Lecture 7: Cancer Hallmarks and Ca Signalling:

Why is Ca²⁺ Described as the Most Versatile Signal in the Body?

• It is used in:

  • Muscle contraction

  • cell division

  • Secretion

  • sensory function

  • nerve transmission

  • brain function

  • blood pressure and heart function

• Spatiotemporal shaping of Ca2+ signalling allows for the regulation of a wide range of physiological responses in a range of tissues throughout the body

What are Hanahan’s and Weinberg’s Hallmarks of Cancer?

• They are key hallmarks of cancer → specific phenotypic responses of cancer cells that define them as cancer cells

• This includes:

  • Sustained proliferative signalling

  • Evading growth suppressors

  • Migration/invasion and metastasis

  • Enabling replicative immortality

  • Inducing angiogenesis and vascularisation

  • Resistance to cell death.

  • Metabolic reprogramming (Added in 2011) → confers growth advantage

• Remodelling of Ca machinery, Ca signalling and homeostasis has a critical role in each of these hallmark responses

  • Currently, no drugs are designed that target this.

How Can Enviromental Factors Cause or Increase the Risk of Cancer?

• Environmental factors like smoking, alcohol, ageing and hormonal changes can have genomic effects (mutations) and epigenetic effects (microRNAs, DNA acetylation) and adaptive changes (altered gene expression → oestrogen sensitive breast cancer or testosterone sensitive breast cancer)

• This can lead to increased or uncontrolled cell proliferation, reduced cell death, increased migration/invasion, tumour vascularisation and invasion, which leads to metastasis, which increases morbidity and death

  • Remodelling of Ca signalling machinery is involved here

What is Metastasis?

• Process where cancer cells break free from their natural tissue constraints and enter the circulation, invading the blood vessels and microcirculation, before attaching to another organ

• Certain primary cancers have a propensity to metastasise in other organs → preventing this may act as a cure

  • e.g. pancreatic (& colon cancer) metastasis in the liver due to the connection via portal circulation supply

  • e.g. melanoma metastasis in the brain due to common embryological origin → melanoctes derived from neurons → have common features and factors which are then recognised by cancer cells, facilitating metastesis

Which Ca Channels Are Implicated in Regulating Cell Proliferation and Cell Cycle

• These Ca channels generate spatial Ca entry signals (localised signals) to activate calcineurin, allowing for NFAT signalling, driving proliferation

  • Store-operated Ca²⁺ channels (SOCE), consisting of ER STIM1 and Orai1

  • Arachidonate-regulated Ca²⁺ channels (ARC), consisting of PM STIM1 and Orai1/Orai3 heteropentamers),

  • TRPC6 AND TRPV6

  • Non-SOCE (plasma membrane SPCA-regulated Orai1)

• These channels are co-localised with calcineurin

Why is the Spatial and Temporal Properties Of Ca Important

• The specific spatiotemporal patterns of Ca²⁺ signals (amplitude, frequency, duration, location) differentially regulate gene transcription and transcription factors via

  • calcineurin → NFAT pathway

  • CAMKII → CREB pathway

  • immediate early genes (Jun, Myc and Fos).

• Ca is important in regulating many transcription factors, especially in cell proliferation and cancer

How does calcium regulate transcription factors and the cell cycle in cancer cells?

• Calcium is crucial for regulating transcription factors involved in cell cycle control.

  • NFAT: Sustained Ca²⁺ activates calcineurin, which dephosphorylates NFAT and allows NFAT to enter the nucleus via the nuclear pore complex and activates cell cycle genes that control M→G1 transition.

  • CREB is activated by Ca²⁺ and also regulates M→G1 transition.

  • Retinoblastoma protein (Rb1) is activated by Ca²⁺ and a key factor for cell cycle regulation.

  • CAMKII is activated by Ca²⁺ and regulates G2→M transition, centrosome-chromosome segregation, and triggers cell division.

• Different spatiotemporal properties of Ca signalling and the frequencies and patterns of Ca²⁺ oscillations can selectively regulate these transcription factors.

