Lecture 8: Ca Signalling as a Theraputic Targets for Cancer

What are Important Considerations When Theraputically Targeting Ca2+ Machinery in Cancer?

• The versatile and ubiquitous nature of Ca signalling means targeting with novel therapeutics can produce unacceptable adverse effects regardless of specificity

• The ideal strategy is to target individual components and regulator proteins of Ca2+ signalling machinery that are either uniquely expressed or gain a new function in cancer cells

What Aspects of Ca2+ Signalling Machinery Could Be Targeted Therapeutically in Cancer

• TRPM7: important for migration invasion → anti-metastatic drug target

• SOCE (Orai1 and ER STIM1): upregulated in cancer

• ARC (Orai1/Orai3 & PMSTIM1)

• SPCA2 (Orail1 & PM-SPCA2 interactions)

• PMCA

How Does SOCE Upregulation in Cancer Create a Paradox for Targeting Ca²⁺ Signalling in Therapy?

• Store-operated Ca channel components Orai1 and ER STIM1are upregulated in many cancers

  • This upregulation promotes cell proliferation, migration/invasion, tumour metastasis and angiogenesis, promoting cancer

• This creates the functional cancer paradox, as SOCE is important for regulating cell proliferation and migration, but can also lead to Ca-dependent cell death

  • Targeting these channels may lead to apoptosis resistance, facilitating cancer development further

Why May ARC (Orai1/Orai3 & PM STIM1) Channels Be Targeted Therapeutically in Cancer?

• The combination of Orai1 and Orai3 expression can alter the assembly of ARC channels, which influences multiple hallmarks of cancer, including cell migration, proliferation, and survival.

• This alteration in channel function contributes to the development and progression of cancer.

What is the function of SPCA2 in normal cells, and how does its role change in cancer?

• Normally expressed in the Golgi, regulating Ca²⁺ and Mn²⁺ transport and plays an important role in growth factor receptor trafficking.

• In cancer, SPCA2 is expressed at the plasma membrane, where it binds to and activates Orai1, mediating non-SOCE and regulating many cancer hallmarks, making it a potential cancer-specific target → cancer specific phenotype and unqiue function in cancer cells

What is the role of PMCA (plasma membrane Ca²⁺ ATPase) in cancer metabolism, and how could it be targeted therapeutically?

• PMCA is involved in regulating metabolic changes in cancer cells, contributing to the Warburg effect (shift from mitochondrial metabolism to glycolysis).

  • This shift occurs due to mutations in mitochondrial enzymes, oncongenic signalling activation of and/or an upregualtion of glycolytic enzymes

• In cancer cells, PMCA has its own glycolytic ATP supply, which can be targeted therapeutically.

• Inhibiting oncogenic glycolytic enzymes that supply PMCA’s ATP could lead to Ca²⁺ overload and selective cancer cell death.

How is TRPM7 shown to be an anti-metastatic drug target?

• The channel suggested as a target for anti-metastatic drug development as it mediates migration in nasopharyngeal carcinoma cells, which could be inhibited using TRPM7 inhibitors.

• Experimental Evidence → use of siRNAs to knock down TRPM7 expression (genetic approach)

  • Two different siRNAs targeting TRPM7 effectively stopped cell migration compared to a control (scrambled siRNA)

  • Gap closure assay: TRPM7 inhibition showed reduced migration of cells into the gap over 16 hours.

  • Scratch assay: Showed TRPM7 knockdown inhibits migration, though it has confounding factors due to cellular injury.

  • Western blotting: Confirmed TRPM7 protein knockdown after siRNA treatment.

Why are Gap Closure Assays and Scratch Assays Used to Assess Migration?

• Both assays assess cell migration by creating a cell-free gap and monitoring how cells move to fill it over time.

• Gap closure assay: a plastic insert is placed in a well, and cells grow around it.

  • Removal of the insert creates a defined gap.

  • Migration is measured by observing cells moving into the gap over a time period

  • Advantage: minimal cell injury, giving a cleaner measure of migration.

• Scratch assay: a scratch is made through a cell monolayer to create a gap.

  • Cell movement into the scratched area is monitored.

  • Limitation: causes cellular injury, which can release mediators that may confound migration results.

How do Ca²⁺ channel inhibitors also support the role for TRPM7 in cell migration?

• Inhibition of Ca²⁺ entry through TRPM7 using 2-APB and La³⁺ reduced cell migration, whereas inhibiton of SOCE-mediated Ca entry with SKF96365 had no effect on migration

  • 2-APB: Poorly selective inhibitor → Inhibits Ca²⁺ entry via TRPM7 (and SOCE).

