How do Cancers Begin/ How does air pollution promote lung cancer development?

Questions to ask for understanding cancer development

• what are the pre-cancer events?

• what drives the transition from normal to cancer?

• what genomic events occur early vs late in tumor evolution?

Early Studies on Clonal Expansion in (apparently) Normal Tissues

• clonal expansion can occur in tissues that look normal under the microscope; somatic evolution begins way before cancer is clinically visible

• 1986/1987: presence of skewed chromosome X inactivation (not 50:50), documented in healthy individuals with a normal blood count (particularly older people)

  • one X chromosome is randomly inactivated in each each cell early in development (supposed to happen); normally results in 50:50 mixture

    • 50% of cells inactive maternal X, 50% inactive paternal X

  • considered if age was a factor

• 1994-2001: TP53 (tumor suppressor) in apparently normal epithelium

  • finding TP53 mutations in normal tissues suggested cancer-associated mutations before a tumor even forms

Early Studies on Clonal Expansion in (apparently) Normal Tissues FIGURE

Clonal Expansion in normal tissues in various organs: Skin + Oesophagus

• looking at pre-malignant state

• in the skin and oesophagus, clones carrying driver mutations (associated w/ oncogenicity) emerge as early as infancy and increase in their number and size with ageing

  • accelerated by exposure to UV light, alcohol and/or tobacco (lifestyle impact)

  • clones increase in number w/ age

• mutations accumulate over time → some mutations give cells a growth advantage → cell divides more → its descendants form a clone → clone expands

Clonal Expansion in normal tissues in various organs: Skin + Oesophagus FIGURE

Clonal Expansion in normal tissues in various organs: Colorectal Epithelium

• in the colorectal epithelium, clonal expansion occurs only occasionally throughout adult life

• during the long-standing inflammation caused by ulcerative colitis, almost the entire colorectal epithelium is remodelled by numerous clones harboring driver mutations

  • i.e chronic inflammation alters microenvironment → more permissive for alterations to drive oncogenesis

• a schema of crypt fission is also depicted (the colon is organized into structures called crypts)

  • crypts that develop mutations will split (fission) and now both daughter crypts carry the mutation

Clonal Expansion in normal tissues in various organs: Colorectal Epithelium FIGURE

Positively Selected Clones in Normal TIssues

• Cancer often arises from clones that were already expanding in normal tissue long before a tumor formed (already had growth advantage)

• driver mutations are acquired at any time during the lifespan, even in infancy and during fetal development

• driver mutations contribute to tissue remodelling by positively selected clones, one of which ultimately develops cancer

  • affected cells gain fitness advantage → divides more than neighbors → its descendants replace surrounding cells → normal tissue becomes a mosaic of competing clones

  • one of these clones may eventually acquire a mutation that leads to malignancy

• driver mutations can continue to persist in tissues w/o being detected by the immune system

• mutations that affect an essential gene in combination with factors* and microenvironment harbors oncogenesis

Positively Selected Clones in Normal Tissues

Histologically Normal Tissues Harbor Cancer-Associated Mutations

• we accumulate mutations as we age

Mutational Signatures

• mutational signatures provide clues as to how mutations were acquired (what damaged the DNA?)

  • mutational signatures: a count of types of mutations you see in a tumor

  • can reveal environmental exposures, inherited DNA repair defects, predict therapy response and show ongoing mutational processes

• tri-nucleotide context is considered (i.e neighboring bases considered (upstream/downstream))

• Mutational signatures decode the evolutionary history of a tumor by analyzing the pattern and context of mutations, revealing the biological processes that generated them

• if a particular signature is highly represented in a tumor, that mutational process was very active in the tumor’s history

Mutational Signatures FIGURE

Mutational Signatures provide clues as to how mutation were acquired FIGURE

• we see diff base changes that we can attribute to diff cancers

Mutational Processes → Mutational Signatures

• mutational processes result in distinct mutational signatures

We Accumulate Mutations Throughout Life

• mutations can come from diff sources

• we assign mutation to diff mutation signatures

How do Cancers Begin?

The TRACERx 421 Cohort

• large prospective study of 421 patients with early-stage non-small cell lung cancer

• purpose:

  • To track tumour evolution over time using multi-region sequencing.

  • To study intratumour heterogeneity.

  • To distinguish clonal vs subclonal mutations.

  • To understand how heterogeneity impacts relapse, metastasis, and survival.

The TRACERx 421 Cohort: Tumor Phylogenies Figure

Smokers: SBS4 Mutational Signature

• majority of smokers lung cancers harbor the SBS4 mutational signature

• in the TRACERx421 cohort, majority of smokers’ lung cancers harbored SBS4

• tumors from never smokers shows little to no SBS4 contribution

Smokers: SBS4 Mutational Signature FIGURE

Lung Cancers in Never Smokers

• lung cancers in never smokers have 3x fewer mutations and are frequently mutated in EGFR (epidermic growth factor receptor; always on to receive signal to divide and grow)

• in the figure, we see a small trunk = less clonal mutations which led to initiation of cancer

Problem: Not all smokers have the SBS4 mutation

• 7-8% of smokers with a long smoking history have no smoking mutations (SBS4) detected

• smoker lung cancers (w/o smoking signature) are similar to never smoker lung cancers (the next figure)

  • question: how are these cancers (few mutations + EGFRm) initiated

Problem: Not all smokers have the SBS4 mutation (2nd Figure)

Air Pollution

• 99% of people who live in places where air pollution levels exceed WHO Guidelines

• the incidence of lung cancer in never smokers appears to associated with average PM2.5 levels

  • pm2.5: small particles that can deeply penetrate the lungs, enter bloodstream, cause inflammation, lead to respiratory issues

Is there a relationship b/w EGFRm lung cancer and PM_2.5 exposure?

