Disease Models of Cancer
TNBC: 6 subtypes based on GEP
- 2 basal-like (BL1, BL2) Activate: cell-cyle/ DNA damage response - immunomodulatory (im) Activate: immune, NfkB, JAK/STAT - mesenchymal (M) Activate: EMT, ECM, wnt, ALK - mesenchymal stem-like (MSL) Activate: EMT, wnt, EGFR - luminal androgen receptor (LAR), decreased relapse-free survival Activate: androgen receptor
Oncotype DX
- 21 gene qRT-PCR - 16 cancer genes, 5 reference genes - Prognostic
Culture methods and xenografts
- 2D cell cultures - 3D cell cultures: > Mammosphere assay/ cultures > Organoids - Xenograft tumours - Allograft tumours
Theros-Breast Cancer Index
- 3 genes qRT-PCR, homeobox genes, IL-17 and unknown transcript - Prognostic
PAM50
- 50 gene qRT-PCR - ER-ve and ER+ve - Second gen?
ER and breast cancer patients
- 70-80% of BC patients have ER+ tumours > 60% of which are also PR+
Neoadjuvant therapy
- Administration of therapeutic agents before a main treatment - E.g. neoadjuvant hormone therapy prior to radical radiotherapy for adenocarcinoma of the prostate
Infiltrating DC
- Also called invasive ductal carcinoma - Most common: cancer has spread
ERBB2/Neu/HER2
- Amplified in 25-30% of breast cancers - Encodes RTK, can heterdimerise with other 3 receptors, preferred dimerisation partner of other ErbB receptors
Yun et al 2015: Activation of AKR1C1/ERbeta induces apoptosis by downregulation of c-FLIP in prostate cancer cells
- Androgen binding to androgen receptors plays important role in growth and development of normal prostate and disease - Adrogen deprivation therapy is used for locally advanced PC but the outcome is transient, resulting in progression to castrate-resistant PC (CRPCA) - no effective curative options - ADT: causes apoptotic cell death of cancer cells - Ratio of androgens to estrogen may contribute to CRPCA development: > Role of ERbeta and ERalpha in PC cells and human prostate cancers > Ectopic expression of ERbeta KO mice develop hyperplasia and PIN lesions - Important role for c-FLIP: > aberrantly expressed in high grade and CRPCA tumours > epidemiological study: high soy diet, reduced PCA incidence --> estrogen matbolites with antitumourigenic activity > downregulation of c-FLIP by 2ME-2 (estrogen metabolite) reported to inhibit tumour growth in vitro and in vivo Findings: AKR1C1 inhibits apoptosis through c-FLIP suppression vi ERBeta. Thus, it contributes to castrate-resistant prostate cancer. - ERbeta competes with Sp1 and Sp3 to downregulate c-FLIP -
LAR
- Androgen receptor - Responded to biculutamide (AR inhibitor)
Additional targeting strategy
- Anti-body conjugates - Trastuzumab emstansine
Xenograft mice
- Athymic mice FOXN1 gene mutation, reduced or lack of thymus, T cell deficiency. Only established cell lines - rare to accept tumours - NOD/SCID mice NOD innate immunity deficiency, SCID eliminates adaptive immunity, PRKDC gene mutation (lack VDJ recombination in B and T cells) - NSD mice (NOD/SCID/Gamma) _ IL2gammaReceptor ull - blocks NK cell differentiation, robust, can accept tumours
TNBC targeted therapies
- BRCA1 - FGFR - PI3K - EGFR1 - mTOR1 - MET - Src
Models commonly used to investigate breast cancer
- Breast cancer cell lines - Xenografts - Genetically engineered mice (GEM) - Complexity and heterogenity of breast cancer means no individual recapitulates all aspects fo the disease, so an integrated and multi-systems approach is necessary
TRAIL in endocrine resistant breast cancer: SUMMARY
- Breast tumours that acquire resistanct to anti-hormone therpay are likely to become sensitive to TRAIL-mediated killing - Endocrins resistant bCSCs are most susceptible to TRAIL - Degradation of cFLIP is one cause of this susceptibility
Tumour heterogeneity
- Cancer is not a single disease - Tumour arise from different tissues - Have different cell-of-origin - Genetically/phenotypically heterogeneous - Treatments don't work on all tumours, or all cells within a tumour - Difficult to predict tumour response and prognosis - Resistance to therapies - Intra and inter-tumour heterogeneity
GEM vs carcinogen-induced mouse models
- Carcinogen-induced mammary tumours in rats more frequently hormone-dependent: model for hormone action in breast cancer
Homotypic vs heterotypic cultures
- Cell cultures tend to be homotypic and do not allow examination of tumour-stromal cell interactions - Stroma establishes paracine interactions which facilitate tumour progression during tumour growth - Heterotypic co-culture systems have been developed: allow selective examination of interactions between tumour and stromal cells, including macrophages, fibroblasts and endothelial cells. But: modelling interactions in vitro is difficult, which is why in vivo models are important
BL1/BL2
- Cell cycle/ DNA damage responses - Respond to cisplatin
Do cell lines contain tumour-initiating cells?