  • La³⁺: Non-selective Ca²⁺ channel blocker that binds to Ca²⁺ binding domains with high affinity (pM), preventing Ca²⁺ entry.

  • SKF96365: SOCE inhibitor

• Suggests migration depends on TRPM7-mediated Ca²⁺ entry, not SOCE.

  • Evidence is supportive but not definitive proof due to poor drug specificity.

  • siRNA knockdown of TRPM7 provides stronger evidence.

Why is TRPM7 Said to Be A Useful Prognostic Biomarker

• TRPM7 expression correlates with poor clinical outcomes in breast cancer.

• The Kaplan–Meier survival curves plot cumulative survival against recurrence-free survival and distant metastasis-free survival

• Patients are stratified into high vs low TRPM7 expression groups using immunohistochemistry of resected breast tumours, with expression correlated to survival

  • It indicate that TRPM7 overexpression is associated with reduced overall survival, increased recurrence and distant metastasis

What is the effect of TRPM7 knockdown on proliferation and viability in breast cancer cells?

• TRPM7 knockdown does not affect cell proliferation or viability in human breast cancer cells (MDA-MB-231).

• When TRPM7 expression was reduced using shRNA (genetic knockdown where translation is blocked), there was no significant difference observed in the MTS assay between control and TRPM7-knockdown cells, in the

  • MTS assays are used to assess proliferation and viability over time.

    • Measures metabolic activity via reduction of MTS to coloured formazan product by viable cells using photospectrometry

• TRPM7 has little role in cell proliferation or cell death

  • Its main contribution to poor patient outcomes is through enhanced migration and invasion, not increased growth or resistance to cell death.

How does the luciferase bioluminescence assay assess metastasis in vivo?

• Breast cancer cells are engineered to overexpress luciferase (reporter gene).

• Cell lines overexpressing luciferase are delivered via tail vein injection into mice, and the cells circulate and form metastases in tissues (e.g. lungs).

• Luciferin injection is converted by luciferase into oxyluciferin, producing light.

• Emitted bioluminescence from metastatic cells is detected using in vivo imaging (bioluminescence imaging system)

• Allows real-time, non-invasive monitoring of metastatic spread over days to weeks.

• Signal intensity (photon flux) correlates with tumour burden.

What does the luciferase metastasis assay reveal about TRPM7 function in breast cancer?

• TRPM8 overexpression and subsequent shRNA-mediated knockdown in MDA-MB-231 breast cancer cells reduces metastatic potential in vivo.

• After tail vein injection:

  • Control cells show strong lung bioluminescence over time.

  • TRPM7-knockdown cells show significantly reduced lung metastasis → less luminescence

• Quantification of photon flux over up to 30 days confirms reduced metastatic burden.

• Conclusion: TRPM7 promotes metastasis, not proliferation.

  • Reduced TRPM7 expression interferes with in vivo metastatic spread.

How Was STIM1 Shown to Influence Cervical Cancer Progression?

• siRNA knockdown of STIM1 → reduced cell proliferation and growth and induced cell cycle arrest in cervical cancer cells

• STIM1 knockdown resulted in more cells present at the G2/M phase → cells arrested prior to mitosis/ cell division and so stop growing

  • Cell proliferation and cycle arrest were assessed using flow cytometry of propidium iodide-stained cells

  • Growth is assessed by counting cells under a microscope

How Was Cell Cycle Analysis Performed Using Propidium Iodide Staining?

• Used to assess cell cycle progression by staining the nucleus of cells.

  • PI binds to DNA and can enter cells with damaged membranes (typically used to measure necrosis, but also applicable to live cells after fixation with a fixative)

• Live cells suspended in animation, then stained with PI, allowing the dyes entry into the nuclei

• Cells passed through flow cytometer → counts cells and detects different nuclear populations

• Generates population histograms of cells in different stages of the cell cycle, measuring size of nuclei by how light is scattered

How does the cell migration assay using Matrigel and crystal violet assess the metastatic potential of cells?

• Cells are cultured on Matrigel, a basement membrane matrix, to simulate the extracellular environment.

• Cells are stained with crystal violet to quantify migration and visualise the cells that have migrated through the Matrigel.

• Purpose: This assay helps evaluate the metastatic potential of cells, e.g., hepatocellular carcinoma (HCC) cells, by measuring how far they migrate under different conditions.

How does the drug SKF96365 affect SOCE and cell migration in metastatic hepatocellular carcinoma cells?

• It is a poorly selective SOCE inhibitor with several effects:

  • It inhibits the Ca²⁺-dependent K⁺ channel by hyperpolarising the membrane potential.

  • Calcium entry via SOCE activates the Ca²⁺-dependent K⁺ channel, which increases the driving force for further Ca²⁺ entry.

