Frontiers in Physiology

Inheritance of Cancer

Only up to 10% of cancers are inherited

Are there any curable cancers?

• Cancer can go into remission.

• Prognosis (if a patient will ge better or worse) is typically measured using a 5-year survival rate

Classification of Cancers

Carcinoma, sarcoma, myeloma, leukaemia, lymphoma, and mixed types

Carcinoma

Malignancy of epithelial origin or cancer of the internal or external lining of the body ( two major types: adenocarcinoma and squamous cell carcinoma)

Sarcoma

Malignancy of the supportive and connective tissues (e.g. bones, tendons, cartilage, muscles): osteosarcoma- bone cancer, glioma- neurogenic connective tissue in brain, chondrosarcoma- cancer of the cartilage

Myeloma

Cancer develops in the plasma cell in bone marrow

Leukaemia

Cancer of the blood cells: myeloid leukaemia- cancer of the myeloid and granulocytic cells, lymphocytic leukaemia- cancer of the lymphoid cells

Lymphoma

Cancer of the glands or nodes of the lymphatic system

Cancer Non-Preventable Risk Factors

• Biomedical Factors - genetic susceptibility and hormonal factors in females

• Ageing

• Ethnicity

• Gender

Cancer Preventable Risk Factors

• Lifestyles Factors: smoking, alcohol consumption, physicial inactivity and obesity, chronic infections, diet

• Environmental Factors: sunlight, radiation, occupational exposure, pollution

Normal Physiology vs Cancer

• Large, variably shaped nuclei

• Many dividing cells

• Disorganized arrangement

• Variation in size and shape

• Loss of normal features

Hallmarks of cancer cells

Sustaining proliferative signalling, evading growth suppressors, avoiding immune destruction, enabling replicative immortality, tumor-promoting inflammation, activating invasion and metastasis, inducing or accessing vasculature, genome instability and mutation, resisting cell death, and deregulating cellular metabolism.

What drives cancer initiation and progression?

• Alterations to the DNA (genetic and epigenetics)

• Alterations to the RNA molecules

• Leading to the loss or gain of functions of proteins that promote cancer initiation and progression

DNA Mutations in Cancer

• Different types of cancers are affected by different combinations of DNA mutations.

• One mutation is not sufficient to cause cancer.

Epigenetic Alterations in Cancer

• DNA methylation

• Histone acetylation

• Non-coding RNA

• These cause changes in cell proliferation, cell cycle control, apoptosis, metastasis, invasion, DNA damage, and senescence

How is cancer is a heterogenous disease?

• This means that there are often subtypes and it differs from one patient to the next

• The cancer affects the same cells in the same location but the cause and behaviour is different

• The subtypes can indicate prognosis

Tumor Heterogeneity

Tumor heterogeneity also exists within a single tumor - one cells may respond more to drugs and others may be more resistant

Why is cancer so difficult to manage?

• Hundreds of potential genetic and non-genetic alterations

• Heterogeneity of cancer

• Tumour microenvironment – determine how they grow, invade, metastasise, interaction with immune cells

• Combinatorial therapy is often needed to treat/manage cancer

• There is still much to be learnt about precision therapy for cancer

Mutations in DNA/RNA that Cause Cancer

• DNA: mutations such as point, frameshift, etc. and epigenetic alterations

• RNA: coding RNA alterations such as alternative splicing and RNA modifications and non-coding RNA alterations such as miRNAs, etc.

Accumulation of Mutations to Cause Cancer

• Mistakes that cause cancer (e.g. mutations) accumulate over time.

• Each mutation provides a selective advantage → clonal expansion.

• Involves multiple changes in cellular properties, uncontrolled growth and proliferation, evade apoptosis, deregulation of cell cycle, cell-cell interaction.