- Cell surface markers: CD44+CD24- and ESA+ to isolate and characterise > but relationship to clinical outcome is not clear - Some labs have identified subpopulations within cell lines that have distinct tumour-initiating phenotypes - MCF-S forms mammosphere exhibits CD44+CD24- expression profile, enriched by 1000-fold in tumour-initiating capacity compared with parental cells - More studies that utilise different subtypes of breast cancer are required to determine whether subtypes contain subpopulations of tumour-initiating cells, and whether they display identical or distinct cell markers > Recently promising approach: Aldeflour (stem cell marker) in FACs analysis along with IHC using ALDH1 antibody
Principal BC models
- Cells > Non-tumorigenic cell lines > Breast cancer cell lines - Mouse > Transplants: cell lines/ primary tumour > Genetically engineered mice
Piggot etl al 2018: PTM that occurs during endocrine resistance and results tumour susceptibility to apoptosis-inducer TRAIL potentially offers a novel stratified approach to targeting endocrine-resistant breast cancer
- Cells in primary-derived xenograft models of endocrine resistance were investigated for susceptible to TRAIL. - Tumour viability, CSC viability (tumourspheres), tumour growth kinetics and metastatic burden were assessed. - BC cell lines with TAMR or FASR more sensitive to TRAIL than endocrine-sensitive controls. - TRAIL eliminated CSC-like activity in TAMR cells - prolonged remission of xenografts - Primary culture: TRAIL significantly deplete CSCs in 85% ER-resistant compared with 8% endocrine-naïve. - Systemic administration of TRAIL in ER-resistant patient-derived xenografts reduced tumour growth, CSC-like activity and mestastases. - Acquired TRAIL sensitivity correlated with a reduction in intracellular levels of cFLIP and an increase in Jnk-mediated phosphorylation of E3-ligase, ITCH, which degrades cFLIP - Novel mechanism of acquired vulnerability to an extrinsic cell death stimulus in ER-resistant breast cancers, which has both therapeutic and prognostic potential
Analysis: Necropsy and staining
- Collect as much data as possuble about tumours, phenotype, animal etc - do not know what information may be of interest - DOB and DOD - Tumour material - paraffin-embedded blocks for analysis - Survival curves
Conditional Systems
- Cre-LoxP (P1 phage): deletion/inversion/translocation - Flp-Frt (yeast)
2D vs 3D culture conditions
- Culture conditions in 2D in vitro cell culture differ from breast microenvironment - 3D reconstituted basement membreane (rBM) cultures have been used - 3D culture environment does not promote global changes in gene-expression patterns - cell lines more similar to themselves than other cell lines grown in same culture conditions, but some signal transduction genes were correlated with cells in 3D environment
2D cultures
- Cultures grown on plastic - Lack exposure to ECM components present in vivo - Exposed to non-physiological substratum, lack heterotypic cell-cell interactions and do not recapitulate 3D tissue architecture
Constitutional or conditional deletion?