  • By inhibiting the K⁺ channel, SKF96365 prevents further Ca²⁺ influx.

• It reduces the migration of metastatic HCC cells (like siRNA knockdown of STIM1).

  • Both the drug and siRNA exhibit similar effects, suggesting the drug’s action is primarily mediated through SOCE inhibition, despite its other non-selective target

What is the Cellular Invasion Assay and How Does It Work?

• Cells are plated onto a Matrigel-coated porous membrane (transwell filter) → forms 2 chambers

• Upper chamber contains low serum (1%); lower chamber contains high serum with growth factors as a chemoattractant.

• Cells invade by breaking down the Matrigel and migrating through the pores of the membrane.

• Cells that reach the lower chamber attach to the bottom of the filter.

• These cells are then stained with crystal violet, DAPI, or Hirsch stain and counted.

• Measures the invasive potential of cells, simulating metastasis

How do SKF96365 and STIM1 knockdown affect invasion of metastatic hepatocellular carcinoma cells?

• Both SKF96365 (SOCE inhibitor) and siRNA knockdown of STIM1 reduce invasion of metastatic hepatocellular carcinoma cells, as shown in the transwell invasion assay

• Demonstrates that invasion depends on SOCE-mediated calcium entry, and inhibiting SOCE (pharmacologically or genetically) blocks this process.

How does the SOCE inhibitor SKF996365 affect breast cancer cell migration, and how is this demonstrated in a gap closure assay?

• SKF96365 reduces the migration of breast cancer cells.

• In a gap closure assay, assessing cell migration, SKF96365 was tested on two breast cancer cell lines.

  • It was compared to a voltage-dependent Ca²⁺ channel (VDCC) blocker, which had no effect on migration, highlighting SKF96365's selective effect on SOCE.

• Demonstrated that SOCE inhibition, but not VDCC blockers, inhibits migration of MDA-MB-231 breast cancer cells.

How is the Breast Cancer Xenograph Model Be Used to Study Metastesis?

• MDA-MB-231 cells (breast cancer) expressing:

  • Thymidine kinase (for tumour growth assessment)

  • GFP (for histological visualisation)

  • Luciferase (for monitoring metastasis via luminescence)

• Cells are injected into SCID mice (immunodeficient, lack T and B cells) via the tail vein.

• These mice are then treated with

  • Control siRNA

  • STIM1 siRNA

  • Orai1 siRNA

• Compared to control, there is reduced metastasis in STIM1/Orai1 siRNA knockdown → reduced tumour detection

How does siRNA knockdown of Orai1 and STIM1 affect metastasis in MDA-MB-231 breast cancer cells in vivo?

• siRNA knockdown of Orai1 and STIM1 reduces metastatic potential of MDA-MB-231 breast cancer cells in vivo.

• Experimental setup:

  • MDA-MB-231 cells expressing thymidine kinase, GFP, and luciferase reporter genes.

  • Treated with control siRNA or Orai1/STIM1 siRNA.

  • Cells were injected into the tail vein of SCID mice.

• Metastasis detection:

  • Bioluminescence imaging (via luciferase reporter) to monitor metastasis.

  • Histological analysis of lungs to confirm metastasis.

What are SCID Mice?

• Mice homozygous for a severe combined immune deficiency mutation

  • Lack functional T and B cells

  • No immune response → allows injected cancer cells to assess metastasise

How does histological assessment of mouse lung tissue demonstrate the role of Orai1 and STIM1 in breast cancer metastasis

• Histological analysis of mouse lung tissue shows extensive infiltration of GFP-positive breast cancer cells (control siRNA), indicating metastasis.

• This is absent/less severe in mice injected with cells where Orai1 or STIM1 was knocked down using siRNA.

  • GFP-expressing cells in the lungs are indicative of metastasis.

  • Knockdown of STIM1 or Orai1 in breast cancer cells prevents metastasis to the lungs, highlighting their importance in metastasis.

• Similar effects seen with SOCE inhibitor SKF96365 → shows the important of SOCE in metastasis

How Was SOCE Effects on Cervical Tumour Growth Investigated In Vivo?

• Xenograft model generated of SiHa cervical cancer cells injected under the skin of female SCID mice (no functional T/B cells, preventing immune rejection of xenografts).

  • Tumour size measured with callipers and luciferase reporter.

• SOCE inhibition treatment (applied every 3 days starting day 6 post-inoculation):

  • Control vehicle (n = 6)

  • SKF96365 (2.5 mg/kg) (n = 6)

  • 2-APB (50 μg/kg) (n = 6)

• Tumour growth and blood vessel number quantified

  • Rich blood supply → facilitates metastasis

What are the effects of SOCE inhibition on cervical tumor growth and angiogenesis in vivo?