  • High-frequency Ca²⁺ oscillations are especially important for CAMKII activity regulating cell cycle control.

How Does Ca2+ Control The Cell Cycle and Centromere Cycle?

• Ca²⁺ plays an important role in controlling the cell and centromere cycle via

  • Ca²⁺-dependent activation of retinoblastoma-1 (RB1) regulates G1/S phase transition,

  • CaMKII regulates G2/M phase transition

  • CREB and NFAT can regulate M/G1 transition

What is the Ras/ERK Signalling Pathway?

• A signalling pathway that regulates gene transcription via spatiotemporal Ca²⁺ signals from SOCE, ARC, TRPC6, TRPV6 and Non-SOCE)

• RasGTPases couple growth factor receptors (e.g. EGFR) to downstream functional responses, including PI3K and PKB/Akt activation

• This activates the ERK pathways, regulation cell proliferation, differentiation and cell survial through the differential activation of genes

How Can Ca2+ Signals Differentially Regulate Ras?

• Ras bound to GDP → inactive

• Ras bound to GTP → active

  • GTPases act as molecular switches moving from active to inactive, hydrolysing GTP → GDP

• Ca2+ signals can differentially regulate Ras activity via GEFs and GAPs:

  • CAPRI (GEF) activated by the amplitude of Ca²⁺ signals (sustained Ca increase)

    • converts Ras-GDP → Ras-GTP

  • Ras-GRF (GAP) Activated by localised Ca²⁺ entry

    • Inactivates Ras-GTP

  • RASAL, (GAP) activated by high frequency of Ca²⁺ oscillations

    • Inactivates Ras-GTP

Where are Ras-GTPases Mutations Seen Seen?

• Many cancers have mutant Ras-GTPases:

  • K-Ras found in pancreas, colon, lung cancers; constiutively activated

  • m-Ras found melanoma

• Temporal and spatial properties of Ca²⁺ signals can differentially regulate these GTPases, affecting proliferation and survival.

What roles do Bak, Bax, and tBid play in Ca²⁺-mediated mitochondrial apoptosis?

• Ca2+ mediates intrinsic cell death at the mitochondria and ER

• Apoptosis is regulated within the mitochondria via pro- and anti-apoptotic proteins Bak and Bax

  • When dissociated, Bak and Bax are anti-apoptotic

• Bak and Bax oligomerise (pro-apoptotic state) to form a pore, releasing cytochrome C

  • tBid, a pro-apoptotic protein, binds to and promotes Bax-Bak oligomerisation, pore formation and Cyt.C release

• Once released from the matrix, cytochrome C forms an apoptosome complex, allowing for the activation of executioner caspases 3/-7 3/-7

• Activation of executioner caspases is regarded as the point of no return → cell death is activated

How Do Anti-Apoptotic Proteins Prevent Apoptosis

• BCL-2 and BCL-XL, which share a similar structure and function, can prevent tBid/Ba/Bak interaction and subsequent Bax-Bak oligomerisation

• This prevents the release of cytochrome C into the cytosol

• Point of no return is not reached → apoptosome complex dissociates/ not formed and prevents caspase 3/7 activation

How Do Pro-Apoptotic Proteins Promote Apoptosis?

• Bad binds to Bcl-2 or Bcl-XL, preventing their interaction with tBid/Bax/Bad complex, promoting apoptosis

  • This prevents Bcl-2/Bcl-xL from inhibiting Bax-Bad oligomerisation

• Bad prevents the anti-apoptotic activity of these proteins, allowing for Bak-Bax oligomerisation, allowing for cytochrome C release and apoptosome complex formation

• Anti- and pro-apoptotic proteins are regulated by growth factor signalling downstream of Ras

• Mechanism: PI3K convert PIP2 → PIP3, which phosphorylates Akt, contributing to Bad phosphorylation and its anti-apoptotic function.

How Does 14-3-3 Facilitate the Anti-Apoptotic Function of Bad?

• Growth factor receptor signalling activates PKB/Akt, MAPK, and PKA, which phosphorylate Bad.

