Cell cycle deregulation in cancer Flashcards
what are the 6 essential hallmarks required for cancer to become malignant?
- ability to grow autonomously (without growth signals/factors)
- disregard cytostatic signals
- ignore apoptotic signals
- stimulate angiogenesis
- invade and metastasise
- become immortal - can keep dividing
what controls cell proliferation?
growth factors
what are growth factors (GFs)?
GFs are relatively small proteins released by some cells, which travel through intercellular space and convey messages to other cells
- they are mitogens = ability to induce cell proliferation
what happens if normal cells are in the presence or absence of GFs?
presence: the cells divide
absence: the cells remain quiescent and do not divide
almost all normal cells require signals to grow and divide
what happens if cancer cells are in the presence or absence of GFs?
cancer cells proliferate no matter the presence of absence of GFs
- cancer cells do not need GFs to proliferate, they do this autonomously
what is growth vs quiescence?
the growth vs quiescence decision determines whether cells proliferate and divide or stay in a resting state
- The decision of growth v/s quiescence must be taken in consultation with neighbouring cells within the tissue
what determines the growth vs quiescence decision?
growth factors
what normal cells are an exception to growth signals being needed for proliferation?
embryonic stem cells: mouse ES cells grown in vitro appear able to drive their own proliferation through internally generated signals
- Only example of WT cells able to generate a benign tumour (teratoma) when injected in an adult organism = tumorigenic
why are external growth signals required for proliferation?
- To maintain the precise structure of the tissue in which the cells are located
- So that cell proliferation occurs across the tissue, not just individual cells, in order to maintain organ function
- If not controlled – organ function compromised
what leads to the sustained proliferative signalling of cancer cells?
- the cell cycle machinery is influenced by cancer-associated proteins (oncogenes and tumour suppressors) which disrupt normal control mechanisms
- sustained proliferative signalling
- cancer cells don’t need presence of GFs to grow
how do growth hormones work in normal cells?
growth hormone leads to signalling cascade, leading to proteins being translated to trigger growth
in what ways may cancer cells induce uncontrolled cancer proliferation?
- Cancer cells may produce growth factors by themselves autocrine proliferative stimulation
- Cancer cells send signals to stimulate surrounding normal cells to produce growth factors – increase tumour size
- Cancer deregulation of growth factor receptor signalling elevated level of receptors or ligand-independent firing
- Constitutive activation of signalling DOWNSTREAM of growth factor receptors
- Increase activity of signalling cascade - Disruption of negative feedback mechanisms that attenuate proliferative signalling
what causes uncontrolled cell proliferation?
the deregulation of the cell cycle
what are the 5 phases of the cell cycle?
- G0/quiescent - cells do not proliferate
- gap-1 (G1) - cells either decide to stay in G0 or progress to S-phase
- S-phase - DNA is replicated
- gap-2 (G2) - preparation for mitosis
- mitosis
what cells can be in G0/quiescence phase?
terminally differentiated cells are in a permanent quiescent state
other cells are transiently in a quiescent state and can re-enter the cell cycle
what is the longest phase of the cell cycle?
G1 - main point where cells decide to stay in G0 or continue onto cell cycle
what occurs during mitosis?
duplicated DNA is equally divided into 2 daughter cells, and cytokinesis splits their cytoplasms to form 2 individual daughter cells
what is the growth vs quiescence decision?
- There is a discrete window to consult the extracellular environment: from the onset of G1 phase to an hour or 2 before the G1-to-S transition.
- G1 decision-making machinery apparent in the responses of cultured cells to extracellular signals
when does the growth vs quiescence decision occur?
toward the end of G1
what occurs during the growth vs quiescence decision?
- Serum and growth factors removed before the cells have completed 80-90% of G1 -> cell fail to proceed further and revert to G0 state
- Serum and growth factors removed in the final hr of G1 -> G1 completed, so proceed to S, G2 and M phase
Cells can only respond to the signals in their environment called a certain point - This is called the restriction point (R point)
what is the restriction point (R point)?
- A weighty decision must be made towards the end of G1: here a cell must make up its mind whether it will go to S, remain in G1 or go to G0 (retreating from active cycle) -> made at a transition called R point
- If a cell should decide at the R point to continue advancing through the cell cycle, it commits itself to proceed into S and complete a programmed series of event (s, G2 & M) that enable it to divide into 2 daughter cells, even if growth factors are not present any longer
what other factors can influence the growth vs quiescence decision?
anti-mitogenic factors such as TGFb are able to impose their growth inhibitory effects only during this period in early and mid-G1
how is the growth vs quiescence decision and R point changed in cancer?