Normal Cell Proliferation

• Growth factor binds to receptor on cell membrane

• Transiently activates signal-transducing proteins

• Signal is transmitted from cytoplasm to nucleus

• Transcription of genes to promote cell division

• Cell enter and progress through the cell cycle

Sustaining Proliferative Signalling

Gain of function mutations: proto-oncogenes to oncogenes that promote cell growth

• Growth factors: secreted by tumour or surrounding stroma cells (e.g. EGF)

• Amplification of growth factor receptor (e.g. HER2)

• Constitutive activation of signal transducing molecules (e.g. RAS)

• Overexpression of nuclear transcription factors (e.g. MYC, FOS)

Evading Growth Suppressors

Loss of function mutations of tumour suppressor genes

• Retinoblastoma (RB) : stop normal cell’s passage through the restriction point at G1 phase

• p53: Guardian of the genome – arrest cell cycle upon the detection of DNA damage

• APC: negative regulator of WNT signalling that activates G1 effectors.

Resisting Cell Death

• Normal cells: DNA damage, hypoxia, disruption to metabolic pathways apoptosis (programmed cell death)

• Cancer cells: mutations/ altered gene expression

  • Disrupt key players of apoptosis

  • Increased ability to withstand intrinsic stresses

Key Players of Apoptosis

• Proapoptotic proteins = BAX, BAK, promote mitochondrial permeabilisation

• Antiapoptotic proteins = BCL2, BCLXL

• BH3 domain only proteins = BAD, BID, PUMA; enhance BAX, BAK

• Inhibitors of apoptosis proteins (IAPs), inhibit Caspase-9, stop apoptosis

• Revise apoptosis pathway (lecture 2)

Enabling Replicative Immortality

Normal somatic cells:

• Have a finite number of replication cycle: ~70 replications

• This limit is called the Hayflick’s limit

• Shortening of the ends of chromosomes (telomeres) after each mitotic cell division

• Telomeres too short cellular senescence.

Cancer cells:

• Lengthening of telomeres

2 Mechanisms of Telomere Lengthening

• Increase telomerase

• Alternative lengthening of telomeres

Activating Invasion and Metastasis

• Cause of 80-90% of cancer-related death

• Initiated by the breakdown of cell-cell adhesion (E-cadherin)

• Degradation of extracellular matrix (MMPs)

• Increase motility or chemotaxis

• Secretion of factors that stimulate cancer cells to migrate

Inducing or accessing vasculature- angiogenesis

• Continuously growing and requires oxygen, nutrients, more efficient ways to get rid of waste.

• Needs to develop blood vessels - Vascular Endothelial Growth Factor (VEGF)

Deregulating Cellular Metabolism

• Increase in growth factor signalling

  • Increase in glucose uptake

  • Build up glycolytic intermediates for DNA, RNA, protein synthesis

• RAS signalling

  • Increase the activity of glucose transporters and glycolysis enzymes enhance glycolysis

  • Increase factors that stimulate protein synthesis

• MYC signalling

  • Increase expression of genes involved in anabolic metabolism and cell growth (glutaminase) - for mitochondrial utilization of glutamine glycolysis

Avoiding Immune Destruction

• Secrete immune suppressive factors (e.g. TGF-β) to paralyse cytotoxic T cells

• Recruitment of immunosuppressive cells including myeloid-derived suppressor cells (MSDCs)

• Evasion via checkpoint molecules

Evasion Via Checkpoint Molecules

• Normal cells

  • PD-L1/PD-1 shut down T cells to prevent autoimmunity

• Cancer cells

  • Evolve this ability to express these checkpoint molecules to escape recognition and killing by T cells.

  • Express a high level of checkpoint molecules

Tumour-Promoting Inflammation

• Tumour promoting inflammatory cells: macrophages, neutrophils, B-cells

• MDSCs: secreted factors promote angiogenesis, promoting tumour growth.

• Subsets of myeloid progenitor cells, typically involved in wound-healing: major source of the angiogenic, epithelial, stromal growth factor and matrix-remodelling enzymes

Genome Instability and Mutation

• Genomic instability: high tendency/ frequency of mutations or other genetic changes during cell division.

• Caused by:

  • DNA damage/ DNA replication stress induced by oncogenes

  • Involves the mutations of DNA repair genes.

