Garbisa, S. (1982) Role of collagenases in tumor cell
invasion.Cancer Metastasis Rev. 1, 277–288

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 PROTEASES AND TUMOR CELLS

(251503021 – MONAMI)
Tumor cells behave like a metabolic parasite for the organism or they drain its energy. They divide
relentlessly, forming solid tumors or flooding the blood with abnormal cells. Tumors arise with great
frequency, especially in older animals and humans, but most pose little risk to their host because they are
localized and of small size. Cell division is a normal process used by the body for growth and repair.
Healthy cells stop dividing when there is no longer a need for more daughter cells, but cancer cells
continue to produce copies. We call such tumors benign; an example is warts. It is usually apparent when
a tumor is benign because it contains cells that closely resemble, and may function like, normal cells. The
surface interaction molecules that hold tissues together keep benign tumor cells, like normal cells,
localized to appropriate tissues. A fibrous capsule usually delineates the extent of a benign tumor and
makes it an easy target for a surgeon They are also able to spread from one part of the body to another.
The spread of tumor cells and establishment of secondary areas of growth is called metastasis; most
malignant cells eventually acquire the ability to metastasize. Thus the major characteristics that
differentiate metastatic (or malignant) tumors from benign ones are their invasiveness and spread.

The capacity of solid tumors to invade surrounding tissue and to metastasize is correlated with the
formation and degradation of structural elements in the vicinity of the tumor cells. Evidence has
accumulated that proteases play a crucial role in tumor cell invasion and metastasis. Proteases in normal
cells are very important in carrying out important biological processes, but transformed tumor cells are
the cause of heavy destruction. The secretion of some specific proteases in tumor cells also make
prognosis very difficult.

Proteases belong to a group of evolutionarily conserved enzymes which are activated in response to the
stimuli in normal cells. They can regulate a variety of different cellular processes such as gene expression,
differentiation, and cell death. Briefly, proteases can be classified into six groups by their mechanisms of
action: serine, cysteine, threonine, aspartate, glutamic acid proteases, as well as metalloproteases. The
threonine and glutamic acid proteases were not discovered until 1995 and 2004, respectively, and
glutamic acid protease is the only subtype not found in mammals so far. The mechanism for cleaving a
peptide bond with a protease typically involves a serine, cysteine, or threonine residue (typically a
histidine is close-by which can activate these residues) or a water molecule (in aspartate, metallo- and
glutamic acid proteases) as the nucleophile in the active site. In many instances, tumor cells induce the
expression of proteolytic enzymes in nonneoplastic neighboring cells, hijacking their activity to favour
tumor expansion.

The tumor cells must arrive in circulation, arrest, extravasate, and invade the local environment and grow
to set up distant metastasis. In all these steps, starting from tumor initiation, growth and metastasis and
finally invasion into some other site involve all classes of proteases. Proteolytic activity in tumors is
regulated in a complex manner, as both genetically unstable cells and stromal cells, such as fibroblasts,
endothelial cells and inflammatory cells are involved. In vitro studies and studies using animal models
have clearly shown protease dependency of many processes in carcinogenesis. The actions of proteases
on tumor cells are as shown.

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Cysteine proteases

Mammalian cysteine proteases form a diverse group of proteolytic enzymes characterized by an active
site cysteine residue. They can be localized in the lysosome or the cytosol , and are secreted in some cell
types under pathological conditions. Cysteine proteases mediate general functions such as intracellular
protein catabolism and specialized functions such as selective activation of signalling molecules
(interleukin, enkephalin, protein kinase C) or extracellular protein degradation. The family of cathepsin
cysteine proteases can degrade both intracellular and extracellular matrix (ECM) proteins. Cathepsins
have been shown to function intracellularly as well as extracellularly, which puts them in a unique
position and contrasts them with most other proteases such as metalloproteases or serine proteases. The
extracellular activity of cathepsins allows cancer cells to attack nearby tissues, blood and Lymph vessels
and metastasize to outlying tissues. The action of cathepsins is regulated by the equilibrium between their
endogenous inhibitors and activation of their inactive forms. The action of cathepsins is regulated by the
equilibrium between their endogenous inhibitors and activation of their inactive forms. ). Cystatins are a
group of reversible, tight-binding competitive inhibitors for cysteine peptidases such as cathepsins B, H,
and L. The activity and concentration of cystatin seem varies in different cancer tissues, but its
interactions with cathepsin B has been widely investigated.