In cancer, cell cycle is similar to normal cells (late G1-to-M phases), but the R-point decision-making machinery is changed/deregulated
what methods can be used to study the cell cycle?
- flow cytometry
- immunofluorescence
- FUCCI system
what is the process of flow cytometry in studying the cell cycle?
- Cell cycle is analysed by measuring DNA content inside each individual cell
- Cells are treated with a fluorescent dye that labels DNA quantitatively
- As the DNA content doubles during the S phase, the intensity of fluorescence increases in proportion.
what does flow cytometry show about the DNA content at different phases of the cell cycle?
- As the DNA content doubles during the S phase, the intensity of fluorescence increases in proportion.
- Thus, cells in G0 and G1 phase (before S) have half the fluorescent signal as those in G2 or M phase
- Quiescent cells have small peak for G2-M, but in dividing cells G2-M peak is much higher, as more DNA is present and they are undergoing proliferation
what are the limitations of flow cytometry?
- can only run single cells - cannot analyse whole tissues
- samples must be fixed, so can only be used in vitro, not in vivo
how can immunofluorescence be used to study the cell cycle?
- The progression through the cell cycle can be measured by staining for proteins that are specifically expressed in different phases of the cell cycle
- Can use combination of stains to determine where in the cell cycle the cells are
which protein stains correlate to which phase of the cell cycle in immunofluorescence?
BrdU = bromo-2-deoxyuridine replaces thymidine during DNA synthesis -> short pulse identifies cells in S phase
Cyclin B1 = G2/M marker
Histone H3 = role in mitotic chromosome condensation, phosphorylated during mitosis
which cell cycle phases are positive for which protein stain in immunofluorescence?
- Cells in G1 will be negative for all markers
- Cells in S will be positive for BrdU and negative for rest
- Cells in G2/M will be positive for cyclin B1 and negative for rest
- Cells in mitosis will be positive for Histone H3 and negative for rest
what are the limitations of immunofluorescence?
- not quantitative - qualitative image analysis
- samples must be fixed, so can only be used in vitro, not in vivo
how can mitosis be studied using immunofluorscence?
- Mitosis can be been monitored using immunofluorescence & epifluorescence microscopy
- Although not quantitative, this method allows the visualisation of the different sub-phases of mitosis.
- Useful when designing drugs which interfere with mitosis, microtubule poisons and anti-mitotic drugs are in clinical trials for a variety of cancer types
what does the FUCCI analysis of the cell cycle enable?
visualisation of the cell cycle in vivo
why is it difficult to analyse proliferation in living organisms?
Difficult to analyse proliferation in living organisms because traditional cell cycle markers rely on immunofluorescent detection -> tissue fixation
what does FUCCI stand for?
Fluorescence Ubiquitin Cell Cycle Indicator
what are the 2 components of the replication control system of eukaryotes? how are these expressed in FUCCI?
- Licencing factor Ctd1 (hCTD1) -> peaks in G1 before the onset of DNA replication, and declines abruptly after the initiation of S phase = RED
- If cells are red they are in G1 - CTD1 inhibitor Geminin (hGem) -> expressed at high levels during S and G2 phase, low levels during mitosis and G1 = GREEN
- If cells are green they are in S and G2
what is dual FUCCI?
in zebrafish, both reporters expressed from a single transgene – no need to cross zebrafish
how is FUCCI used in a transgenic mouse?
- Mouse expressing hCdt1 crossed with mouse with hGem marker, to produce hCdt1-hGem mouse
- Every somatic cell nucleus exhibited either red or green fluorescence.
- E13 FUCCI transgenic embryo were fixed and coronal sections of the brain were prepared.
- Red and green fluorescence was examined in every section using confocal laser scanning microscopy
- Red and green expressing cells were identified in the developing cerebral cortex.
- Bright red cells -> post-mitotic neurons (hCdt1 accumulation after cell cycle exit)
what does FUCCI analysis allow?
This technology allows for in vivo analysis of spatial and temporal patterns of cell-cycle dynamics, owing to the brightness of the fluorescence and the high contrast between the red and green colour
what fluorescence do the different cell stages express in FUCCI analysis?
Early G1 = no/low red fluorescence
Late G1 = strong red fluorescence
G1/S = red and green fluorescence (yellow)
S/G2/M = green fluorescence
how is the cell cycle linked to stem cell differentiation?