    • e.g MYH, base excision repair

  • Telomere damage – loss of protection to the ends of chromosomes chromosome fusion

  • Epigenetic dysregulation – global DNA hypomethylation activation of oncogenes

  • Centrosome amplification

Centrosome Amplification

• Centrosome: cellular structure that organises microtubules during cell division

• Important for the segregation of chromosomes

• Lead to altered karyotypes

Hallmarks of Cancer: New Additions

• Nonmutational epigenetic reprogramming

• Unlocking phenotypic plasticity

• Polymorphic microbiomes

• Senescent Cells

Unlocking Phenotypic Plasticity

Escaping terminal differentiation, becoming stem/progenitor cell-like (e.g. in acute myeloid leukaemia, accumulation of myeloid progenitor cells/ poorly differentiated cells.

Polymorphic Microbiomes

Gut microbiome can modulate susceptibility and pathogenesis of human cancer (e.g. colorectal-cancer)

Senescent Cells

Hide away, stay dormant and therapeutic resistant

Mutation vs Polymorphism

• Mutation : rare change in the DNA and are associated with a higher risk of developing a disease. Occur in < 1% of the general population.

• Polymorphism : DNA sequence variation, which is common (>1%) in the general population and typically not associated with any disease (e.g. Single nucleotide polymorphisms).

• Both mutation and polymorphism are genetic alterations.

• Disease causing genetic alterations = mutations

Causes of Mutations

• Inherited from parent(s)

• Carcinogens: UV radiation, chemicals, viruses, smoking, etc.

• Random mistakes that occur during the cell cycle

Types of Mutations

• Point mutation: change in a single DNA base and includes missense, nonsense, and silent

• Frameshift mutation: insertion or deletion that shifts the DNA sequence

Point Mutation Types

• Missense: change in a single bp causing an amino acid change

• Nonsense: change in a bp causing a stop codon

• Silent: change in a bp that doesn’t change the amino acid

Gene Mutation Overview

• A change in the nucleotide sequence

• Small scale

• Can sometimes be corrected

• Only a slight structural change

• Comparatively low influence

Chromosomal Mutation Overview

• A change in the nucleotide sequence of a segment of a chromosome

• Large scale

• Difficult to correct

• Major structural change

• Can be lethal

Types of Chromosomal Mutations

• Translocation: breakage and removal of a large segment of DNA from one chromosome, followed by the segment's attachment to a different chromosome.

• Inversion: DNA segment of the chromosome was turned 180° and reinserted.

• Deletion: loss of a piece of DNA segment from a chromosome.

• Duplication: production and inclusion of one or more copies of any segment of the chromosome.

Somatic vs Germline Mutations

Somatic mutations

• Occur in non germline tissue

• Cannot be transferred to an offspring

Germline mutations

• Present in egg or sperm

• Are heritable

• Cause cancer family syndrome (e.g. Familial Adenomatous Polyposis Syndrome)

• All somatic cells in the offspring are affected

Inheritance of Cancer Causing Mutations

• Inheriting a cancer causing mutation doesn’t guarantee that the person will get cancer

• With hereditary cancers, the first mutation is inherited and already present at birth. Additional mutations can build up over time, leading to cancer.

Cancer Promoting Missense Mutations

• Gain of function mutations: proto-oncogenes to oncogenes that promote uncontrolled cell growth

• Example BRAF V600E (50% of all Melanoma)

• Loss of function mutations: tumour suppressor genes

• Example: p53 (guardian of the genome)

Cancer Causing Nonsense Mutations

• Loss of function: tumour suppressor genes

• Inducing premature stop codons – TGA, TAA, TAG

Cancer Causing Silent Mutations

• Loss or gain of function due to alternative splicing, changes in RNA stability, and translation

• Ex. KRAS - RNA stability

Cancer Causing Frameshift Mutations

Deletion at codon 1309 of APC in colorectal cancer - very frequent in individuals with Familial Adenomatous Polyposis Syndrome

Cancer Causing Translocation

• An abnormality of chromosome 22, whereby a part of chromosome 9 is transferred and inserted into it : t(9;22) (q34; q11)

• Hallmark of chronic myeloid leukaemia (CML)

• Resulted in the BCR-ABL fusion gene

• Constitutively active protein tyrosine kinase that promotes cell proliferation and suppresses apoptosis

• Inhibits DNA repair, causing genomic instability

Multistep Progression of Cancer

Example of Multistep Progression of Cancer

• Adenoma-carcinoma for colorectal cancer is well understood that these mutations occur to cause cancer

Driver vs Passenger Mutations

• Driver mutations: Mutations that are necessary for the development, growth and survival of cancer cells

• Passenger mutations: Neutral genetic changes that do not provide any selective advantage to the development, growth and survival of cancer cells.