- Depends on embryonic or early adult lethality or tumour development (e.g. in just one organ) - Conditional approaches are required for organ-specific targeting
Human breast cancer cell lines
- Dozens, 10 most used cell lines - Most common research material - Different properties: hormone/receptors/cell type/ aggressive - Majority originate from late stage tumours (-ve point) - Similar to primary tumours, but additional genomic aberrations had occurred, different categories of subtypes
Biomarker discovery
- Drug response experiment - Genomic and genetic characteristics on cell lines are experimentally determined via high-throughput technologies - Normalisation of data for further analysis - Identify subset genes highly associated with drug activities - After narrowing candidate genes to a few hundred, statistical classification modelling to construct a multivariate prediction model - Then clinical setting
DCIS
- Ductal Carcinoma in situ - Precancerous breast lesion that indicates a higher risk for invasive ductal breast cancer
2. Selective estrogen receptor downregulators
- E.g. Fulvestrant - SERs tend to be used for pre-peri menopausal women - downregulates and promotes degradation of ER - 100x greater affinity for ER and TAM - More potent than Tamoxifen and works in a different way, lose receptor inside cell - second line therapy after tamoxifen resistance - Used as a 2nd line therapy after TAM resistance - Sensitises ER+ BC to chemo (prevents ER upregulation) - Recommended for metastatic BC either alone or in combination with AI
Conditional activation
- E.g. PI3K H1047R, Ras G12V or G12D, Her2/Neu - Typically heterozygous - Mutant oncogene, knocked onto the endogenous locus - Blocked from being expressed by a STOP cassette, prevents expression. But STOP site is flanked by LoxP site. - Add Cre recombinase, STOP cassette is removed and gene expression occurs
3. Aromatase inhibitors continued
- E.g. letrozole, anastrozole, exemestrane - Aromatase expressed in ovary (pre-menopause), testis, placenta, brain, bone, adipose - Only effective along in post-menopausal women because ovaries upregulate oestrogen in pre-menopausal women due to increase in GnRH production (-ve feedback) - In premenopausal women, can be used with GnRHA AI+ OFS = 70% complete/partial responses AI + TAM = 50% CR/PR
M/MSL
- EML, ECM, wnt, ALK, EMT, wnt, EGFR - NVP/BEZ235 (PI3K/mTOR inhibitor) and dasatinib (abl/scr inhibitor)
Breast cancer cell line models of TAM acquired resistance
- ER+ cell line gorwn in low concentrations of Tamoxifen or Faslodex for 2 years minumum -> TAMR, mesenchymal-like -> FASR
Prognosis of receptor stratification
- ER+ have better prognosis than triple negative (cells driven by other processes so more aggressive)
Why make GM mouse tumour models?
- Early events in tumour formation - Biology of tumours - Genetic epistasis and target validation - Tissue-specificity of oncogenes/TSGs - Immunotherapy - Pre-clinical testing
Ostrogen receptor signalling
- Estrogen receptor alpha - tends to be the dominant marker for ER+ disease - Estrogen receptor beta
MammaPrint
- Expression of a set of 70 genes can be used for recurrence and outcome assessment in breast cancer - Microarray - Prognostic: 10 yr metastasis - FDA approved
Metastasis and breast cancer models
- Field is developing - Example of bone metastasis in 2005 - Orthotopic injection of human breast cancer cells into mammary fat pads of NSG (immunodeficient) mice resulted in metastasis in different organs by Lorns et al 2012
Do tumour from GEM contain tumour-initiating cells?
- GEM and allograft models in breast cancer have identified tumour-initiating cells of BC: > Zhang et al (2008): Tumour initiating cells were identified in syngeneic/allograft null mouse tumour model - In vitro and in vivo data supporting cancer stem cell model hypothesis - Tumours arising from this model retain ER and anti-ERs inhibit formation of these tumours - Mammary epithelial cells from p53 null tumours were FACs sorted based on CD29 and CD24 markers, then transplanted into mammary fat pads of mice - Small population of tumour cells had stem-cell like properties
Future of personalised medicine
- Genomics - Nanopore,PacBio - Cost? - Insurance? - NICE
Examples of constitutional vs conditional deletion: Brca1 and Brca2
- Germline +/- KO viable and no phenotype - Germline -/- KO lethal - Must use conditional approaches
Phenotyping
- H&E (staining) - IHC (immunohistochemistry - standard set for proliferation and apoptosis) - IHC - model/tissue specific stains/FACS (flow cytometry and fluorescence-activated cell sorting) analysis for models of leukaemia - Stage/grade/histological classification - Need a team: pathologist, bioinformatician, statistician
HER2
- Heregulin receptor - Cell-surface receptor - Tyrosine kinase - Heterodimerises with other receptors within the family (preferred receptor for the others to heterodimerise with)
Do GEM recapitulate human breast cancer?
- Histology of most tumour from GEM does not resemble the common types of breast cancer - Many similarities between mouse and human tumours and differences - Because of diversity of human breast cancer and species differences, individual GEM models of breast cancer cannot recapitulate all aspects of human disease
Prognostic and predictive biomarker example
- Hormone receptor status: subdivided into ER+, HER2+, ER+HER2+, or triple negative (ER-HER2-PR-)
Human Tumour Xenograft
- Human tumor cells are transplanted, either under the skin or into the organ type in which the tumor originated, into immunocompromised mice that do not reject human cells - Low transplantation efficiencies, possibly due to need for immunocompromised mice - Removes need for requirement for cells to invade and intravasate --> cannot investigate metastatic process in these models
Disadvantages of xenografts
- Immunocompromised mice - immune system contributes to tumours - Main BC subtypes not represented? - Cell lines not representative of the most common tumours - Subcutaneous flank tumours are not correct microenvironment (humanise fat pad for xeno - Co-evolution of stroma tumour not recapitulated - Metastasis models predominantly found only in lung and liver (but also lymph nodes, liver, bone and brain) - Therefore, testing drugs for tumour regression has limited potential
Personalised medicine
- Improving treatment through the stratification (segmentation) of patient population into homogeneous drug response groups - The integration of personal genetic information with predictive/ prognostic markers of treatment response and survival
Do xenografts mimic human breast cancer?
