L6, Infectious Agents and Cancer

Question 1

What percentage of cancers are reportedly attributed to infectious diseases? Which are the two most prevalent?

Answer 1

• Around 15% of cancers
• H.pylori and HPV

Question 2

List 6 cancers with strong infectious drivers:

Answer 2

• Cervical cancer (HPV)
• Liver cancer (Hep. B and C)
• Lymphomas (viruses)
• Stomach cancer (H.pylori)
• Rarer cancers cause by parasitic infections
• HIV enables several cancers including Karposi’s sarcoma

Question 3

Early discoveries in tumour virology (1910-1981)

Answer 3

• Rous: transmission experiments for avian sarcomas, discovered RSV
• Identification of papillomavirus in rabbit
• Discovery of murine leukaemia retrovirus
• Adenovirus and SV40 induce tumours in rodents
• First human tumour virus (EBV -> Burkitt’s lymphoma)
• Origin of Src oncogene
• AIDS-related cancers -> KS and non-Hodgkin’s lymphoma

Question 4

Rous sarcoma experiment

Answer 4

• Removed sarcoma from breast muscle of affected chickens
• Broke down and ground the tissue with sand
• Collected filtrate from fine-pore filter (retaining only small viral particles and removing cell matter)
• Injected filtrate into young chickens who eventually developed sarcomas

Question 5

How can viruses directly drive carcinogenesis? (x3)

Answer 5

• DNA damage response and subsequent mutation due to integration of viral DNA into cellular DNA (retroviruses; HPV, EBV etc)
• Modified viral oncogenes after integration into host cell DNA
• Modified host genes integrated into viral genome act as oncogenes

Question 6

How can viruses indirectly drive carcinogenesis? (x5)

Answer 6

• Detection of virus by PAMPs/DAMPs can induce chronic inflammation
• Oxidative stress (ROS/RNS, mitochondrial stress, ER stress pathway)
• Immunosupression by virus (HIV-1/2)
• Prevention of apoptosis
• Induction of chromosomal instability and translocations

Question 7

Structure of RSV:

Answer 7

• env: surface glycoprotein (facilitating uptake through interacting with host)
• gag: protein coat
• pol: reverse transcriptase
• lipid bilayer acquired from infected cell

Question 8

Retrovirus lifecycle:

Answer 8

• Entry into cell and shedding viral envelope
• Reverse transcriptase makes DNA/RNA and then DNA/DNA double helix
• Integration of host DNA copy into host chromosome
• Assembly of many new virus particles, each containing reverse transcriptase, into protein coats

Question 9

RSV monolayer experiment:

Answer 9

Studying how virus conveys cancer

• Cell monolayer infected with RSV particle
• Changed behaviour of cell; foci lose contact-contact inhibition so are no longer being signalled to cease growing once monolayer complete

Question 10

Temperature mutant RSV experiments:

Answer 10

• Normal morphology cells infected with ts RSV mutant at 37 degrees -> produced transformed morphology
• At increased temperature (41 degrees), transformed morphology was lost -> normal morphology
• Shifting temperature back down restored cancerous phenotype
• As such, the cancerous phenotype requires presence of active viral proteins

Question 11

Proposed model for source of v-src:

Answer 11

• RSV has extra gene, src in genome compared to ALV
• Host cell chromosomal DNA contains c-src which integrates with dsDNA provirus (from ALV virion), once transcribed as v-src is packaged into capsid as RSV

Question 12

Give the role of the Src oncogene: Mechanism of activation?

Answer 12

Src produced a protein kinase which signals for proliferation

• In inactive form, C-terminal tail is phosphorylated
• C terminal tail dephosphorylation activates activation loop, triggering autophosphorylation of src-activator
• Activation loop phosphorylated and catalytic cleft thus exposed -> ACTIVE -> signals proliferation
• In normal cells this is initiated in certain conditions whereas in v-src oncogenes it is constitutively activated

Question 13

What makes v-Src oncogenic compared to the gene in a healthy cell?

Answer 13

• Lacks Tyrosine-527 on c-terminal tail; not inactivated by phosphorylation
• Constitutively active

Question 14

How does ALV induce cancer?

Answer 14

• Insertional mutagenesis
• ALV: Slowly transforming retrovirus
• Provirus inserts randomly into host genome
• Very occasionally, inserts upstream of c-myc (1 in 10 million)
• Induction of cancer is a rare event

Question 15

HPV and cervical cancer: Statistics

Answer 15

• 4th most common cancer in women worldwide
• 86% of cases in the developing world
• Issue of transmission; compared ot breast cancer, much higher prevalence in 18-44

Question 16

HPV vaccinations:

Answer 16

• Vaccine programmes introduced in early 90s
• Cohorts with higher vaccination rates have historically better incidence rates (*partly predicted values)
• Depends on lifetime of vaccination -> how long does protection last (expected around 10 years)?