• Phosphorylated Bad dissociates from the mitochondria and binds to 14-3-3 protein.

  • Dissociation of Bad from BCL-XL and BCL-2

• This binding prevents Bad from inhibiting BCL-XL and BCL-2, allowing them to exert their anti-apoptotic effects and block Bak-Bax oligomerisation.

• Bad phosphorylation is anti-apoptotic; 14-3-3 facilitates this protective effect.

What are the Opposing Effects of Reactive Oxygen Species on PKB?

• Global ROS elevation directly inhibits PKB → inhibition of Bad Phosphorylation → Promotes Apoptosis

• Local ROS supply inhibits PTEN (lipid phosphatase) → ↑ PKB activation → ↑ Bad Phosphorylation → Apoptosis Inhibited (Dephosphorylation of Bad Prevented

  • PTEN converts PIP3 → PIP2, acting as the off switch for PKB activation

    • PIP3 accumulates

How Does Ca2+ Overload Promote Apoptosis?

• Sustained Ca2+ overload can activate calcineurin, a protein phosphatase, which dephosphorylates Bad

• Dephosphorylated Bad dissociates from 14-3-3 and sequesters the anti-apoptotic proteins Bcl-2/Bcl-xL, promoting apoptosis.

• 14-3-3 proteins, when unbound, bind to and inhibit PMCA, exaggerating Ca2+ overload

• The dissociation of phospho-BAD from 14-3-3 exacerbates the Ca²⁺-dependent apoptosis.

How Does Ca2+ Overload and ROS Production Lead to Necrosis

• Excessive Ca²⁺ uptake via the mitochondrial Ca²⁺ uniporter (MCU) leads to reactive oxygen species (ROS) generation.

• This high Ca²⁺ can be driven by SOCE, contributing to Ca²⁺ overload

• This high Ca²⁺ and ROS activate the mitochondrial permeability transition pore (mPTP), leading to a loss of mitochondrial membrane potential (ΔΨm) and mitochondrial depolarisation.

• The loss of ΔΨm impairs ATP synthesis via ATP synthase, causing ATP depletion.

• ATP depletion prevents maintenance of membrane potential and ion homeostasis, causing cell swelling, membrane blebs, bursting, and release of cellular contents as transporter, pH regulatory and volume regulatory mechanisms are no longer supported

• This leads to necrosis and inflammation

How can Ca2+ initiate intrinsic Cell Death at the ER?

• Reduced Ca2+ uptake and depletion of Ca2+ stores in the ER causes ER stress.

• This activates the unfolded protein response → promotes apoptosis by promoting the cleavage of Pro-caspase 12 (resides on the ER membrane) into caspase-12

• Caspase 12 detaches from the ER membrane and activates Caspase 3 (Executioner caspase) → facilitates apoptosis

• Paradoxically UPR can activate pro-survival gene transcription e.g. BCL providing protection against apoptosis

How Do BCL-2, Bax, and Bak Regulate Ca²⁺ Proteins?

• BCL-2: binds to SERCA and IP₃R, decreasing Ca signalling, preventing Ca²⁺dependent apoptosis

  • BCL-2 inhibition of SERCA reduces ER Ca²⁺, modulating apoptosis

• Bax and Bak promote Ca²⁺ release from IP₃R, promoting Ca^2+-dependent apoptosis

  • Bcl-xL, Bax, and Bak can also bind and inhibit IP3Rs → preventing Ca²⁺dependent apoptosis

• Ca²⁺ overload can activate calpain, cleaving IP₃R, reducing Ca²⁺ release

How Does Oxidative and Metabolic Stess Contribute to Ca²⁺ Dependent Cell Death?

• Oxidative and metabolic stress in the nucleus leads to the accumulation of NAD+

• NAD+ can be converted to ADP ribose via PARP/PARG pathways

• ADP ribose is important in DNA damage repair

• ADPR can activate TRPM2 channels and faciliate further Ca²⁺ contributing to Ca²⁺-dependent cell death

How is Apoptosis Controlled in Cells:

• In any cell, there is a constant balance between pro- and anti-apoptotic mechanisms, mediated by Ca signalling

• Calcium has paradoxical mechanisms controlling cell death

  • Balance between excessive Ca signalling vs reduced Ca signalling

  • This can either promote apoptosis (tumour suppressive mechanism) or iniate an anti-apoptotic mechanism leading to apoptosis resistance and cancer

How can the Pro-Apoptotic Mechanism Suppress Tumour Formation?