Approach to confirm that a genetic alteration is causing cancer

1. Identify the genetic alteration/mutation

2. Validate the role of the genetic alteration/ mutation in vitro and in vivo

3. “Rescue” or correct the genetic alteration/ mutation to stop the cancer

How to identify the genetic mutation causing cancer?

Gene sequencing

How to validate the effect of a mutation in vitro?

1. Derive a new cancer cell line from patient with the mutation

2. Insert the mutation in an existing cancer cell line (e.g with CRISPR)

3. Determine changes in cancer growth and behavior.

How to validate the effect of a mutation in vivo?

• Use mouse xenograft models

1. Inject cancer cells and engraft them in mice

2. Measure tumour growth, size, and volume, followed by histology

How to “rescue” cancer in vitro and in vivo?

Using small molecule inhibitors, DNA editing, antisense oligonucleotides, viral-loaded siRNA, and short hairpin RNAs.

Epigenetics

• Changes to the DNA and gene expression that do not involve any changes to the DNA sequence itself

• Include modifications to the DNA, histone proteins, and RNA

DNA Methylation

The addition of methyl groups to DNA (mainly on cytosine residues). Predominantly at CpGs (CG in the code) in mammals.

CpGs

• Most CpGs in the DNA are methylated (because DNA methylation silences retroviral repeat sequences that need to maintain genomic stability)

• The exception being CpG rich regions called CpG islands.

• CpG islands are present in 50-60% of mammalian gene promoters

• Unmethylated cytosines in CpG island allows binding of transcription factors to the promoter of genes to enable the gene to be actively transcribed/expressed.

• Methylated cytosines in the promoter region can lead to gene silencing important for tissue specific gene expression

How can DNA methylation go wrong in cancer?

• DNA hypermethylation

• DNA hypomethylation

DNA Hypermethylation

Can induce silencing of tumour suppressor genes (TSGs). This occurs by hypermethylation at the promoter sites of TSGs that prevent transcription factor binding.

DNA Hypomethylation

• Local effects:

  • Activation of oncogenes (e.g. Cyclin D2 in colorectal cancer).

  • Loss of imprinting that drives the proliferation of cancer cells (e.g. IGF2/H19 locus in colorectal cancer).

• Global effects:

  • Activation of repetitive sequences increases susceptibility to chromosome breakage and therefore genomic instability.

  • Reactivation of retrotransposon disrupt the normal gene structure and function/ chromosomal rearrangement

MLH1 methylation in sporadic microsatellite unstable colorectal cancer

• MLH1: a mismatch repair (MMR) gene

• MLH1 promoter methylation occurs in about 10-20% of all sporadic colorectal cancers (that exhibit microsatellite instability)

• MLH1 is a tumour suppressor gene

• MLH1 methylation leads to a loss of tumour suppressor function

• MLH1 methylation can be transmitted from a parent to an offspring

Chromatin exists in which two states

• Heterochromatin (closed): highly condense, gene poor, transcriptionally silent

• Euchromatin (open): less condense, gene-rich, more easily transcribed.

How can histone modification imbalance cause cancer?

Over modification of histone proteins causes chromatin to condense and can silence tumour suppressor genes. The opposite can cause activation of oncogenes.

Chromatin remodelling occurs when histone modification go awry and can cause:

• DNA replication stress

• Metastasis

• Tumour immunity

• Angiogenesis

• Senescence

• DNA damage response and repair

Two Classes of Histone Modifications

• Repressive 

  • Closed chromatin

  • Transcription repressed

• Active

  • Open chromatin

  • Transcription activated

Interplay Between Cancer and Epigenetics

1. Cancer development and progression involve a combination of genetic and epigenetic changes.

2. Genetic mutation can occur in genes that regulate DNA methylation

   1. DNMT3A – de novo methyltransferase 3A is responsible for methylating DNA.

   2. Mutations of DNMT3A occur in approximately 20% of acute myeloid leukaemia cases

3. Alteration of genes that regulates histone modification/chromatin remodelling is frequent in cancer

Approaches to Study Epigenetics Dysregulation in Cancer

Bisulfite Sequencing and Detecting Histone Modifications

Bisulfite Sequencing

• DNA methylation detection

• Bisulfite Conversion

  • Sodium bisulfite converts unmethylated cytosine to uracil

  • Cannot convert methylated cytosine

• Process

1. Extract DNA from cancer sample

2. Bisulfite conversion

3. PCR amplification

4. Sequencing, restriction digestion, quantitative PCR, etc

Detecting Histone Modifications

1. Antibodies specific to a histone modification are used to immunoprecipitate the histone-DNA complex

2. DNA is purified

3. DNA is analysed by microarray, next generation sequencing, or quantitative PCR

How to manipulate DNA methylation and histone modifications to study effect in cancer?

1. Using inhibitors (e.g. 5-Aza-deoxycytidine for DNA methylation – global effect).

2. Deleting proteins that regulate DNA methylation/histone modifications – global effect.

3. CRISPR-based epigenome editing approaches - target specific

Coding RNA

mRNA → protein

Types of coding RNA alterations in cancer

• General increase or decrease of mRNA encoding specific genes (e.g. oncogenes, tumour suppressor genes, metastasis-promoting genes)

• Alternative splicing

• Polyadenylation and RNA export

• Modifications to mRNA (epitranscriptomics)

Increase in mRNAs encoding oncoproteins

1. Increase in transcription factor activity

2. Increase in transcription co-activator activity (e.g CBP and p300)

3. Increase in gene enhancer activity such as high c-Myc

4. Aberrant genetic and epigenetic changes

Decrease in mRNAs encoding tumor suppressors

1. Loss of transcription factor activity

2. Loss of transcription co-activator activity

3. Decrease in gene enhancer activity

4. Aberrant genetic and epigenetic changes

5. Aberrant regulation by non-coding RNAs

Alternative Splicing

• A cellular process in which exons from a single gene are joined in different combinations, leading to different, but related, mRNA transcripts

• Alternatively-spliced mRNAs are called isoforms

• A key process in promoting protein diversity and allowing tissue specific gene function (e.g. brain may require isoform 1 of gene X whereas skin requires isoform 2 of gene X

Intron Retention (IR)

• Inclusion of an intron in the mature mRNA

• The type of alternative splicing that is least studied and understood.

• Intron retention is coupled with nonsense-mediated decay (NMD) to repress genes that will otherwise inhibit granulocyte differentiation.

• It is a previously overlooked mechanism of gene expression control

• IR inactivates tumour suppressor genes

Modifications to mRNA (epitranscriptomics)

Regulate mRNA splicing, maturation, localization, translation, and degradation

N6-Methyladenosine (m6A) RNA modification

• Addition of the methyl group to the N6 position of adenosine.

• Most pervasive and prevalent “reversible” internal modification to mRNAs.

• Occurs co-transcriptionally (i.e. added to mRNA when transcription is happening in the nucleus).

• Regulates all aspects of mRNA metabolism: transcription, splicing, stability, export, translation.

Proteins that regulate in m6A RNA modification

• writers : deposit m6A on mRNAs- METTL3 is the catalytic enzyme

• erasers: remove m6A on mRNAs

• readers : recognise m6A to direct splicing, degradation, export, translation

METTL3 and m6A RNA Modification

• AML is the type of cancer showing highest levels of METTL3 overexpression

• METTL3 overexpression leads to increased m6A on mRNAs encoding:

  • MYC- promotes cell cycle progression, maintenance of stem cells

  • BCL2- prevent apoptosis (programmed cell death)

  • PTEN- regulates cell division

• A strategy proposed to kill AML cells is to deplete METTL3

VIRMA

• The largest protein in the m6A writer complex

• Acts as a scaffold to hold the other proteins in place and allow METTL3 to function efficiently

• Exists as two different isoforms:

  • Full-length (FL)

  • N-terminal isoform (N-term)

VIRMA Cancer Hypothesis

• VIRMA is aberrantly regulated/expressed in human cancers.