The cell cycle state of stem cells determines cell fate propensity
- the phase that the cells are in is important for cell lineage decisions
how can the cell cycle and stem cell fate be analysed?
Generated FUCCI human embryonic stem cells and used FACS sorting isolate cells at different stages of the cell cycle -> induction of differentiation
- Stimulated the cells with different factors to form one of the 3 germ layers – stem cells will take a certain fate depending on which stage of cell cycle they are in
what cell fate is induced when the stem cells are triggered to differentiate in early G1?
Early G1 stimulation differentiated to endoderm or mesoderm, cannot differentiate to neuroectoderm
what cell fate is induced when the stem cells are triggered to differentiate in late G1?
Late G1 stimulation differentiated to neuroectoderm, not endoderm or mesoderm
what cell fate is induced when the stem cells are triggered to differentiate in S/G2/M?
Cells in S, G2, M did not differentiate into the germ layers
what is metastasis?
Metastasis = formation of secondary tumours in secondary sites
- Multi-step process, starting with the invasion of cancer cells into surrounding tissues
- Cancer cells in primary tumour acquire ability to invade into surrounding tissues and blood vessels
- ~90% of cancer death are due to metastasis
does the cell cycle phase determine invasive ability of a cancer cell?
yes :D
how can the impact of the cell cycle on cancer metastasis be analysed?
Invasive breast cancer cell line expressing the FUCCI reporters:
- Cells grown as 3D organoids embedded in extracellular matrix
- Can monitor if the cancer cells can leave the organoid and migrate into the ECM
- red cells in G1
- green cells in G2
which cell cycle phase is linked to cancer invasive ability?
- Cancer cells that can undergo metastasis first are in G1 phase and G2 phase cells behind
- Red cells always at the front
- The most invasive cells are the cells G1
why are the cells that are in G1 more invasive?
Cancer cells in G1 express protein which degrades the ECM, allowing the rest of the cells to then migrate through
why is it difficult to both inhibit the proliferation and metastasis of cancer?
To stop proliferation of cancer, cells in G1 phase must be blocked
- However, while this will prevent tumour growth, it will enable the cancer cells to become metastatic and spread around the body
how is progression through the cell cycle controlled?
by cyclin/CDK complexes
what are CDKs?
cyclin-dependent kinases: kinases deployed by the cell cycle machinery to help the cells progress from one phase to the next
what regulates CDKs?
accessory proteins called cyclins
- in the absence of cyclins, CDKs are inactive
- if cyclins are present, CDKs are active and can phosphorylate downstream effectors
what are cyclin/CDK complexes responsible for?
- CDKs/cyclin complexes are responsible for sending out the signals to the proteins that carry out the work to move the cells through the cell cycle (via phosphorylation)
- Activity of CDK must only be present under specific phases of cell – ensure correct direction of progression
what mechanisms are used to regulate CDK activity?
- abundance of cyclin subunits
- cyclins activate the catalytic activity of CDKs by 400,000 fold e.g. cyclin A/CDK2 - Cyclin/CDK association - cyclins help substrate recognition of the complex in the cell
- CDK targets can only bind to CDK if cyclin is present - activating/inhibiting phosphorylation events
- if one side of of CDK is phosphorylated, it is active
- if ATP-binding site of CDK is phosphorylated, it is inactive - CKI proteins inhibit CDK: INK4 family and CIP/KIP family
- when CKI proteins are removed, the ATP binding site is no longer phosphorylated, so CDK can activate
how are CDK/cyclin complexes inhibited?
when the cyclin is phosphorylated and ubiquitinated
what do cyclins ensure the coordination of?
- The synthesis of individual cyclins coordinates the sequential completion of DNA replication and cell division
- Collapse of cyclin levels as the cell progresses through the cell cycle occurs via degradation (ubiquitination-dependent)
- Cyclin proteins are removed from cells via degradation to inhibit the complex
- therefore, the cell cycle can only progress in one direction
how do the cyclin/CDK levels vary during the cell cycle?
- Cyclin D/cdk4/6: high levels in G1
- Cyclin E/CDK2: low levels throughout most of G1, rapid increase after the R point
- Cyclin A/CDK2: levels increase in concert with the entrance in S phase
- Cyclin B/CDC2: levels increase in anticipation of mitosis
what are CKIs and what are the 2 families of them?
CKIs are CDK-inhibitors
2 families:
- INK4
- CIP and KIP
What is the role of CKIs?
they bind to CDK/cyclin complex and inactivate them to inhibit cell cycle progression
which part of the cell cycle is deregulated in cancer?
the restriction point
which cyclin/CDK complexes are deregulated in cancer?
