Invasion of ECM

Degradation of ECM
Tumor cells may accomplish this by secreting proteolytic enzymes or by inducing
stromal cells (e.g., fibroblasts and inflammatory cells) to do so.
Proteolytic enzyme. Matrix metalloproteinases (MMPs), cathepsin D, and urokinase
plasminogen activator are overexpressed in tumors and have been implicated in tumor cell
invasion. Example: MMP-9, a gelatinase that cleaves type IV collagen found within the epithelial
and vascular basement membrane, also stimulates the release of VEGF from ECM-sequestered
pools and generates collagen and proteoglycan cleavage products with chemotactic,
angiogenic, and growth-promoting effects.
Inducing stromal cells. Examples are the following, fibroblasts and inflammatory cells.
Attachment of tumor cells to ECM proteins
Tumor cells demonstrate complex changes in the expression of integrins, which you will
recall are transmembrane proteins that participate in adhesion of cells to other cells and to
ECM.
Locomotion is the final step of invasion
This is the propelling tumor cells through the degraded basement membranes and zones
of matrix proteolysis. Migration is a multistep process that involves many families of receptors
and several signaling pathways that eventually impinge on the actin cytoskeleton. Cells must
attach to the matrix at their leading edge, detach from the matrix at their trailing edge, and
contract the actin cytoskeleton to ratchet forward.

Vascular Dissemination, Homing, Dissemination

Once in the circulation, tumor cells are vulnerable to destruction by a variety of
mechanisms including mechanical shear stress, apoptosis due to anoikis, and innate and
adaptive immune defenses. Circulating cells that establish metastases are much more likely to
migrate as multicellular aggregates than as single cells. Clumping of tumor cells in the blood is
promoted by homotypic interactions as well as heterotypic interactions between tumor cells and
blood elements, particularly platelets
1. Location and vascular drainage of the primary tumor
The first is a matter of simple anatomy thus colon carcinomas are far more likely
to give rise to metastases in the liver, the first organ downstream of the tumor, than to
metastases elsewhere.

2. Tropism of particular kinds of tumor cells for specific tissues

Tumor cells may express adhesion molecules whose ligands are found
preferentially on the endothelial cells of the target organ. Some cancer cells express
chemokine receptors, which may guide tumor cells to tissues expressing chemokines,
much in the way chemokines normally act as attractants for cells of the immune system.
Some tissues may provide a favorable “soil” for the growth of tumor seedlings.
According to this “seed-soil” hypothesis, originally proposed by Paget, the ability of
tumor cells originating from a particular site to adapt to a foreign environment may be
limited to certain tissue types.
 3. Escape from tumor dormancy
Even when metastatic cells take root and survive within distant tissues, they may
fail to grow.