- In vivo environment - Uses cell lines that may not represent most common breast cancer types - Xenografts must be established in immunocompromised mice: absence of intact immune system may affect tumour development and progression - Increasing evidence of roles of immune system in early breast cancer and metastasis - Xenografts commonly generated by subcutaneous injection of tumour cells into the flank of the mouse, and the microenvironment may alter growth/ metastatic potential of the engrafted cells - Orthotopic translplantation into mammary gland provides a more favourable microenvironment - Mammary fat pad of mice may not reproduce complex interactions between epithelial and stromal compartments in human breast cancer - Massague et al: Xenograft assays are relevant to human breast cancer - Xenograft models are inefficient and access to clinical samples is limited
Disadvantages of cancer cell lines
- Karyotypically unstable - Acquisition of additional mutations makes uniformity of cell lines uncertain - Provenance - Homotypic: tumour-stromal interactions are not taken into account in simple monocultures - co-cultures are becoming more popular
Implementation: Breeding Strategies
- Legal requirementsL local ethics review, project license with specific endpoint which must not be exceeded, training and personal license - Chromosome location (for multiple alleles) - Numbers (power) - Time
LCIS
- Lobular carcinoma in situ - Abnormal cells form in the milk glands of the breast that are not cancerous but patient has increased risk of developing invasive breast cancer.
Molecular subtypes of breast cancer
- Luminal A - Luminal B - ERBB2 - Basal-like - Normal
Conditional gene expression
- Mammary-specific gene promoter (msgp) - Stage-specific - Exogenously inducible m-s transgene m-s KO m-s inducible KO m-s inducible transgene/KO
(Spontaneous) metastasis assays
- Metastases arise from orthotopic tumours - Tumour cells must be competent in all steps of metastasis - Tumour cells injected into vasculature via intracardiac or intravenous injection - bypassing crucial steps of metastasis
Lorns et al 2012: Mouse model for study of human breast cancer metastasis
- Metastasis in mouse models using human breast cancer cells was sporadic and at low rates - Lorns et al Used NSG (immunodeficient mice) which are highly susceptible to metastasis formation - Injection of aggressive human breast cancer carcinoma cells orthotopically into mammary fat pads - Human breast cancer cells frequently metastasise in severely immunocompromised NSG murine hosts - immune environment partly determines metastatic ability of the human breast cancer cells
Xenograft/allografts purpose
- Model breast cancer biology, tumour microenvironment angiogenesis, metastasis - Perpetuate and expand primary human tumours (high throughput screening) - Tumours have short life ex vivo
Cell lines applications
- Monolayer of normal and BC cells to look at cell behaviour/responses - 3D basement membrane (matrigel), culture of normal cells, architecture, polarity, differentiation into structures, start to see structures that mimic the mammary gland - 3D basement membrane culture of BC cells - neoplastic progression/ responses - Non-adherent mammosphere culture: tumour initiating cells studies - Testing cancer drugs, compariso of cell subtype responses to GEFITINIB, FACs, Proliferation Foxo3a
3D Culture
- Morphology and behaviour of breast epithelial cells in 3D reconstituted basement membrane accurately mimic breast structure and function - When breast cancer cells are grown in 3D cultures they fail to become growth arrested, lack polarity, display aberrant architectures and may become invasive
Differences between human breast cancer and GEM breast cancer models
- Mouse tumours metastasise primarily to the lungs (and liver), but getting progress (e.g. bone in 2005) - Contain less fibrosis and inflammation - Nearly all are hormone-independent (~50% of human breast cancers are hormone dependent)
GEM models with unique histologies compared to other GEM
- Myc - HER2 - Ras Distinct phenotypes are significant and suggest initiating oncogene profoundly affects tumour phenotype in mice and potentially humans
Structural organisation of nuclear receptors
- N-terminal domain - DNA binding domain - Hinge region - Ligand binding domain - C-terminal domain
Are cell lines representative of human breast cancer?
