Kinases Flashcards
Describe two different molecular techniques that have been used to enhance our understanding of the pathological dysregulation of a specified molecule.
- Specified molecule introduction (1) (1/6) (FAK; general; activation; cancer)
- Focal Adhesion Kinse (FAK) = cytoplasmic PTK
- activated by matrix & integrin receptors
- critical role in integrin-mediated signal trans.
- In integrin-mediated cell adhesions:
- FAK = activated via disruption of an auto-inhibitory intramolecular interaction b/w it’s N-T FERM domain & the central kinase domain
- Autophos @ Y397 = HM of FAK activity
- Activated FAK forms complex w/ Src family kinases (SFK)
- Initiates multiple downstream sig path via phos of other prot
- Integrin signalling through FAK has been shown to promote cell migration in many in vivo and in vitro studies
- Consistent w/ these roles (in migration & angio) ↑ XPS’ion and/or activation of FAK = found in variety of cancers.
- Small molecule inhibitors for FAK activity may represent novel therapies
Describe two different molecular techniques that have been used to enhance our understanding of the pathological dysregulation of a specified molecule.
- Specified molecule introduction (1) (2/6) (guide for therapeutics)
- FAK = observed to have resistance to platinum-chemotherapy, other treatments = vital
COMBINATIONAL EFFECTS OF CISPLATIN AND FAK INHIBITION
- CisPlatin can ↑FAK p-Y397, so FX’s of low-dose CP treatment in presence/absence of a FAK-I were measured (tumorsphere formation, ALDH activity, cell viability)
- Try & tackle the problem that CP causes - can go back to usual role
- Cell viability & ALDH activity = measured (functional marker of various tumour types)
- CP treatment - ↑ tumoursphere formation & ALDEFLOUR activity (assay used to ID & isolate cells w/ ↑ ALDH activity, indicative of tumour growth): suggests low dose CP serves as an activation-type stress of FAK
- FAKi treatment - ↓ tumoursphere formation & ALDEFLOUR activity comp to control OVCAR3 & KMF cells
- FAKi ≠ directly cytotoxic, only CP + FAKi decreased tumoursphere viability
- single agent CP/FAKi treatment ≠ alter growth/viability in 2D culture
- 3D conditions: FAKi ↓ FAK Y397-p &↑ % of KMF/OVCAR3 cells in G1 phase of cell cycle
FINDINGS INDICATE:
- ability of FAKi to decrease 3D cell proli & exert a combinational activity w/ low-dose CP to promote apoptosis make it a potential therapeutic combination
Describe two different molecular techniques that have been used to enhance our understanding of the pathological dysregulation of a specified molecule.
- Specified molecule introduction (1) (3/6) (difficulty in treatment, why do we need others?)
- often over-XPS’ed & activated in several advanced-stage solid cancers
- FAK promotes tumour progression & metastasis
- High-grade Serous Ovarian Cancer (HGSOC) tumour microenvironments are enriched w/ matrix proteins that are FAK activators
- FAK knockdown & FAKi studies suggest a imp role for FAK in promoting invasive tumour growth, but downstream targets of FAK = varied & may be tumour/stroma-context dependent
- phenotypes ass with FAK KO can be distinct from FAKi phenos, b/c kinase-inactive FAK retains its important scaffolding roles
- Several ATP-competitive FAKi have been developed
- Some HGSOC cells can resist the DNA damage & stress induced by platinum chemotherapy & therefore = resistant to treatment
Describe two different molecular techniques that have been used to enhance our understanding of the pathological dysregulation of a specified molecule.
- Molecular techniques (1) (4/6) (new in vivo murine epithelial ovarian cancer model)
- created a new in vivo model using ID8-IP cells that had gains in Kras, Myc, & FAK genes (KMF-cell)
- murine KMF cells contain several gains/losses in genes common to the top 20 set of genes that are altered in HGSOC
Describe two different molecular techniques that have been used to enhance our understanding of the pathological dysregulation of a specified molecule.
- Molecular techniques (1) (5/6) (serial sections; immunohistochemical staining, quantitative image analysis)
- ↑degree of Pax8 & pY37 FAK co-localised staining in primary biopsy samples
- pFAK had both cytoplasmic & nuclear localisation
- pFAK staining was:
- Higher in ovarian tumour vs surrounding stromal cells
- Remained high in non-necrotic tumour samples, obtained after multiple rounds of neoadjuvant CT
- Pre & Post-CT, FAK lvls trended upwards in tumours that survived CT
- establishes an association b/w FAK signalling & tumour chemo-resistance.
Describe two different molecular techniques that have been used to enhance our understanding of the pathological dysregulation of a specified molecule.
- Molecular techniques (1) (6/6) (analysis of anchorage-independent PromoCell media)
- used this method to help devise a mech that may explain why ovarian cancer cells are particularly resistant to CT
- during tumoursphere formation and/or in response to CP (cisplatin; CT drug) stimulation, there is adhesion-indep non-canonical FAK activation
- FAK pY397 = marker associated w/ adhesion & increased T stiffness
- during staining they observed pY397 FAK staining, not expected b/c pFAK was rapidly lost when cancer cells were removed from the 2D cultures
- Explanation: when the culture time was increased, OVCAR3 cells cultered in anchorage-independent PromoCell media resorted their phosphorylation of FAK
- These cells had clustered to form tumourspheres
- CP treatment of these cells increased FAK pY397 & pβ-catenin
- β-catenin = FAK SBT that promotes β-catenin activation in endo cells
- The above supports idea that adhesion-independent non-canonical FAK activation may occur during
- tumoursphere formation, or
- in response to CP treatment - making them difficult to treat