- Cyclin D with CDK4/6
- Cyclin E with CDK2
what are cyclin Ds?
- D-type cyclins convey messages from the extracellular environment to the cell cycle clock in the nucleus
- Levels of D do not vary dramatically as the cell advances through the cell cycle
- Several pathways downstream of GF receptors stimulate accumulation of D.
- D half-life ~30min
- Because the levels of D fluctuate together with the levels of extracellular mitogens, D constantly inform the cell cycle clock of current conditions in the environment around the cell
how are cyclin Ds regulated?
- D-type cyclins are controlled by extracellular signals: growth factors + integrin-mediated ECM attachment
- Different growth factors and cytokines converge to promote expression of cyclin D1
- Integrins also stimulate expression of cyclin D1 to promote cell cycle progression
- Removal of GFs -> rapid collapse of cyclin D1 levels
which cyclin D is the most studied?
- Cyclin D1 is crucial in growth, and is deregulated in cancer
how is cyclin D deregulated in cancer?
- Cancer-associated mutations in cyclin D1 results in constitutive nuclear localisation & impaired degradation of cyclin D1
- Cyclin D1 has been found to be overexpressed in breast, lung cancer, melanoma & oral squamous cell carcinomas
- Oncogenic activation (e.g. Ras) leads to increased cyclin D1 expression
- Cyclin D1 KO mice do not form mammary tumours upon oncogene expression - for oncogenes to drive tumour formation, cyclin D1 must be present
what does overexpression of cyclin D promote?
- cell proliferation
- increased anchorage-independent growth (doesn’t need attachment to basement membrane to grow)
- reduced chemotherapy sensitivity
- elevated survival in presence of cisplatin (chemotherapy drug)
- still requires serum to form a tumour
what does downregulation of cyclin D promote? what does this show?
- reduction of tumour growth and reduced proliferation
- induction of apoptosis, due to G1 arrest in absence of cyclin D
shows that cyclin D is essential for the growth of cancer cells
- for tumour formation to occur, cyclin D must be present
does inhibition of cyclin D by shRNA lentiviruses cause regression?
Targeting cyclin D1 could be good to limit tumour growth when you already have a tumour (not regression, tumour is just growing less)
what is a Kaplan Meir survival curve?
non-parametric statistical method used to estimate survival function correlating to the level of a protein
- Each downward step in the lines represents an event (e.g. death) experienced by a patient in the corresponding group
what does overexpression of cyclin D correlate with?
lower overall survival in gastric cancer patients:
- High level of D1 leads to lower survival of patient (low cyclin D1 leads to higher survival)
- Cyclin D1 is overexpressed in poorly differentiated gastric tumour -> poor prognosis
- Cyclin D1 overexpression is associated with increased invasion and metastasis
- Cyclin D1 can have a prognostic role in gastric cancers
what is the role of cyclin E in the cell cycle?
After the R point, E-type cyclins (E1 and E2) associate with CDK2 phosphorylation of substrates required for entry in S phase
how is cyclin E deregulated in cancer?
- Cyclin E gene amplification was observed in 15 different tumour types, leading to cyclin E overexpression
- E1 is overexpressed in 40% of ovarian high-grade serum carcinomas (HGSCs), as a result of gene amplification in half of the cases cell cycle progression & resistance to therapy
how does High Grade Serum Ovarian Carcinoma progress?
- High Grade Serum Ovarian Carcinoma (HSOC) originates in the fallopian tube epithelium (FTE)
- P53 mutations occur in the early stages of tumorigenesis, leading to cellular metastasis
- Primary tumour is formed in the ovary, and can then form secondary tumours in peritoneum
- STIC = non-invasive tumour, characterised by high proliferating ability
what does overexpression of cyclin E promote?
- Cyclin E1 overexpression in vitro strongly promotes cell growth of primary tumour cells
- Cyclin E1 overexpression promotes clonogenic growth and loss of contact inhibition
- Cyclin E1 has an oncogenic role in ovarian carcinoma
- Cells overexpress mutant p53 as well
- CCNE1 (cyclin E gene) overexpression drives phosphorylation of CDK2 = CDK activation
what does cyclin E overexpression correlate with?
Patients with Cyclin E1 overexpression correlates with poor ovarian cancer survival (HGSOC)
- Cyclin E1 amplifications are associated with increased mRNA expression
- Protein overexpression are associate with poor survival of ovarian cancer patients
- Cyclin E overexpression occurs in early cancer lesions -> specifically involved in HGSOC development
what is produced when cyclin E is cleaved?