• SOCE inhibitors SKF96365 and 2-APB reduced tumour size, tumour weight, and number of blood vessels in treated groups.

• Tumours appeared lighter and had a brown colour due to necrosis.

• Treatment reduced blood vessel formation, crucial for tumour growth and metastasis.

• Inhibition of store-operated Ca²⁺ entry (SOCE) suppresses cervical tumour growth and tumour angiogenesis in vivo

How does the orthotopic model assess the role of STIM1 overexpression in tumour growth and angiogenesis?

• Tumour cells are injected beneath the skin at two separate sites in animals:

  • Control cells

  • Cells with STIM1 overexpression

• Animals act as their own control for comparison.

• Tumour mass and vessel number are recorded

  • STIM1 overexpression → larger tumours with more blood vessels.

  • Control group shows reduced tumour size and vessel number.

• VEGF production is measured

  • Produced by the tumour to promote angiogenesis and provide a rich blood supply that facilitates metastasis.

• Orai1 and STIM1 (SOCE) are important for tumour growth and metastasis, as their overexpression increases both tumour vascularity and metastasis potential.

What is the effect of STIM1 overexpression on tumour growth and angiogenesis (in Orthotopic Model)?

• Enhanced tumour angiogenesis and growth were seen

• SCID mice inoculated with cervical cancer cells at bilateral dorsal sites:

  • Control (mock-transfected) cells.

  • STIM1-overexpressing cells

• After 21 days there were increased tumour vessel numbers and tumour weight

  • significance leve: P < 0.01, n = 6

• ELISA reveaed increased VEGF-A secretion → STIM-1 regulates VEGF-A production

  • P < 0.01 vs wild type (n = 5)

What is the effect of STIM1 knockdown on tumour growth and angiogenesis (in Orthotopic Model)?

• Reduces tumour growth and angiogenesis.

• SCID mice inoculated with:

  • control shRNA

  • shSTIM1-transfected cell

• After 15 days, there was decreased tumour size, fewer blood vessels, and reduced tumour weight in the STIM1 knockdown group.

  • P < 0.01, n = 6

How is STIM1 expression altered in early-stage cervical cancer compared to normal tissue?

• A paired analysis (n = 24): compared carcinoma tissue (T) vs. adjacent nonneoplastic epithelia (N) using immunoblotting revealed higher STIM-1 in tumour tissue compared to normal

• Quantitative analysis using STIM1 in normal squamous epithelia (control) as a baseline revealed higher STIM1 levels in tumour cells, when expressed relative to the control

• Immunofluorescence results revealed increased STIM1 in cancer tissues and decreased E-cadherin ↓ (epithelial marker).

How does STIM1 expression correlate with tumour size and metastasis in cervical cancer?

• Higher STIM1 expression correlates with larger tumour size (n = 24).

• STIM1 levels are significantly higher in tumours with local pelvic lymph node metastasis.

• Lymph node cancer cell detection (higher STIM1 expression) is a good indicator of metastasis.

What is the SOCE Cancer Paradox?

• There is extensive evidence from cell line and in vivo models that SOCE not only contributes to cancer hallmarks (cell proliferation, migration, and angiogenesis), but also promotes cancer

• There is also evidence that SOCE also stimulates apoptosis via Ca entry and overload, leading to mitochondrial-dependent apoptosis, which would inhibit cancer

• This generates a functional paradox, as targeting hallmarks that promote cancer may also act to inhibit cell death and, as a result, promote cancer

  • Inhibiting cell death can promote cancer by allowing rapid tumour growth with increased cell turnover.

What is the Functional Consequence of The SOCE Cancer Paradox?

• Inhibition of Orai1 and STIM1 not only suppresses cancer-promoting hallmarks but also inhibits apoptosis, potentially promoting cancer progression.

• This inhibition of cell death may have a greater effect on tumour progression than inhibiting cell proliferation.

• When targeting SOCE in cancer therapy, it’s important to consider the potential for apoptosis inhibition, as it could lead to unintended promotion of cancer.

• Suggests that the inhibition of Ca channels inhibits apoptosis and can promote cancer

How does Orai1 affect apoptosis in prostate cancer cells?

• Orai1 knockdown (siRNA) or overexpression of non-functional mutant Orai1 reduces apoptosis in prostate cancer cells.

• This is because SOCE normally drives apoptosis via Ca²⁺ entry.

• Inhibition of Orai1 or Orai3 through loss-of-function mutations prevents Ca²⁺ influx, thereby inhibiting apoptosis, a hallmark of cancer.