- No single cell line is truly representative but a panel of cell lines show heterogeneity in primary breast cancers - Differences detected in cell lines and primary tumours (e.g. copy number abnormalities in
ER
- Nuclear receptor - Estrogen receptor
ER alpha
- Nuclear receptor - Transcription factor - Helix 12 position determines co-factor binding - Estrogen-bound ER directly interacts with coactivators p300 or CBP at the AF-1 domain and with Src1, GRIP1, or AIB1 at the AF-2 domain - Tamoxifen-bound ER recruits co-repressors NCOR1 and SMRT to AF-domain - Mutations in amino acids have been shown to change co-factor binding, e.e. ER-351 mutation can weaken the binding of co-repressors in the presence of tamoxifen, but can still recruit other co-represors and co-activators to the ligand-indepedent AF-1 domain - Growth factor-mediated phosphorylation events promote ER activity
TP53 (p53)
- One of the most frequently mutated genes in breast cancer - Encodes p53 - guardian of the genome - Tumour suppressor gene - Regulates cell cycle
Oestrogen production
- Ovaries are the largest source of oestrogen in premenopausal women - Oestrigen derived from androgens
TEToff
- Presence of tetracycline, exoression from a tet-inducible promoter is reduced - Tetracycline-controlled transactivator - No tetracycline: tetR protion of tTA will bind tetO sequences and the actovation domain promotes expression - Tetracycline present: tetracycline binds to tetR, which precluded tTA binding to the tetO sequences and subsequent increase in expression of activation domain, resulting in reduced gene expression --> TETon is the opposite
Xenograft tumour
- Produced by injecting tumour cells into the flank or into mammary fat bad or immunocompromised mice - Tumour formation usually occurs rapidly and reproducibly - Large cohorts of tumour-bearing mice can be generated - Xenografts of hormone-dependent cell lines allow examination of hormone regulation in breast cancer - So widely used as preclinical models - Used to investigate metastasis, as part of metastasis assays
Gene expression profiling (GEP)
- Prognostic indicator
Implementation: Mixed vs pure backgrounds
- Pros and cons - Real life vs standardiation - Transplantation - Backcrossing (cross to parent) and speed congenics (congenics=differs from particular inbred stain by a specific gene, locus/genetic region) - Caution - modifiers
Prognostic biomarker
- Provide information on relapse risk and prognosis/ development of disease - Estimate/forecast effects of a particular treatment - Clinical outcome
Predictive biomarker
- Provides information about effect of a therapeutic intervention - Enables sub-stratification of patients for certain therapies - Treatment response
CSCs
- Recent studies have highlighted the potential role of CSCs in metastasis - Disaggregatng cells and sorting into difference groups (Flow cytometry or FACS) found that transplanting cell populations back into mice - only some grew back in the tumour - these are the CSCs - 3-4% of cells in tumour (on average) have the capacity to form a new tumour
Modelling Heterogenity in BC
- Relevance to human breast tumour biology 'elements of the disease' - Heterogeneity of BC: intra and inter > Genetic/epigenetic basis of disease > Disease kinetics/ metastasis > Drug response/ relapse, poor prognosis > Cancer stem cell hypothesis
Classic biomarkers
- Reliable and accurate measurement of a biological or pathological process or a pharmacological response to therapy
2. Selective estrogen receptor modulators (SERMS)
- SERMs > E.g. Tamoxifen - decreased 10yr risk overall > partial non-steroidal agonist in liver, bone, uterus, competitive receptor agonist in breast, brain > 5 yr recurrence down 50-80% > 5 yr death, down 20% 15 yr recurrence > overall death from 33% to 24% > Recommended for 10 yr in pre-peri-menopausal women
Xenograft advantages
- Short latency - High throughput - Reproducible bulk processing - Model ER+ human tumours (unlike GEM) - In vivo environment and behaviour e.g. metastasis
Mammary ducts
- Sit in stromal tissue and fat - Grape analogu - small alveoli are where milk is produced - Muscle cells around each alveoli which contract milk out following oxytocin stimulation -
Intraductal Papilloma
- Small, benign tumour which forms in milk duct in the breast
Mammosphere formation assay
- Sphere forming assay to estimate breast cancer cell potential to behave like stem cells - Specific density and number of cells are plated in low adherence plates within a different medium - Conditions prevent differentiation and allow self-renewal of stem or progenitor cells