1. Introduce more full-length and N-terminal VIRMA (N-term) into cancer cells

2. Measure m6A changes

3. Measure phenotypic changes to cancer cells in terms of their proliferation and growth

VIRMA Results

• VIRMA FL but not N-term promotes proliferation and clonogenicity (more colonies) of cancer cells in vitro

• Increased expression of VIRMA FL but not N-term promotes tumour growth in MDA-MB-231 xenografts

• Overexpression of FL but not N-term VIRMA increases m6A methylation on mRNAs

Types of Non-Coding RNA

• Divided into small (<200 nt) or long non-coding RNAs (lncRNAs) (>200 nt).

• Possess structural/housekeeping or regulatory function.

• Best studied is microRNAs (miRNAs).

• lncRNA

miRNA

• 18-25 nucleotide long single- stranded RNA

• Regulates gene expression post-transcriptionally

• Exists in animals and plants

• According to miRBase version 22.1, there are 38589 miRNAs discovered to date.

• Can be derived from introns, exons, other non-coding RNAs (e.g. snoRNAs).

• Once processed, miRNAs will be incorporated into RNA-induced silencing complex (RISC).

miRNA Canonical Function

• Mature miRNA in RISC will bind (with partial complementarity) to sequences in the 3’ untranslated region (3’UTR) of target genes.

• Triggering

1. mRNA cleavage

2. Translation repression

3. mRNA deadenylation

miRNA Non-Canonical Function

• Mature miRNA bind regions of the genome/transcriptome other than 3’ UTR:

1. Promoter region- trigger gene activation or silencing

2. lncRNA- disrupt the function of lncRNA

3. pri-miRNAs- prevent normal miRNA biogenesis

miRNA deregulation in cancer

• Onco-miRs – target tumour suppressor genes

• Tumour suppressive miRs- target oncogenes

• Role in metastasis: miRNAs can be involved in regulating epithelial to mesenchymal transition (EMT), invasion and angiogenesis at the secondary site.

Example: miR-200 family and cancer metastasis

• High expression of E-Cadherin is critical to maintain the epithelial state.

• ZEB1/2 are transcription regulators that repress the expression of E-Cadherin and promote EMT.

• ZEB1/2 are cognate target of miR-200 family members (miR-200a,miR-200b, miR-200C, miR- 141, miR-429).

• miR-200b targets Quaking 1 (QKI)

• QKI is a critical regulator of blood vessel formation- mice lacking QKI gene die in utero because blood vessels cannot form.

• In cancer (e.g. lung cancer), miR200b level decreases, allowing QKI to be expressed at high levels to promote angiogenesis

circRNA

• Single-stranded RNA, which unlike linear RNA, forms a covalently closed continuous loop.

• Generated due to back-splicing of mRNAs - exons join together to form a circle

• To date, over 180,000 circRNAs have been identified in humans

Functions of circRNA

• Transcriptional regulation: interact with transcription factor/RNA pol II transcription complex to promote transcription.

• Splicing regulation: compete with the splicing of linear mRNAs leading to altered gene expression.

• Post-transcriptional regulation: act as miRNA sponges to interfere with miRNA action.

• Translational regulation: binds to translational initiation-related proteins (e.g. eIF4G and PABP) to suppress translation. Some circRNA contains start codon and competes with linear mRNA to access the ribosome for translation : mRNA traps.

ciRNA in Cancer

• Good cancer biomarkers

• Can be associated with different cancer hallmarks

Example: circRNA-10720 and cancer metastasis

• High expression of E-Cadherin is critical to maintain the epithelial state.

• Twist1 is another transcription regulator that represses E-Cadherin.

• In hepatocellular carcinoma (liver cancer), Twist1 upregulates the expression of circRNA-10720, which is derived from Cul2 gene.

• circRNA-10720 sponges miRNAs that targets vimentin.

• Vimentin is a filament protein that is crucial for the establishment of the mesenchymal phenotype.

• Given that miRNAs targeting vimentin are sponged by circRNA-10720, vimentin is released from miRNA-mediated repression and becomes highly expressed in metastatic cancer cells → promotes EMT and metastasis