What experimental approaches show that Orai1/SOCE regulates apoptosis?

• siRNA knockdown of Orai1 + thapsigargin:

  • Thapsigargin depletes ER Ca²⁺, which normally activates SOCE.

  • siRNA knockdown prevents SOCE, inhibiting apoptosis.

• SOCE activation:

  • Using IP3, EGTA, and BAPTA to manipulate Ca²⁺ levels and demonstrate that Ca²⁺ entry drives apoptosis.

• Use of mutant Orai1/Orai3 (loss-of-function mutations):

  • Prevents Ca²⁺ influx and inhibits apoptosis.

How is SOCE functionally demonstrated in relation to Orai1 and STIM1?

• Fluorescent tagging using YFP-STIM1 and CFP-Orai1 to demonstrate their translocation upon activation, showing SOCE assembly.

• Ca²⁺ currents are measured:

  • Knockdown of Orai1 or STIM1 → reduced currents (inhibition of SOCE).

  • Overexpression of Orai1/STIM1 → increased currents, confirming robust SOCE function.

Why might ARC channels and Orai3 be better therapeutic targets than SOCE and Orai1 in cancer?

• SOCE (Orai1/STIM1) is activated by STIM1 and ER Ca²⁺ store depletion.

  • Increased Orai1 expression (as seen in cancer) increases SOCE, which can drive apoptosis

  • Targeting Orai1 alone may inadvertently promote apoptosis resistance (problematic)

• ARC channels (Orai1 + Orai3) are regulated by plasma membrane STIM.

  • Orai3 overexpression means more Orai1 subunits are required for ARC channel assembly, at the expense of SOCE channels, reducing SOCE → Orai1 is diverted away from SOCC assembly

    • Overexpression promotes cell proliferation, migration, invasion, and metastasis

• Targeting Orai3/ARC can inhibit cancer hallmarks and overcome apoptosis resistance.

  • Orai3 overexpression decreases SOCE and leads to apoptotic resistance.

What is the Role of Orai3 in ARC Channels in Cancer Progression?

• Orai3 is upregulated in breast, lung, and prostate cancers and contributes to tumorigenesis

• ARC channels (Arachidonate-Regulated Ca²⁺ Entry): facilitate store-independent Ca²⁺ entry channels

  • Formed by heteropentameric subunits of Orai1 + Orai3

  • Increased Orai3 → more ARC channels → promotes cancer progression

How does Orai3 contribute to prostate cancer progression?

• Orai protein ratios are altered in prostate cancer vs. normal tissue.

• Orai3 is strongly upregulated.

• Overexpression of Orai3 in PC3 cells leads to:

  • Increased ARC-mediated, store-independent Ca²⁺ entry

  • Increased NFAT-mediated cell proliferation

• Orai3 knockdown (siRNA) in xenograft models → reduced tumour growth

What is the proposed mechanism by which Orai3 contributes to cancer progression?

• Increased Orai3 and/or arachidonic acid (from the tumour microenvironment) recruits Orai1 into Orai1/Orai3 ARC channels.

• This increases ARC-mediated NFAT activation and cell proliferation.

• This leads to a decrease in Orai1 available for SOCE channels, leading to apoptosis resistance.

How Are ARC Channels Regulated?

• Controlled by PM STIM1 (not ER STIM1)

• STIM1 was originally identified as stromal interacting molecule-1, a cell adhesion–related protein

• ARC channels are activated independently of ER Ca²⁺ store depletion

• Arachidonic acid, produced following growth factor activation, activates the channe;

What is the suggested role of ARC channels in cancer cell migration and progression?

• Arachidonic acid promotes breast cancer cell migration via FAK phosphorylation

• Plasma membrane STIM1 and ARC channel may sense the tumour microenvironment, promote cancer cell migration, progression, and metastasis

• Suggests ARC channels could play a critical role in cancer development and metastasis

What is the Effect of Orai3 Knockdown on Breast Cancer Progression

• siRNA Knockdown of Orai3 reduces cellular proliferation

• This causes cell cycle arrest, shown by the altered distribution of cells in G1/S and G2/M phases

• This leads to a reduction in cell invasion

• Indicates Orai3 is required for cell cycle progression and invasive behaviour

How does Orai3 knockdown affect breast tumour growth in vivo?

• Tested using xenograft / orthotopic breast cancer models

• Cancer cells were injected into the mammary fat pad

• Orai3 knockdown tumours are significantly smaller than controls

  • Reduced tumour volume and weight

• Demonstrates Orai3 contributes to breast tumour growth in vivo

What did measurements of ARC and CRAC currents reveal about Orai3 in prostate cancer?