1. Ovarian function suppression (OFS)
- Surgery replaced by chemical inhibition - Gonadotrophin releasing hormone agonists (GnRHa) act by downregulating pituitary GnRH receptors, thereby suppressing the release of LH and FSH - Upregulating top end of the pathway desensitising the pituitary gland to excess - Pituitary downregulates/ switches off response to hormone
TRAIL in breast cancer
- TRAIL only effective in ER-/mesenchyman - New data suggests that most CSCs are senstive to TRAIL irrespective of the tumour subtype
cFLIP/TRAIL in BC
- TRAIL preferentially targets bCSCs - cell line studies in vitro - bCSCs can be eliminated by TRAIL if cFLIP is inhibited - cell lines in vitro and in experimental metastasis xenografts - There is heterogeneity is response to TRAUL between CSC and non-CSC cells - irrespective of the subtype of breats cancer - but experiemtns only in cell line models currently
Piggott et al 2011: Suppression of cFLIP (apoptosis inhibitor) selectively eliminated breast cancer stem cell activity in response to TRAIL (anti-cancer therapy)
- Therapeutic targeting of bCSCs is hampered by heterogeneity and resistance to exiting therapeutics - Suppression of cCLIP by siRNA in 4 BC cell lines Sensitivity to TRAIL determined by complementary cell apoptosis assays (also a novel heterotypic cell assay) - Tumour initiating potential of cancer stem cell subpopulations determined by mammosphere culture, aldeflour assay and in vivo transplantation - siRNA of cFLIP resulted in partial sensitisation of TRAIL-resistance cancer cell line to pro-apoptocic effects of - TRAIL irrespective of phenotype - 10-30% cancer cell populations remained viable after TRAIL/cFLIP treatment - Mammosphere and aldefluor assay: pro-apoptotic stimulus selectively targeted the functional bCSC pool - Eliminating cell renewal TRAIL/cFLIP therapy could prevent metastatic disease progression in a broader range of BC subtypes - Athymic mice
Examples of constitutional vs conditional deletion: Tp53 and Pten
- Tp53 -/- viable and live to adulthood, get leukaemia/sarcoma - Tp53+/- KO viable and live to adulthood, combine with e.g. Brca1 -Pten -/- KO lethal - Pten +/- KO viable, spectrum of disorders (neurological/mammary tumour)
Stratification of breast cancer: Transcriptomics
- Transcriptomic - gene expression profiling - Subdivision of breast cancer into disease subgroups by gene expression maps onto original hormone status chorts - gene expression profiles identify existin and new gene networks and signalling pathways implicated in breast cancer - Luminal A, Luminal B, Basal-like, HER2-enriched, Normal-like
Current indications
- Trastuzumuab used 1 year (no additional benefit after this) as adjuvant or following chemo = 40% improvment in DFS - 98% 3 yr survival for small tumour node negative HER2+ - Trast + Pert + Chemo --> in metastatic setting - Trast + Pert --> in non-metastatic setting - Trat + Pert 6 months neoadjuvant
TNBC
- Triple negative breast cancer - 17% of BC - 75% basal-like - 5-10% bear BRCA1 mutation (90% BRCA1 are TNBC) - High risk of metastasis (lung, liver, brain, bone) - 15% TNBC develop brain metastasis, not well studied as few animals form brain mets - Short latency to metastasis, poor prognosis - Primary treatment: > Neoadjucant chemo: 17% pathological complete response > adjuvant combination chemo: 5yr survival 70%
Similarities between human and GEM breast cancer models
- Tumour formation resulted from multiple mutations - Tumours from GEM contained regions that resembled human breast cancer - Genes associated with human cancer induced cancer in mice
Prognostic marker examples
- Type/Grade - Nodal Status - Lymph/vasc invasion - Tumour size - Age - Ethnicity -Comorbidity
Tumour initiating cells
- Undergo self-renewal in mammosphere assays and when transplanted as xenografts give rise to tumours with differentiated cell types present in original tumours
Implementation: Genotyping
- When breeding animals you have to identify the ones you need - Design of PCR strategies - Cre and neoR cassettes
Genomic analysis
- Whole genome, exome seq or targeted - Biological drivers? - Similarity to human disease
Breast cancer cell lines