Evasion of Immune Surveillance

Malignant tumors express various types of molecules that may be recognized by the
immune system as foreign antigens It appears that protein antigens that elicit CD8+ cytotoxic T-
cell responses are most relevant for protective antitumor immunity. Immune surveillance is the
process by which the body’s immune system constantly scans the body’s cells for emerging
malignant cells, and destroys them.
Tumor Antigens
Protein antigen classification according to source and molecular structure.
1. Neoantigens produced from genes bearing passenger and driver mutations.
As discussed earlier, neoplastic transformation results from driver mutations in
cancer genes; these mutated genes encode variant proteins that have never been seen
by the immune system and may thus be recognized as nonself. Because of the enabling
hallmark feature of genetic instability, cancers often have a high burden of passenger
mutations throughout their genomes. Although these mutations are neutral in terms of
cancer cell fitness and thus unrelated to the transformed phenotype, some by chance fall
in the coding sequences of genes and give rise to protein variants that serve as tumor
antigens (provided the mutation is in an epitope that binds to the MHC molecules of the
individual). Notably, the size of the mutational load in a particular tumor, as deduced
from DNA sequencing of tumor genomes, correlates well with the strength of the host
CD8+ cytotoxic T-cell response and the effectiveness of immunomodulatory therapies.
2. Overexpressed or aberrantly expressed normal cellular proteins.
Tumor antigens may also be normal cellular proteins that are overexpressed or
aberrantly expressed in tumor cells. Examples are the following:
Tyrosinase. This is an enzyme involved in melanin biosynthesis that is expressed
only in normal melanocytes and melanomas. The immune system has little tolerance for
tyrosinase, since it is only produced in small amounts by a very small population of cells.
Cancer-testis antigen. Sperm cells lack MHC-I, so they possess this antigen but
cannot express it on their surface but, tumor cells have MHC-I, so when they express
this on their surface, the immune system can target them.
Melanoma antigen gene (MAGE) family. Originally described in melanomas, but
are now known to be expressed in other cancers as well. Found in 37% of melanomas
and in some carcinomas of the lung, liver, stomach & esophagus.
3. Tumor antigens produced by oncogenic viruses.
In several cancers associated with ongoing active or latent viral infections, the
responsible viruses encode viral proteins that are recognized as foreign by the immune
system.
Cytotoxic T lymphocytes (CTLs) play a vital role in surveillance against virus-
induced tumors by recognizing viral antigens and killing virus-infected cells. The
 importance of this immune mechanism is made evident by the high incidence of virally
induced cancers that is observed in patients with inherited or acquired T-cell
immunodeficiency. Many of these tumors are caused by Epstein-Barr virus (EBV) or
HPV, DNA viruses that carry potent viral oncogenes. CD8+ T-cells in an
immunocompetent individual can recognize proteins produced by infection from latent
DNA viruses (EBV, HPV).
4. Oncofetal antigen
Expressed in high levels in fetal tissues and cancer cells. Once thought to be
cancer-specific in adults, but levels can increase during inflammation, and small
amounts are expressed in normal adult tissues. There is no evidence that the immune
system can target oncofetal antigens in adult cells. Their clinical significance lies in their
use as tumor markers for diagnosis and surveillance.
CEA is found in colon and rectum, prostate, ovary, lung, thyroid, or liver while
AFP is found hepatocellular carcinoma and yolk sac tumor and a specific type of gastric
cancer.

Antitumor Effector Mechanisms

This is the principal immune mechanism of tumor eradication is killing of tumor cells by
CTLs specific for tumor antigens. Cytotoxic T-lymphocyte recognizes tumor neoantigens that
consist of mutated gene products or viral proteins that are endogenously synthesized and
presented in the context of MHC class I molecules.
CTL response to tumor is initiated by recognition of tumor antigens on host antigen
presenting cells (APCs). Dendritic cells and macrophages in the tumor microenvironment ingest
tumor cells or released tumor antigens and migrate to draining lymph nodes. Here, they present
the antigens in the context of MHC class II molecules and, through a mechanism called cross-
presentation, in the context of MHC class I molecules allowing the antigens to be recognized by
naïve CD8+ CTLs. Activation of antigen-specific CTLs also requires costimulatory molecules,
which are upregulated on APCs presumably by “danger signals” released from damaged or
necrotic tumor cells (DAMPs). Once activated by interaction with APCs, tumor-specific CTLs
can migrate from lymph nodes to the tumor and kill tumor cells, directly and serially, without any
assistance from other cell types.