• Apoptosis is mediated by excessive Ca²⁺ signalling via:

  • Calcineurin activation → dephosphorylates Bad

  • Ca²⁺ uptake via MCU → ROS production

  • Unbound 14-3-3 proteins → inhibit PMCA → sustained Ca²⁺ overload

• ROS generation, tBid/Bax/Bak complex formation and Bad inhibition of Bcl-2/Bcl-xL leads to cytochrome C release and Caspase 3/7 activation

• ER stress & UPR lead to pro-caspase 12 activation, caspase 3/7 activation and apoptosis

• This leads to a controlled cell death which acts as a tumour-suppressive mechanism

How can anti-apoptotic mechanisms promote tumour formation?

• ROS can inhibit PTEN → PKB/Akt activation → increasing Bad phosphorylation and a → apoptosis blocked

• Growth factor signalling → PKB, PKA, MAPK activation

• Phospho-Bad binding to 14-3-3, Bcl-2/Bcl-xL expression

• Dampened Ca²⁺ signalling → apoptosis resistance → cancer progression

How can anti-apoptotic mechanisms promote tumour formation?

• Dampened Ca²⁺ signalling reduces pro-apoptotic triggers, promoting cell survival.

• ROS can inactivate PTEN, leading to the accumulation of PIP3.

• The activation of PKB/Akt can lead to the phosphorylation of Bad, blocking apoptosis

• Growth factor signalling via PKB, PKA, and MAPK enhances pro-survival pathways.

• 14-3-3 proteins bind phosphorylated Bad (pBad), preventing it from sequestering Bcl-2/Bcl-xL.

• UPR activation can increase transcription of survival genes (e.g., Bcl-2, Bcl-xL).

• This can lead to apoptosis resistance, which supports tumour formation and progression.

How Does Tissue Context Influence The Balance Between Apoptosis and Proliferation?

• Depending on the cell type, the balance between apoptosis and cellular proliferation may favour tumour formation

• Tissue homeostasis: balance between cell division and cell death

  • Terminally differentiated cells: less likely to die → proliferation favoured → more likely to promote cancer formation

  • In tissues with large cell turnover, where there is rapid proliferation and death, apoptotic resistance can contribute to tumour progression

• High turnover tissues: apoptosis resistance → tumour progression

How Do Cancer Cells Migrate and Interact With the ECM?

• The Leading edge is formed from lamellipodia/invadopodia (specialised membrane protusions) via actin polymerisation, which causes the cell to extend.

  • Membrane stretch occurs and is sensed by ion channels.

  • Invadopodia secrete MMPs, which degrade the ECM.

  • New integrin-mediated contacts form.

  • Low resting Ca2+; Ca2+ spikes reuired for migration

• Integrins are foot-like processes that traverse through the plasma membrane and bind to the ECM via fibronectin

  • They form focal adhesion complexes and link the ECM to the actin cytoskeleton at the leading edge

• Trailing edge requires focal adhesion disassembly and actomyosin contraction to allow rear-end retraction.

  • Loss of rear-end integrin contacts.

What are the key mechanisms involved in focal adhesion complex assembly and cell migration at the leading edge?

• At the leading edge, localised Ca²⁺ entry, amplified by Ca²⁺ release through IP3Rs, activates calmodulin-dependent kinase (CaMKII), proline-rich tyrosine kinase (PYK2), and focal adhesion kinase (FAK), promoting focal adhesion complex assembly.

• Actin fibres assemble in a Ca²⁺-regulated and ATP-regulated manner

• Integrins attach to these actin fibres to facilitate focal adhesion complex assembly and aid migration.