Serine proteases

Serine proteases form a group of proteases which have close relationship with cell growth and
differentiation. They often exist as zymogens and are activated by specific and limited proteolysis, which
in turn regulate the enzyme activities. Normal regulation of serine protease activities is essential for
physiological activities of the cell, and abnormal regulation of these protease activities can lead to
pathological conditions. Matriptase, a type II transmembrane serine protease is involved in angiogenesis,
degradation of extracellular matrix, and in the progression of some epithelial cancers. ). But in normal
cells, it is inhibited by hepatocyte growth factor activator inhibitor-1(HAI-1). During the progression of
human prostate cancer (CaP), there is expression of matriptase and loss of HAI-1 which may be an
important event. Trypsin is one of the best characterized serine proteases. Trypsin exhibits selective
proteolytic activity against the peptide bonds in protein molecules that have carboxyl groups donated by
the amino acids arginine and lysine. For physiological protection against premature activity, as known
from pancreatic physiology, trypsin is secreted as an inactive zymogen (trypsinogen) in the pancreatic
juice, and is activated by conversion to trypsin by an enteropeptidase in the alkaline milieu of the
duodenal lumen. Secondly, trypsinogen may be activated into active trypsin by an enteropeptidase found
in duodenal enterocytes. the antiprotease mediator pancreatic secretory trypsin inhibitor (PSTI) protects
from premature activity. An imbalance in this ‘protease–antiprotease-system’ system plays a
pathophysiological role in the development of pancreatiti and seems to pose an increased risk for
developing pancreatic adenocarcinoma.

Aspartate proteases

Aspartic proteases form a group of enzymes that consist of two lobes separated by a cleft containing the
catalytic site made up of two aspartate residues. Cathepsin–D (Cath-D) is an aspartic endo-protease that is
ubiquitously distributed in lysosomes (Barrette and Cathepsin, 1970). It was considered for a long time

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that the main function of cath-D was to degrade proteins in lysosomes at an acidic pH. Cathepsin D has
been studied over the last three decades, mainly from the perspective of its role in cancer development
and as a suggested independent tumor marker. The direct role of cath-D in cancer metastasis was first
demonstrated in rat tumor cells in which transfection-induced cath-D overexpression increased their
metastatic potential in vivo (Liaudet et al., 1994; 2006). In that rat tumor model, the cath-D mechanism
responsible for metastasis stimulation seemed to have a positive effect on cell proliferation, favouring the
growth of micro-metastases, rather than increasing the invasive potential.

Threonine proteases

Threonine proteases (proteasomes) have the task of eliminating cellular proteins, tagged for degradation
through a complex modification termed polyubiquitinization. It is a process of addition of a series of
ubiquitin molecules to another protein, targeted for degradation. It is responsible for intracellular protein
turnover in eukaryotic cells, including the processing and degradation of short- and some long-living
proteins required for regulation of various cellular functions. Proteasome inhibition leads to the
accumulation of proapoptotic proteins in tumorigenic cells but not normal tissue. Proteasome inhibition
can promote degradation of anti-apoptotic proteins and prevent degradation of proapoptotic proteins,
resulting in programmed cell death in malignant cells.

Matrix metalloproteases

Matrix metalloproteases (MMPs) are a family of nine or more highly homologous Zn 2+ endopeptidase
that collectively cleave most of, if not all, the constituents of the extracellular matrix. MMP expression is
raised in multiple tumor types, and mostly, the increase correlate with decreased survival. MMPs are
responsible for the turnover and remodeling of extracellular matrix proteins. Substrates for the enzymes
range from the fibrillar collagens of bone, skin and interstitial, to the non-fibrillar collagens, laminina.
Matrix metalloprotease secretion and activation seems to result from a specific interaction between
tumour and stromal cells. The breakdown of tissue architecture mediated by these activated enzymes
allows the primary tumour to expand, invade the neighbouring blood vessels and spread to distant sites in
the body. Invasive growth in these secondary sites also appears to be facilitated by the action of matrix
metalloproteases. MMP induction mechanisms appear to be different depending on the characteristics of
the diverse cells, with ability to produce these enzymes.

Despite initial problems with MMPIs, the stimulating results obtained with Marimastat on matrix
metalloproteases in cancer are a proof of principle on the clinical value of these compounds for future
cancer treatment. We therefore conclude that increased activity and altered localization of many proteases
of all the five classes are associated with tumor progression and even secretion of some specific proteases
in tumor cells makes prognosis very difficult.