Mechanisms of Immune Evasion by Cancers

Immune responses often fail to check tumor growth because cancers evade immune
recognition or resist immune effector mechanisms. Since cancer is all too common in persons
who do not suffer from any overt immunodeficiency, it is evident that tumor cells must have
ways to escape or evade the immune system in immunocompetent hosts. Several mechanisms
appear to be operative.
Selective outgrowth of antigen-negative variants. During tumor progression, if tumor
cells express a large number of neoantigens, it is unlikely that all can be lost, but the same goal
may be accomplished by other strategies.
Loss or reduced expression of MHC molecules. Such cells, however, may trigger NK
cells if the tumor cells express ligands for NK cell activating receptors.
Engagement of pathways that inhibit T-cell activation. Tumor cells actively inhibit tumor
immunity by upregulating negative regulatory “checkpoints” that suppress immune responses.
Through a variety of mechanisms, tumor cells may promote the expression of the inhibitory
receptor CTLA-4 on tumor-specific T cells. C
Secretion of immunosuppressive factors. TGF-β is secreted in large quantities by many
tumors and is a potent immunosuppressant. Including IL-10, prostaglandin E2, certain
metabolites derived from tryptophan, and VEGF, which can inhibit the movement of T cells from
the vasculature into the tumor bed.
Induction of regulatory T cells (Tregs). Tregs are immunosuppressive that contribute to
”immunoevasion”.

Genomic Instability
Genetic aberrations that increase mutation rates are very common in cancers and
expedite the acquisition of driver mutations that are required for transformation and sub-
sequent tumor progression.
Mutagenic agents – rarely occur due to:1) normal cell’s ability to repair DNA damage, 2)
oncogene-induced senescence and 3) immune surveillance. Remember the role of p53 which
arrests cell division to provide time for repair of DNA damage caused by environmental
mutagens and induce apoptosis in irreparable cells.
Defect in DNA repair gene/ protein has increased risk of developing cancer including
certain kinds of sporadic cancers. Not oncogenic but abnormalities greatly enhance the
occurrence of mutations in other genes. Genomic instability occurs if both copy of DNA repair
gene is lost but haploid insufficiency of a subset of genes may also promote cancer.

1. DNA Mismatch Repair Factors

This act as “spell checkers” during the process of DNA replication. Hallmark of
mismatch-repair defects is microsatellite instability. One of the diseases with this type of
defect is Hereditary nonpolyposis colon cancer (HNPCC) syndrome. Autosomal
dominant disorder characterized by familial carcinomas of the colon (one defective
gene); Affects predominantly cecum and proximal colon. Problem arises after a loss of
function mutation on the normal gene. MSH2 and MLH1 is the most commonly affected
gene and this usually affects the cecum and proximal colon.
2. Nucleotide Excision Repair
UV radiation causes cross-linking of pyrimidine residues, preventing normal DNA
replication. Such DNA damage is repaired by the nucleotide excision repair system.
Several genes are involved in nucleotide excision repair. Inherited loss-of-function
mutations in any of these genes gives rise to a syndrome called xeroderma
pigmentosum that is marked by an extraordinarily high risk of skin cancers, specifically
squamous cell carcinoma and basal cell carcinoma.
3. Homologous Recombination
Genes mutated in Ataxia Telangiectasia is important in recognizing and
responding to DNA damage caused by ionizing radiations. Another is Bloom Syndrome,
who develop neural symptoms, aside from predisposition to broad spectrum of tumors.
In Fanconi anemia, there is a mutation of any one of these genes can result in
the phenotype. BRCA2, which is mutated in individuals with familial breast cancer, is
also mutated in a subset of persons with Fanconi anemia. Fanconi anemia proteins and
 BRCA proteins form DNA damage response network whose purpose is to repair certain
types of DNA damage using homologous recombination repair pathway. Defects in this
pathway leads to activation of salvage nonhomologous end joining pathway, formation of
dicentric chromosomes, bridge-fusion-breakage cycles, and massive aneuploidy.

4. DNA Polymerase
This has a low rate of error, defined as addition of nucleotide that does not match
its partner on the template strand of DNA cancers with DNA polymerase mutations are
the most heavily mutated of all human cancers.
5. Regulated Genomic Instability in Lymphoid Cells
Special type of DNA damage plays a central role in the pathogenesis of tumors
of B and T lymphocytes. Early B and T cells both express a pair of gene products, RAG1
and RAG2, that carry out V(D)J segment recombination, permitting the assembly of
functional antigen receptor genes after encountering antigen mature B cells express a
specialized enzyme called antigen-induced cytosine deaminase (AID), which catalyzes
both immunoglobulin gene class switch recombination and somatic hypermutation errors
during antigen receptor gene assembly and diversification are responsible for many of
the mutations that cause lymphoid neoplasms.