• PM_2.5 air pollution has become increasingly implicated in lung cancer

• air pollution is associated w/ LCINS (lung cancer in never-smokers)

  • many never smoker lung cancers are EGFR mutant

  • epidemiological studies suggest that PM_2.5 exposure may contribute to the development of EGFR-mutant lung tumors

• therefore EGFRm lung cancer isn’t caused solely by random mutations or genetic predisposition; environmental exposures may act as mutagenics or inflammatory trigger

Is there a relationship b/w EGFRm lung cancer and PM_2.5 exposure? FIGURE

Does Pollution Trigger Tumor Iniitation of Mutant Clones? Inconvenient Truths

• TRACERx: 7-8% of smokers with a long smoking history have no smoking mutations detected

• BRAF V600E in melanoma is not a UV light induced mutation

• Normal healthy tissue harbors mutant clones with a cancer driver mutations w/ no evidence of cancer

• 17/20 Environmental Carcinogens tested in mice, without directly causing DNA mutations

Classical Mutation Model vs Tumor Promotion Model

Tumor Promotion Model:

• cells that harbor pre-existing mutations (acquired somatically, random mutations)

• carcinogen provides a condusive microenvironment to promote pre-exsting mutations to initiate (instead of inducing more mutations)

Tumor Promotion Mice Experiment

• DMBA: carcinogen known to induce mutations

• TPA: tumor promoter (induces conducive environment)

• Standard Initiation-Promotion Model: you need both mutation (initiation) and promotion close together to get tumors (mutation or promotion alone is not enough)

• Delayed Promotion Model: Initiated (mutated) cells can remain dormant for months to years; only need a later promoting event to expand into tumors

Tumor Promotion Mice Experiment FIGURE

Air Pollution (PM) in Mouse Models

• air pollution (PM) promotes cancer in mouse models w/ pre-existing mutations

Does PM2.5 drive an environmental carcinogen signature and DNA mutagenesis?

• mutation profiles obtained from WGS of mouse tumours look the same (no significant diffs)

• tumour mutation counts do not significantly differ b/w control and pollution exposed mice

  • air pollutants accelerate age mutations (could be considered a promotional mutation)

How does Pollution Initiate Cancer without Directly Causing DNA Mutations

• RNA seq of mouse tumors: tdTOM (control) and EGFR-L858R (oncogenic driver mice)

• exposed to PBS control (TC) or air pollution (EA)

• testing if air pollution changes gene expression or interact w/ the EGFR mutation

Principal Components Analysis (PCA)

• reduces thousands of gene expression variables into a few major axes that capture the biggest differences between samples

• X-axis = PC1 and Y-axis =PC2; each dot is one sample (one mouse tumor)

  • PC=principal component

  • PC1: Captures the largest source of variation in the dataset

  • PC2: Captures the second largest source of variation

• samples that cluster together are similar in gene expression, share many characteristics

• samples that are far apart are transcriptionally diff

• value of PCA is not important, it's how its grouped

PCA: RNA Seq of Mouse Tumors

PC1: 38% variance EGFRm drives variance

PC2: 19% variance Air pollution drives variance

Pollution induces an AT2 transcriptional Signature

• air pollution induces an AT2 transcriptional signature in lung tumors

  • causes changes in the microenvironment that promotes progenitor-cells for lung tumors

• air pollution also affects immune cells (macrophages), not just AT2 cells

  • can lead to suppressing anti-tumor responses and promoting AT2 cell proliferation

• RNA-seq of tumors revealed dysregulated pathways

Exploring the effects of pollution exposure on human lungs

• PM2.5 exposure levels equivalent to those frequently encountered in major Asian cities

• Bronchial brushing taken from healthy individuals 24 hrs after exposure to 300µg/m³ for 2 hours

• compared to brushings from same individual after filtered air

IL1B

• IL1B is upregulated in both human and mouse pollution exposure experiments

• blocking IL1B inhibits pollution induces cnacer in EGFRm mouse model

Clinical Trial of Canakinumab (anti-IL1B drug)

• Clinical Trial of Canakinumab (anti-IL1B drug) reduced lung cancer incidence

• suggests IL1b/air pollution-induced tumor growth may be prevented

Do EGFR and KRAS mutations exist in normal tissue?

Yes, these mutated cells can exist for years and be silent and don’t always form tumors

• latent = present but inactive

  • cells w/ a driver mutation, not yet cancerous or expanding agressively, being kept in check by the environment

• A tumor promoter must act on latent cells to become cancerous

• tumor promoter: doesn’t necessarily cause new DNA mutations, but stimulates proliferation or survival of existing mutated cells

Latent Cells and Tumor Promoters Figure

Anthracosis

• the asymptomatic, milder type of pneumoconiosis as caused by the accumulation of carbon in the lungs due the repeated exposure to air pollution or inhalation of smoke or coast dust particles

  • carbon deposits (anthracotic pigment)

• EFRm clonal expansions are associated w/ anthracosis

  • anthracotic pigment (associated w/ pollution) is not associated w/ presence of EGFRm in normal tissue (acquiring the mutation), but the VAF of those mutations (expansions of those clones)

    • VAF = varient allele frequency, the proportion of DNA sequencing reads that carry a specific mutation at a given position

Mutations w/ Age in Non Smokers

• mutations increase w/ age in normal lungs from never smokers

Lung Cancer Promotion by Air Pollutants Figure

A Vision towards cancer prevention