Cleavage of cyclin E results in the expression of low molecular weight cyclin E -? stable protein, higher affinity for CDK2
what does cyclin E cleavage correlate with in breast cancer?
Cyclin E cleavage correlates with poor prognosis in breast cancer
- Cleaved form of cyclin E1 was only observed in cancer leading to cell cycle deregulation & resistance to therapy
- Cleaved cyclin E may be a good protein to target cancer
- Cohort of 515 UK breast cancer patients: patients positive for low molecular weight cyclin E showed worse prognosis in all breast cancer subtypes analysed
what kind of extracellular signals promote cell proliferation?
growth factors/mitogens
what kind of extracellular signals inhibit cell proliferation and cell cycle progression?
Transforming growth factor-beta (TGF-beta) inhibits cell proliferation and cell cycle progression in normal cells
how is TGFb signalling implicated in cancer?
Early stages of tumour formation: TGFb arrests the growth of many cell types and blocks cell progression
Later stages of tumour progression: TGF-beta contributes to tumour invasiveness
what is TGF beta signalling pathway involved in?
TGFb signalling pathway is involved in many cellular processes (cell growth, cell differention, apoptosis & cellular homeostasis)
what i the process of TGFb signalling?
- TGFb superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor.
- The type I receptor then phosphorylates receptor-regulated SMAD2/3 (R-SMADs) which can now bind SMAD4 (common SMAD or coSMAD).
- R-SMAD/SMAD4 complexes translocate and accumulate in the nucleus where they act as transcription factors
- Smad phosphorylation unmasks a nuclear localisation signal -> nuclear translocation of the SMAD complex
- Interaction of Smad with transcriptional co-activators and repressors
- SMAD7 = inhibitory SMAD, interacts with TGFbR1 to inhibit SMAD binding & promoted TGFbR ubiquitination & degradation
how does TGFb control cell cycle progression?
TGFb strongly increases the levels of p15-INK4B (CKI), leading inhibition of cyclin D-CDK4/6 complexes, so cells can’t reach the R point
Activation of TGFb signalling causes weak induction of p21-Cip1 (CKI).
-Stronger induction of P21-CIP upon DNA damage, causing cell cycle to be halted until the genome is repaired
- This ensures a cell does not progress into S-phase & copy damaged DNA
how do mitogenic factors promote cell cycle progression?
Mitogenic factors mute the action of CKI to favour cell cycle advance:
- Akt phosphorylates p21-Cip1 in the nucleus, leading to translocation of P21 out of nucleus and into the cytoplasm
- Akt phosphorylates p27-Kip1 in the cytosol and prevents P21 nuclear translocation
- therefore, cyclin/CDK complexes are no longer inhibited by CKIs in the nucleus, so can remain active and promote cell cycle progression
how do mitogens and TGFb compare in controlling cell cycle progression?
Mitogens act in an opposite fashion to TGFb
- mitogens inhibit CKIs and promote cyclin/CDK complexes to promote cell cycle progression
- TGFb activate CKIs to inhibit cyclin/CDK complexes, thus inhibiting cell cycle progression
what is an example of a mitogenic factor?
PI3K pathway, activated directly or indirectly by growth factor receptors Akt = kinase activated downstream of PI3K
what is promoted when constitutively active Akt is expressed in cells?
- CKI p21 is excluded from nucleus and localised in the cytoplasm, meaning cyclin/CDK complexes in the nucleus can remain active and promote cell progression
what is promoted when Akt is downregulated?
When dominant negative Akt is expressed, p21 can localise in the nucleus and inhibit CDK-cyclin complex, thus inhibiting cell cycle progression
how does Akt correlate with breast cancer?
Akt activation induces cytoplasmic p27-Kip1, so cyclin/CDK complex in nucleus is no longer inhibited
- These effects on intracellular localisation appear to have clinical consequences.
- Low grade (less advanced) human mammary carcinomas: low levels of active Akt, so p27 in the nucleus -> able to exert anti-proliferative functions
- High-grade tumours: stronger Akt activation, so cytoplasmic p27
- Patients bearing primary tumours with nuclear p27 (green) have a relatively good prospect of long-term, disease-free survival over a period of 6 years after the diagnosis
- Patients with nuclear + cytosol localisation suffer significant relapses, many leading to death.
does correlation with clinical outcome prove causation?
correlation with clinical outcome does not prove causation: i.e. that p27 mis-localisation causes clinical progression
- BUT important as prognostic marker