Cancer-enabling Inflammation
Proposed cancer-enabling effects of inflammatory cells and resident stromal cells
include the following:
1) Release of factors that promote proliferation
2) Removal of growth suppressors
3) Enhanced resistance to cell death
4) Angiogenesis
5) Activation of invasion and metastasis
6) Evasion of immune destruction

Dysregulation of Cancer-associated Genes

Chromosomal Changes
This section discusses chromosomal abnormalities and epigenetic changes that
contribute to carcinogenesis and then briefly touch on the role of noncoding RNAs.
Chromosomal changes lead to the dysregulation of genes with an integral role in the
pathogenesis of that tumor type. Identification of chromosomal changes through karyotyping –
the morphologic identification of metaphase chromosomes. The following are the importance of
studying chromosomal changes: 1) genes in the vicinity of recurrent chromosomal breakpoints
or deletions are very likely to be either oncogenes or tumor suppressor genes and 2) certain
karyotypic abnormalities have diagnostic value or important prognostic or therapeutic
implications.
1. Chromosomal Translocation
 Any type of chromosomal rearrangement—translocations, inversions,
amplifications, and even small deletions—can activate proto-oncogenes, but
chromosomal translocation is the most common mechanism.
Protooncogenes are activated in two ways: 1) By promoter or enhancer
substitution – the translocation results in overexpression of a proto-oncogene by
swapping its regulatory elements with those of another gene, typically one that is highly
expressed, 2) By formation of a fusion gene – the coding sequences of two genes are
fused in part or in whole, leading to the expression of a novel chimeric protein with
oncogenic properties.
Example of this is Burkitt Lymphoma in which there is an overexpression of a
proto-oncogene caused by translocation is exemplified by Burkitt lymphoma. Virtually all
Burkitt lymphomas have a translocation involving chromosome 8q24, where the MYC
gene resides, and one of the three chromosomes that carry an immunoglobulin gene. In
Burkitt lymphoma the most common translocation moves the MYC-containing segment
of chromosome 8 to chromosome 14q32.
Another example is Chronic Myelogenous Leukemia and a subset of B-cell acute
lymphoblastic leukemias which there is a defect in Philadelphia chromosome. This
provides the prototypic example of a chromosomal rearrangement that creates a fusion
gene encoding a chimeric oncoprotein. In this instance the two chromosome breaks lie
within the ABL gene on chromosome 9 and within the BCR (breakpoint cluster region)
gene on chromosome 22.
Also a good example is Acute promyelocytic leukemia associated with a
reciprocal translocation between chromosomes 15 and 17 that produces a PML-RARA
fusion gene.
2. Deletion
This is associated with the loss of particular tumor suppressor genes. Examples
are the following and their defects: 1) retinoblastoma – chromosome 13q14, 2) renal cell
carcinoma – VHL tumor suppressor gene on chromosome 3p, 3) T-cell acute
lymphoblastic leukemias – small deletions of chromosome 1 (juxtapose the TAL1 proto-
oncogene with a nearby active promoter) leading to overexpression of the TAL1
transcription factor and 4) Lung cancers – chromosome 5 causing oncogenic EML4-ALK
fusion gene encoding a constitutively active tyrosine kinase.
3. Gene Amplification
Overexpression of oncogenes may also result from reduplication and
amplification of their DNA sequences. Such amplification may produce up to several
hundred copies of the oncogene in the tumor cell. In some cases, the amplified genes
produce chromosomal changes that can be identified microscopically. Two mutually
exclusive patterns are seen: 1) multiple small extrachromosomal structures called
double minutes and 2) homogeneous staining regions which is derived from the insertion
of the amplified genes into new chromosomal locations, which may be distant from the
normal location of the involved oncogene. The affected chromosomal regions lack a
normal pattern of light and dark-staining bands, appearing homogeneous in karyotypes.
4. Chromotrypsis
The true extent of chromosome rearrangements in cancer is only now coming
into view thanks to sequencing of entire cancer cell genomes, which allows for
comprehensive “reconstruction” of chromosomes from DNA sequences. Genomic
 sequencing has revealed not only many simple rearrangements (e.g., small deletions,
duplications, or inversions) that were not appreciated by prior methods, but also much
more dramatic chromosome “catastrophes” termed chromothrypsis (literally,
chromosome shattering). Chromothrypsis is observed in 1% to 2% of cancers as a
whole, but is found in up to 25% of osteosarcomas and other bone cancers and at
relatively high frequency in gliomas as well. It appears to result from a single event in
which dozens to hundreds of chromosome breaks occur within part or across the entirety
of a single chromosome or several chromosomes.