- Widely used - Proliferation, apoptosis, migration - Easily propagated - Easy to observe behaviour/ biochemistry - Used to study key properties of a cancer cell: proliferation, surivical, migration invasion - Tractable to genetic manipulation - Yield reproducible and quantifiable results - Human cells generally used - Steroid hormone dependence and other aspects of BC not well modelled in mice: human breast cancer cell lines exhibit hormone dependence -> progesterone, oestrogen - Cell lines can be grown as xenografts - allowing examination in vitro and in vivo - Need to account for different variants of the same cell lines which have different phenotypes
Target of TRAIL
- cFLIP implication for breast cancer stem cell therapy - The endogenous TRAIL-mediated apoptosis inhibitor cFLIP is downregulated in endocrine resistant BC cells- - Mechanism of endocrine-related TRAIL resistance is mediated by cFLIP - Endocrine-resistance mediated TRAIL sensitivity greatest in CSCs - Loss of cytoplasmic cFLIP confers sensitivity of CSCs to trail - Suppresion of cFLIP sensitises ER+ and ER- BC CSCs to TRAIL cytotoxicity
Cancer Cell-Autonomous TRAIL-R Signaling Promotes KRAS-Driven Cancer Progression, Invasion, and Metastasis
- mTRAIL-R promotes KRAS-driven lung and pancreatic cancer growth and metastasis - Human TRAIL-R2 promotes tumour growth, migration, invasion and metastasis - Endogenous mTRAIL-R constitutively activates Rac1 in vivo tumours - TRAIL-R2 expression positively correlates with the onset of metastasis in patients - Many cancers harbour oncogenic mutations KRAS -> cancer progression, invasion and metastasis
Luminal B
- p38 MAPK - Sonic Hedgehog - Notch - G1/S checkpoint - Sterol biosynthesis - TGF-beta, FGF
Next gen seq - exome sequences
- sequencing all protein coding region of genes in genome - includes mutation analysis with expression data - Deep sequencing of genomic variation > detects single nucleotide differences > Detects clonality/ heterogeneity - Now 10 subtypes of BC - including targetable mutations - But does it provide clinical benefit? Still problems with tumour heterogeneity and diver/passenger mutations
Luminal A
- val/leu/ile/glu degradation - glu metabolism - VEGF - IGF-1
Analysis of these additional genes...
...has helped to identify key oncogenic drivers, and disease modifiers, that reclassify disease subtypes and provide a molecular mechanism for disease - Luminal, HER2, Triple negative
Inhibiting HER2 in BC
1. Inhibit receptor tyrosine kinase activity (HER2 is an RTK) 2. Inhibit receptor dimerisation HER2 positivity drives CSC-like phenotype - have an increased population of CSC-like cells that exhibit increased tumour initiation and metastasis properties --> Trastuzumab can suppress this subpopulation
Decision making about breast cancer treatment based on:
1. Nodal status 2. Tumour size 3. Tumour type/grade 4. Lymphatic and vascular invasion 5. Tumour hormone receptor and HER2 status 6. Age 7. Ethnicity
Biomarker panel assay
1st gen prognostic signatures: mammaprint, 76-gene signature, oncotype DX - MammaPrint - Oncotype DX -PAM50 - Theros-Breast Cancer Index Advantages: - Reduces overtreatment - Treatment selection (e.g. selecting patient for chemo when classical biomarkers suggest not) - Mammaprint would have altered 34% of patients treatments BUT - Many different therapeutic strategies between clinics makes interpretation and clinical decisions more difficult to normalise, quality differs between different clinics - Sample collection varies dramatically and affects results - 1st generation prognostic signature tests limited for assessing ER- BC
Personalised medicine drugs for breast cancer
Biomarker: BRCA2/2 Drug: PARP inhibitors Compound: Olaparib Biomarker: Estrogen receptor Drug: Selective estrogen receptor modulators Compound: Tamoxifen for ER+ breast cancer. Biomarker: Estrogen receptor Drug: Aromatase inhibitors Compound: Letocole Biomarker: Estrogen receptor Drug: Estrogen receptor antagonist Compound: Fulvestrant Biomarker: ER Drug: mTOR inhibitor Compound: Everolimus Biomarker: HER2+ Drug: Monoclonal antibody Compound: Herceptin/Trastuzamab Biomarker: HER2+ Drug: Tyrosine kinase inhibitor Compound: Tykerb/ Lapatinib
Targeted therapies
Cdk4/6 (palbociclib) HDACs PI3K/mTOR (everolimus) SRC VEGF/angiogenesis (bevacuzimab/avastin) TRAIL (dulanermin) cdk4/6 + TAM = improved relapse free survival in mestastatic BC b + TAM = sensitisation to TAM c + AI = effective in progression free survival of metastatic ER+ patients but only in some patients (personalised medicine)
Stratification of breast cancer
Classical classification systems: - DCIS - LCIS - Intraductal Papilloma - Infiltrating DC