• Orai3 is overexpressed in human prostate cancer tumours compared to Orai1/2 and STIM1.

• CRAC Channel(SOCE) measurements revealed

  • Orai3 knockdown (siRNA) does not affect SOCE-mediated Ca²⁺ currents (I_CRAC) or Ca²⁺ overshoot

  • Knockdown of Orai1 or STIM1 inhibits ICRAC and Ca²⁺ overshoot.

• siRNA Orai3 knockdown inhibits cell proliferation, induces cell cycle arrest, and reduces cell cycle proteins.

• This suggests that Orai3’s oncogenic effects are independent of SOCE/CRAC currents, suggesting it promotes cancer via ARC-mediated pathways rather than classical SOCE.

What are the effects of Orai1 and Orai3 knockdown on prostate cancer cells?

• ARC currents are activated by arachidonic acid (AA) and

• Knocked down by Orai1 or Orai3 siRNA.

• This:

  • Inhibits ARC currents and AA-induced [Ca²⁺]ᵢ increase

  • Inhibits AA-induced prostate cancer cell proliferation.

  • Baseline proliferation is unaffected.

• Knockdown in xenograft → prevents tumour growth and reduces tumour volume

• This suggests that Orai3/ARC channels are critical for AA-driven proliferation and survival, but not for baseline growth.

What is the Effect of Orai3 Overexpression on Prostate Cancer Cells?

• Increases cell proliferation.

• Induces apoptosis resistance.

• Enhances AA-induced apoptosis resistance.

How does the balance of Orai1 and Orai3 channels affect tumor growth and apoptosis?

• There is a balance of Orai1 and Orai3 channels

• The correct balance favours ARC channel assembly.

• This facilitates cancer hallmarks, including apoptosis resistance, by recruiting more Orai1 channels

  • Fewer Orai1 channels available for SOCE channels assembly results in reduced SOCE and less apoptosis.

• Targeting Orai3 is more effective than targeting Orai1 because it disrupts ARC-mediated apoptosis resistance without inhibiting SOCE-mediated cell death.

How is calcium signaling, specifically STIM1 and Orai1, a potential therapeutic target in cancer?

• Ca²⁺ signalling is crucial in cancer therapy, but targeting it can have adverse effects due to ubiquitous expression (e.g., STIM1 and Orai1).

• Orai1 is important for apoptosis resistance in cancer cells.

• Orai3 has unique expression and function → provides a more selective target for therapeutic strategies due to its role in tumour progression without broad systemic effects.

What is the SPCA2 (Secretory Pathway Ca²⁺-ATPase)?

• Located on the Golgi apparatus

• It acts as a Ca pump that transports Ca²⁺ and Mn²⁺ into the Golgi lumen.

• Essential for proper protein processing of newly synthesised protein in the Golgi → traffics IGF-1 receptor

• In breast and prostate cancer, it is said to be trafficked to the membrane and binds Orai1 to mediate store-independent Ca²⁺ entry.

• This process is crucial for cell proliferation, tumour growth, and cell migration/invasion.

What role does SPCA1 play in basal-like breast cancer?

• Upregulated

• Increases processing and trafficking of IGF-1R (Insulin-like Growth Factor 1 Receptor).

• Promotes cell growth and proliferation, contributing to tumor progression

What role does SPCA2 play in breast cancer?

• Overexpressed and mislocalised to the plasma membrane

• It directly binds to Orai1 at the N-terminal domain and activates Ca2+ entry independent of STIM1 or store depletion

• This results in increased Ca²⁺ entry, NFAT nuclear translocation and increased (Ca2+ dependent) prolifereation

What are the Effects of SPCA Silencing and Overexpression in Cancer Cells?

• SPCA2 silencing decreases resting Ca²⁺, proliferation, anchorage-dependent growth, and tumour growth in xenograft models.

• SPCA2 overexpression: causes the opposite effects (increases Ca²⁺, growth, and tumour progression).

• Mutant SPCA2 (no ATPase activity) still increases resting Ca²⁺ and growth, suggesting tumorigenic effects independent of ATPase function.

What are the therapeutic implications of targeting SPCA2 in breast cancer and other cancers?

• SPCA is normally absent from the plasma membrane in healthy cells.

• Targeting the SPCA2-Orai1 interaction could be a novel therapeutic strategy for breast cancer.

• Uncertainty whether this store-independent Ca²⁺ entry pathway occurs in other cancers, but it presents a potential future drug target.

How does SPCA2 knockdown and overexpression affect breast cancer cell proliferation and invasion?

• SPCA2 knockdown inhibits the proliferation and invasion of breast cancer cells.