• SOCE (STIM1/Orai1), TRPM7, TRPV2, IP3R, and ARC channels play key roles in Ca²⁺ regulation for migration and invasion

How do ion channels like Orai1, TRPV2, and TRPM7 contribute to cell migration and invasion?

• Orai1 channels, activated by Ca²⁺ ATPase on the plasma membrane in some cancers can drive migration and invasion by promoting Ca²⁺ entry.

• TRPV2: Ca2+ entry regulates the release of MMPs to break down the ECM and focal degradation creating a path for cell movement.

• TRPM7: Ca2+ entry facilitated by membrane stretch and regulates migration by phosphorylating myosin-IIA heavy chain via a-kinase domain, inhibiting actomyosin contraction, and facilitating cell spreading.

What role does focal adhesion kinase (FAK) and proline-rich tyrosine kinase (PYK2) play in cell migration?

• FAK and PYK2 are crucial in focal adhesion assembly, which is necessary for cell migration.

• FAK can inhibit PMCA (plasma membrane Ca²⁺ ATPase), enhancing Ca²⁺ signalling, which supports migration.

• PYK2 also plays a critical role in stabilising the focal adhesion complex at the cell’s leading edge.

Why are Ca²⁺ Flickers Seen at he Leading Edge?

• Experimental evidence shows localised Ca2+ flickers at the leading edge of migrating endothelial cells

• These localised signals activate the Focal adhesion complex via CaMKII and PYK2-mediated phosphorylation of focal adhesion kinase (FAK).

  • This is crucial for cancer cell migration

• CAMKII is differentially regualted by high frequency Ca osciallation → as the Ca spikes become more frequent the leading edge will drive migration

How Does High Sustained Ca2+ at the Trailing Edge Faciliate Cell Migration

• High sustained Ca2+ leads to actomyosin contraction and a disassembly of the focal adhesion complex

• This high Ca2+ is sufficient to activate calpains, which break down the protein componenst of the Focal adhesion complex

• This causes a dissociation of the complex from the integrins, which, along with the contractile machinery wil pull away and cause detachment of the cell membrane from the matrix (trailing mechanism occurs)

How is Ca²⁺ signalling machinery altered in cancer, and why is this important?

• In cancer, Ca2+ signalling machinery is remodelled through the upregulation or downregulation of key components

• This remodelling can contribute to the hallmarks of cancer.

• In most cases, Ca²⁺ machinery is overexpressed, including

  • Orai1 (except in prostate cancer)

  • STIM1,

• STIM2 is typically underexpressed in cancer.

How are TRP channels altered in cancer as part of Ca²⁺ signalling remodelling?

• In cancer, TRPC channels are overexpressed as part of Ca²⁺ signalling remodelling.

• Most TRPV channels are also overexpressed.

• However, TRPV1 is an exception and is reduced in bladder cancer.

  • This is notable because TRPV channels normally play an important role in bladder function.

How is Ca²⁺ transporter expression altered in different cancers?

• In cancers like colon, oral, and breast, Ca²⁺ transporters are generally reduced, but PMCA-2 expression is increased.

• In pancreatic cancer, PMCA-4 is overexpressed, and its overexpression is linked to poor patient survival, making it a potential therapeutic target.

  • Differences between primary and metastatic cancer could explain variations in this finding

• These transporters are linked to various hallmark responses → novel drug target (no Ca transporter targeting drugs on the market)

Why is it difficult to target Ca²⁺ signalling machinery in cancer therapy?

• Unclear whether altered expression of Ca²⁺ signalling machinery is the cause or consequence of cancer, and whether it directly drives cancer progression.

• This uncertainty makes it hard to design specific therapies targeting Ca²⁺ machinery.

Why is targeting Ca²⁺ signalling machinery in cancer therapy difficult?

• Some Ca2+ signalling machinery is ubiquitously expressed and has a widespread function

  • If targeted, the drug will generate adverse effects regardless of drug specificity

• Current focus on targeting Ca signalling machinery that exhibits a unique function or expression in cancer

  • Prefered drug target that would result in fewer or no adverse effects