Epigenetic Changes
This refers to factors other than the sequence of DNA that regulate gene expression . It
includes: histone modification, DNA methylation and alterations that regulate the high order of
DNA. Epigenetic alterations includes: 1) silencing of tumor suppressor genes by local
hypermethylation of DNA, 2) global changes in DNA methylation example is AML mutation in
genes encoding DNA methyltransferases and 3) Relationships in the epigenome.

Noncoding RNAs and Cancer

MicroRNAs (miRs) are small noncoding, single-stranded RNAs, approximately
22nucleotides in length, that mediate sequence-specific inhibition of messenger RNA (mRNA)
translation through the action of the RNA-induced silencing complex (RISC).
Given that miRs control normal cell growth, differentiation,and cell survival, it is not
surprising that they play arole in carcinogenesis. Altered miR expression, sometimes stemming
from amplifications and deletions of miR loci,has been identified in many cancers. Decreased
expression of certain miRs increases the translation of oncogenic mRNAs; such mIRs have
tumor suppressive activity. Conversely, overexpression of other mIRs represss theexpression of
tumor suppressor genes; such mIRs promote tumor development and are often referred to as
onco-mIRs.
Specific examples of contributions of mIRs to cancer arenumerous; the following are
among the best established:
OncomiRs. miR-200 has been shown to promote epithelial- mesenchymal transitions
believed to be important in invasiveness and metastasis. miR-155,originally identified at
the site of retroviral insertions inavian lymphomas, is overexpressed in many human B
cel llymphomas and indirectly upregulates a large number of genes that promote
proliferation, including MYC.
Tumor suppressive miRs. Deletions affecting certain tumor suppressive miRs,such as
miR-15 and miR-16,are among the most frequent genetic lesions in chronic lymphocytic
leukemia, a common tumor of older adults. In this context, it appears that their loss leads
to upregulationof the anti-apoptotic protein BCL-2.

CARCINOGENIC AGENTS AND THEIR CELLULAR INTERACTION

Chemical Carcinogenesis
Initiation
Results from exposure of cells to a sufficient dose of a carcinogenic agent
An initiated cell is altered, making it potentially capable of giving rise to a tumor. This is not
sufficient for tumor formation that causes permanent DNA damage (mutations) – rapid and
irreversible and has “memory” and tumors are produced even if the application of the promoting.
Agent is delayed for several months after a single application of the initiator in which it inflicts
nonlethal damage to the DNA that cannot be repaired. The mutated cell then passes on the
DNA lesions to its daughter cells.
Promoters
Promoters can induce tumors to arise from initiated cells, but they are non-tumorigenic
by themselves. Tumors do not result when the promoting agent is applied before, rather than
after, the initiating agent. The cellular changes resulting from the application of promoters do not
affect DNA directly and are reversible. Promoters enhance the proliferation of initiated cells in
which they contribute to the acquisition of additional mutations.