  • Achieved using shRNA-mediated knockdown (specific to SPCA2, not SPCA1).

• SPCA2 overexpression increases proliferation and invasion of breast cancer cells.

How do Ca²⁺ transport mutants of SPCA2 contribute to tumorigenesis?

• ATP-binding mutant and CA-binding mutant were generated to study protein function.

• Mutations had little effect on the transport activity.

• Suggests that the tumorigenic properties are not driven by the Ca²⁺ transport activity of these mutations.

• Instead, the mutants must bind to Orai1, which regulates cancer-related phenotypes.

How is SPCA2 shown to localise to the plasma membrane and bind to Orai1?

• Use of Myc and HA tag

  • Myc-tagged SPCA2 co-localises with HA-tagged Orai1 in immunofluorescence experiments → detected using Ab

  • Normally located at the Golgi, but shown to traffic to the plasma membrane.

• Use of immunoprecipitation:

  • SPCA2 immunoprecipitates with Orai1 from whole cell lysates and cell surface biotinylated plasma membrane fractions.

  • Biotinylation of surface proteins and separation using streptavidin shows that SPCA2 binds to Orai1 at the membrane.

How does SPCA2 mediate its cancer phenotype?

• SPCA2 reaches the plasma membrane and mediates cancer phenotypes by binding to Orai1 and facilitating Ca²⁺ entry, rather than through its Golgi function.

• This unique mechanism and expression in cancer cells make SPCA2 a potential drug target.

How Can the PMCA Be Targeted?

• Unable to target mechanisms that indirectly regulate Ca2+ signalling, as they are altered during cancer transformation

• In cancer cells, the PMCA is uniquely regulated (rather than having a unique function)

  • Evidence suggests PMCA is fueled with ATP in a distinct manner in cancer cells, offering a potential target for cancer therapy.

How is Glucose Metabolised in Normal, Differentiated Cells?

• In the presence of oxygen, glucose is metabolised primarily in the mitochondria via:

  • Glycolysis → Pyruvate → TCA cycle → Oxidative phosphorylation

• Mitochondria produce ~95% of cellular ATP (~30–32 ATP per glucose).

• Glycolysis alone contributes ~5% of ATP (~2 ATP per glucose).

• In the absence of oxygen, glucose is metabolised to lactate in anaerobic glycolysis (inefficient ATP production).

What is the Warburg Effect?

• The shift from mitochondrial oxidative phosphorylation and metabolism to glycolysis for ATP production, even when oxygen is present.

• Also known as aerobic glycolysis → converts glocuse to lactate

• First proposed by Otto Warburg (1924); awarded the Nobel Prize in 1931.

• Considered a hallmark of malignant tumours.

• Occurs in proliferative tissues and cancer cells

How does metabolism differ in cancer cells compared to normal cells?

• Cancer cells preferentially use aerobic glycolysis, converting glucose to lactate even in the presence of adequate oxygen.

• This is energetically inefficient for ATP yield but supports:

  • Rapid proliferation and tumour growth

  • Survival in hypoxic tumour regions

• ATP production remains essential to fuel ATP-dependent processes, including PMCA-mediated Ca²⁺ extrusion.

Why is the Warburg Effect (Aerobic Glycolysis) Beneficial for Cancer Cells?

• In biosynthesis, glycolytic intermediates can be fed into pathways producing:

  • Amino acids

  • Nucleic acids

  • Fatty acids

• Lactate is toxic and transported out of cells via monocarboxylate transporters (MCTs) with protons, causing extracellular acidification.

• This acidic microenvironment activates matrix metalloproteinases MMPs and facilitates ECM breakdown, allowing for cell migration and invasion.

  • It also acts to suppress immune surveillance, promoting tumour immune evasion.

• It also allows survival in poorly oxygenated tumour cores.

How Does the Warburg Effect Support Ca2+ Homeostasis and Cancer Cell Survival?

• ATP remains essential for ATP-dependent processes, including:

  • PMCA activity, which maintains low resting cytosolic Ca²⁺.

• Glycolysis provides a rapid, local ATP supply to sustain Ca²⁺ extrusion.

• This helps prevent cytotoxic Ca²⁺ overload, promoting cancer cell survival.

• Suggests that metabolic regulation of PMCA may represent a therapeutic vulnerability.

Why is Upregulated Glycolysis critical for cancer cells?

• It provides a privileged ATP supply to maintain ATP-dependent processes like:

  • PMCA: pumps Ca²⁺ out → maintains low restig [Ca²⁺]i

  • Na⁺/K⁺-ATPase: maintains membrane potential, driving Ca²⁺ entry via TRP, SOCE, and ARC channels

• Supports cancer hallmarks: proliferation, migration, and apoptosis resistance, even under hypoxic conditions

How could targeting glycolysis and glycolytic enzymes be a therapeutic strategy in cancer?