Temporal regulation
CreERT2 and FLPERT2 CreERT2 (Cre with oestrogen receptor) absence of tamoxifen- bound to heat shock protein. Once adding tamoxifen, binds, so it can go to the nucleus and recombine DNA Doxycycline: TetON or TetOFF. Involves 2 transgenic elements > TetOFF: TET transactivator bound to tetra or doxycycline, binds response element and blocks transcription, remove doxycycline allows transcription to occur > TET on- opposite system - Ubiquitously expressed promoter use: R26R (drives Cre or Flp for whole body expression, often inducible, otherwise a germline mutation is required) - Tissue-specific promoters - Cell-specific promoters: Colon in (Cre driven by Lgr5 promoter, or Cre driven by Ah-cre promoter which Kos GOI in TACs)
Databasing
Databases have to be detailed and accurate - one mistake and you can waste weeks or months and a lot of money breeding the wrong animals
Examples of genes in GEM of breast cancer
Effects of loss of tumour suppressor genes: Trp53, Brca1, Pten Effects of GOF in oncogenes: Erbb2/Neu/HER2, Myc, Ccnd1, PyMT, Hras - Cre/loxP, TET-inducible transgenes, BAC transgene, retroviral mediated delivery of transgene
Human cell lines: non-tumorigenic MEC lines
Human cell lines - Very few established cell lines - Mostly, short term from mammoplasties Mouse cell lines - More lines (readily available) - Spontaneously immortalised: differentiated (milk) - May be karyotypically unstable and lack complete MED phenotype (cv primaries) - Widely used to study MED functions and early steps tumorigenesis
Applicability to human disease
Important for findings
1. Inhibit receptor tyrosine kinase activity (HER2 is an RTK)
Lapatinib - Binds intracellular ATP pocket - Inhibits autophosphorylation
Breast cancer types
Luminal A (ER+PR+HER2-KI67-) postmenopausal, slow-growing, responds to hormone therapy Luminal B (ER+PR+HER2+KI67+ or ER+PR+HER2+KI67+)- LN metastasis, may respond to chemo Normal breast like (ER+PR+HER2- basal marker+)- well differentiated Basal like (triple negative; ER-PR-HER- basal marker+)- associated with BRCA1, high grade, aggressive HER2 enriched (ER-PR-HER2+) Subdivision of BC into disease subgroups by gene expression maps onto riginal hormone status cohorts
Conditional oncogene activation
Only activate in tissue/ time of choice
Endocrine therapy (ET)
Past standard of care for advanced BC in 1970s: hypophysectomy, adrenalectomy, oophorectomy ET current: 1. Ovarian function suppression (OFS) 2. Selective estrogen receptor modulators and downregulators (SERMS, SERDs) 3. Aromatase inhibitors (prevent production of oestrogen) 4. Combinations with targeted therapies
Other analysis: Penetrance and gross findings
Penetrance: proportion of individuals carrying a particular variant/allele of a gene (genotype) that also express an associated trait (the phenotype)
Personalised medicine key objectives
Prevention: - Early intervention: forecast clinical outcome before symptoms develop Prediction - Avoid adverse side effects...is chemo needed? - Instructs treatment...risk of developing metastasis - select optimal response...type of chemo? Participation - Patients more likely to comply with treatments
Tisse-specific promoters
Prostate: PSA, Probasin Mammary: K14, K5, Blg, WAP, MMTV Brain: GFAP, Nestin Skin: K5, K14 Lung: adenovirus Blood: transplant Stomach: TFF1, Cln18 Pancreas: PDX1 Colon: AhCre, Villin, Lgr5 (dosing)
Targeted therapies in ER+ BC
Purpose: increase sensitivity, overcome acquired resistance
DFS
The length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer
Omics
Transcriptome Genome ncRNAs Proteome etc
2. Inhibit receptor dimerisation
Trastuzumab/ Herceptin - Monoclonal Ab - Binds ECD IV - Blocks ligand - Prevents ligand-independent dimerisation of HER2 - Not so effective at preventing heterodimerisation - Can suppress CSC-like subpopulation Pertuzumab - Monoclonal Ab - Binds ECD II - Inhibits ligand-dependent heterodimerisation - 2nd therapeutic Herceptin + Petuzumab + Docetaxel treatment
3. Aromatase inhibitors
Type I: Steroidal E.g. ecemestane Type II: Non-steroidal E.g. letrozole, anstrozole The drug of choice for advanced metastatic ER+ BC in post-menopausal women (letrozole) - interferes with oestrogen production in pre-monopausal women. However lack of oestrogen results in a feedback mechanism to produce more oestrogen so the drug is more effective once ovaries have shut down. Prevents residual production of oestrogen and no feedback to ovaries