Question 17

HPV characteristics:

Type of virus, key subtypes

Answer 17

• dsDNA virus
• Many subtypes, some innocuous (cause warts)
• Show tissue tropism
• High risk HPVs 16 and 18 cause cervical cancer

Question 18

HPV genome:

Answer 18

• Early genes E1 to 7 (where E6 and E7 are oncoproteins) carry out viral replication
• Late genes (L1, 2) produce capsid proteins

Question 19

HPV life cycle:

Answer 19

• Microabrasions permit virus into tissue
• Virion penetrates to stem cells in epithelial basement membrane -> inhabit differentiating cells and proliferate themselves
• Not initially part of host genome; at an unknown point, virus enters host genome, uses E6 and E7 to drive carcinogenesis whilst reproducing
• Tumour cells do not usually produce virions; this is an exception

Question 20

E7 mechanism:

Answer 20

• High risk E7 binds to Rb and targets it for proteasomal degradation
• Rb no longer binds and inhibits E2F
• E2F target genes upregulated -> cell cycle entry and proliferation

Question 21

E6 mechanism:

Answer 21

• Targets p53 for proteasomal degradation (ubquitin)
• Complex requires both E6 and E6A to target p53

Question 22

Further tumour promoting effects of E6 and E7:

3 key hallmarks of cancer implicated

Answer 22

• E6 derepressed expression of hTERT (telomerase component)-> enabling replicative immortality
• E6 stimulates expression of VEGF (angiogenic factor)
• E6 and E7 stimulate genomic instability

Question 23

H.pylori bacteria in cancer

Answer 23

• Estimated to cause up to 90% of stomach cancers
• Causes chronic inflammation
• Carcinogenesis is driven in part by chronic inflammation and resultant genotoxic stress but also specific of virulence factors e.g. CagA

Question 23

Further infections linked to cancer (3 examples)

Answer 23

• Southeast asian liver fluke causes liver cancer
• Bilharzia causes bladder cancer
• H.pylori causes stomach cancer

Question 24

Cancer and ER stress: Features of cancer cells, role of ER and UPR in cancer, relevance for field...

Answer 24

* Common biochemical characteristics in tumours include: Hypoxia, high [GSH], certain proteinases, peroxide and ER stress * Higher rate of protein synthesis coupled with deregulated proliferation vs chromosome abnormalities in cancer impose stress on the ER * UPR: unfolded protein response, helps to balance protein production with demand; deregulated in cancer * Ensuing inflammation, cell markers and involvement in EMT -> relevant to invasiveness and motility * All of these features are frequently targeted in modern approaches e.g. theranostics in which diagnoses via fluorescence is coupled with therapeutic agents like camptotecin and its derivatives (various benefits, see C363)

Question 25

Role of CagA in H.pylori

Answer 25

* Pleiotropic effects... * Can disrupt the cell cytoskeleton, cell polarity and cell junctions in addition to being mitogenic and pro-inflammatory

Question 26

Role of CagL in H.pylori:

Answer 26

* Allows H.pylori to bind to integrins on the basolateral surface -> integrated into cells * As a result, TypeIV secretion system injects peptidoglycan and CagA into the cell, stimulating an inflammatory response

Question 27

Morphological changes in H.pylori infected cells:

Answer 27

* Hummingbird phenotype * Cell-cell junctions altered * Cell elongation facilitates invasion

Question 28

+ Key feature of HPV positive cervical cancers

Answer 28

* Found to express E6 and E7 (viral oncogenes) * These two components are the only parts of the viral genome that are consistently expressed in cervical carcinomas * These oncoproteins fulfil roles in two stages: tumour initiation (key focus) but also malignant progression (by inducing genomic instability etc) * This is often a slow progress with malignancies arising years to decades after original infection

Question 29

+ How do basal cells divide in epithelial basal layer

Answer 29

* Asymmetric division perpendicular to basal membrane * One daughter retains basal cell characteristics and one is committed to differentiation -> withdraws from cell division cycle, subsequently pushed upwards within stratified tissue

Question 30

+ Why is evading anoikis important in the HPV life cycle? How is this thought to be carried out? | Experimental evidence:

Answer 30

* Differentiated keratinocytes must retain the ability to reproduce DNA and divide beyond the basal membrane * Thought to be regulated by association with p600 protein of E7 through an amino terminal domain -> depletion of p600 known to cause loss of anchorage-independent growth * Just one of many delicate cancer promoting activities of the E6 and E7 viral oncogenes

Question 31

+ Give the name of the two cancer causing liver flukes, where are liver flukes most prevalent? | How is it often contracted?

Answer 31

* **O. viverrini** * C. sinensis * Globally, incidence varies greatly, highest in Northeast Thailand * Typically ingested through raw/fermented fish containing infectious 'metacercaria' of the pathogen

Question 32

+ What type of cancer do liver flukes typically cause? | Technical name + key feature of this cancer type

Answer 32

* Cholangiocarcinoma * Malignant neoplasm of biliary duct system * Difficult to diagnose ('silent killer') due to relatively silent clinical progression

Question 33

+ Key pathological changes of liver fluke infection | O.viverrini distribution among hosts:

Answer 33

* Distribution of O.viverrini is highly dispersed in human hosts; generally light infection but small percentage of people have heavy infection and high risk of disease * Primarily damages the bile duct -> acute inflammation (IL-6) upon infection -> hyperplasia -> adenomatous formation, scarring, fibrosis -> cancer * Chronic infection more prone to carcinogenesis by ROS/RNS induction after initial inflammation * Analogous to liver cirrhosis but found in bile duct (instead of HCC) * Treatment against the liver fluke itself is actually thought to be a risk factor especially upon repeat infections and treatments (praziquantel)

Question 34

+ Governmental intervention against liver fluke

Answer 34

* Mass drug administration (MDA) by Thai government * 1980s * Reduced prevalence of O.viverrini from 80% to 15-20% by 1997

Question 35

+ H. pylori virulence factors:

Answer 35

* CagA -> Act.s ERK1/2 -> IL-8 and NFKB ->...-> atropic gastritis * VacA -> cyt c release in mit -> apoptosis * HtrA -> cleaves junctions (cadherins etc) * CagL -> binding to integrins on basolateral surface