• Inhibiting glycolysis may disrupt ATP supply to the Na⁺/K⁺-ATPase and PMCA, which normally maintains low resting Ca2+, reducing apoptosis resistance

• This may also affect the driving force for Ca²⁺ entry through TRP, SOCE, and ARC channels

• Cancer cells have adapted to hypoxia and may be more susceptible to glycolytic inhibitiors than healthy cells due to their reliance on glycolytic ATP

What is the Effect of Glycolysis Inhibition of PMCA in Pancreatic Cells?

• The PMCA relies on glycolytically-derived ATP.

• Glycolytic inhibition (using iodoacetate [IAA] → inhibits GAPDH, bromopyruvate [BrPyr] → inhibits hexokinase) causes:

  • ATP depletion

  • PMCA inhibition

  • Ca²⁺ overload

  • Necrotic cell death

• PMCA activity is measured by the Ca²⁺ overshoot response, with the clearance phase recorded → glycolytic inhibitors slow/ inhibit Ca²⁺ clearance.

How do mitochondrial inhibitors affect PMCA activity in pancreatic cancer cells?

• Mitochondrial inhibitors include:

  • CCCP (protonophore) → collapses mitochondrial membrane potential

  • Oligomycin → inhibits F1F0-ATP synthase

  • Antimycin → inhibits complex II of ETC

• PMCA activity is measured by the Ca²⁺ overshoot response, with the clearance phase recorded → inhibitors do not affect PMCA activity, Ca²⁺ clearance, or cell death in pancreatic cancer cells.

Why could targeting glycolysis be an effective therapy for pancreatic cancer?

• Pancreatic cancer cells rely on glycolytic ATP for PMCA function.

• Inhibiting glycolysis selectively causes Ca²⁺ overload and necrotic death in cancer cells.

• Healthy cells rely more on mitochondria for ATP, so they may be less affected.

• Targeting glycolysis may therefore be a promising therapeutic strategy.

Which glycolytic enzymes fuel the PMCA (Plasma Membrane Ca²⁺ ATPase) in cancer cells, and why are they important?

• PFKFB3 (phosphofructokinase fructose bisphosphatase-3): major oncogenic isoform of PFK2 → abundantly/uniquely expressed in cancer cell → theraputic target

• PKM2 (pyruvate kinase M2): responsible for ATP production; exclusively expressed in highly proliferative cells and cancer cells → potential therapeutic target

• PFKB1: rate-limiting enzyme with complex regulation

• Several glycolytic enzymes are upregulated in cancer, supporting PMCA function and contributing to the cancer cell metabolic phenotype.

How do glycolytic enzymes at the plasma membrane support the PMCA and Na⁺ pump?

• Glycolytic enzymes form a cluster/metabolome at the plasma membrane, providing a privileged ATP supply for the PMCA and Na⁺ pump.

• This localised ATP production is important for maintaining the driving force for Ca²⁺ channels.

• The PMCA may rely on this sub-membrane pool of glycolytically-derived ATP to function efficiently.

What experimental evidence shows that glycolytic enzymes fuel PMCA activity?

• Inside-out smooth muscle plasma membrane vesicles studies show that glycolytic enzymes associate with the plasma membrane.

• These enzymes create a localised ATP pool that fuels PMCA activity.

• Further research indicates that the PMCA preferentially uses membrane-generated ATP for Ca²⁺ transport.

How is the PMCA-glycolytic ATP system relevant in cancer cells?

• Cancer cells often show aberrant glycolytic enzyme expression (Warburg effect).

• This may provide a privileged glycolytic ATP supply to Ca²⁺ pumps.

• This can represent a potential novel therapeutic target.

What evidence supports the role of sub-membrane glycolytic ATP in cell function?

• Silencing PFKFB3 in endothelial cells disrupts the sub-membrane ATP pool and inhibits migration.

• It’s unclear if this involves Ca²⁺ signalling disruption or operates similarly to cancer cells.

• Highlights the potential importance of sub-membrane glycolytic cascades in cell invasion and cancer progression.

How could targeting glycolytic enzymes serve as a cancer therapy?

• The Warburg effect may be both a survival strategy and the “Achilles heel” of cancer cells.

• Targeting glycolytic enzymes like PFKFB3 and PKM2 could:

  • Cut off ATP supply to Ca²⁺ and Na⁺ pumps

  • Inhibit proliferation, migration, and invasion

  • Selectively kill cancer cells while sparing normal cells reliant on mitochondrial ATP