Reference:

1. https://www.ncbi.nlm.nih.gov/pubmed/9357157
2. https://www.ncbi.nlm.nih.gov/pubmed/10675719
3. https://www.karger.com/Article/PDF/218439
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838618/
5. https://www.ncbi.nlm.nih.gov/books/NBK21590/
6. https://www.ncbi.nlm.nih.gov/books/NBK9553/

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7. https://en.wikipedia.org/wiki/Cancer_cell
8. http://www.academicjournals.org/app/webroot/article/article1380188105

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 THE BIOLOGY OF ANGIOGENESIS

(251503020 – MONIKA)
Angiogenesis is the development of new blood vessels and occurs during normal development and in
some disease states. In normal physiology, angiogenesis has a role in embryogenesis, the female
reproductive cycle, wound healing and bone formation.

In many disease states, angiogenesis is a key target for pharmaceutical intervention. In a number of
pathological states, including tumour development and progression, diabetic retinopathy, rheumatoid
arthritis and psoriasis, blood vessel formation is excessive, and the aim of pharmaceutical intervention is
to inhibit the process. Conversely, in wound healing and coronary artery disease, the aim is to stimulate
new vessel formation. Therefore, angiogenesis is a common factor in a wide range of diseases affecting
many millions of people worldwide, and much research is focused on stimulating or inhibiting the process
of angiogenesis.

Angiogenesis is controlled by a number of growth factors and inhibitors. Well-known angiogenic

(stimulatory) growth factors include basic Fibroblast Growth Factor (bFGF), Granulocyte Colony-
Stimulating Factor (G-CSF), Interleukin-8 (IL-8), Transforming Growth Factors alpha and beta (TGF-
α and TGF-β) and Vascular Endothelial Growth Factor (VEGF).
Angiogenic inhibitorsinclude Angiostatin; Interferons (alpha, beta and gamma), Endostatin, Interleukin-
12 (IL-12) and retinoids.

Angiogenesis is a Multi-Step Process

Therapeutic compounds act on one or more of the key stages to stimulate or inhibit the pathway.
Approved drugs acting on angiogenesis include Avastin (bevacizumab), a monoclonal antibody that binds
biologically active forms of VEGF and prevents its interaction with VEGF receptors (VEGFR-1 and
VEGFR-2), thereby inhibiting endothelial cell proliferation and angiogenesis. Avastin is approved for use
in colorectal cancer, advanced breast cancer and non-small cell lung cancer (NSCLC). Three tyrosine
kinase inhibitors of the EGF receptor or the VEGF receptors are approved as anticancer therapies,
including Sutent(sunitinib) for renal cancer. Regranex(becaplermin) stimulates angiogenesis and is
approved for diabetic foot ulcers. Macugen(pegaptanib), with anti-VEGF properties, is used to treat age-
related macular degeneration.

Angiogenesis models used in research and development should ideally incorporate each key stage, in
order to assess the potential for multiple effects at different points.

Key stages of angiogenesis

Key Stage Markers

Basic Fibroblast Growth Factor (bFGF): a potent

Stage One:Endothelial cell activation in response to stimulatory factor for endothelial cell migration and
angiogenic factors. proliferation. Vascular Endothelial Growth Factor
(VEGF): initiates cell proliferation and migration.

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 Matrix Metalloproteinases (MMPs): MMP1 (a collagenase)
Stage Two:Degradation of the capillary wall by
and MMP2 are expressed during angiogenesis and act to
extracellular proteinases.
degrade extracellular matrix components.

Stage Three:Formation of a branch point in the vessel

Integrins: expressed on newly forming vessels.
wall.

Integrins: allow migrating endothelial cells to interact with

Stage Four: Migration of endothelial cells into the specific components of the surrounding matrix. MMPs and
extracellular matrix towards the angiogenic stimulus. urokinase:aid migration of endothelial cells into the
surrounding matrix.

Angiopoietin (Ang 1): produced by surrounding stromal

Stage Five: Re-organisation of endothelial cells to form
cells; facilitates endothelial cell survival and stabilisation of
tubules with a central lumen.
new capillary tubes.

Platelet Derived Growth Factor (PDGF):produced by

Stage Six:Interconnection of the new tubules to form a
endothelial cells of the new capillaries; recruits pericytes
network (anastomosis).
which stabilize the new vessels.

REFERECES:-

1. https://www.cellworks.co.uk/angiogenesis.php
2. https://www.cancerquest.org/cancer-biology/angiogenesis

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