Lecture 09: Sodium Transporter And Cancer

How are Ion Channels Involved In Hanahan and Weingbergs Hallmarks of Cancer?

• Sustained Proliferative Signalling: K⁺ channels are important in regulating the cell cycle and membrane potential, impacting cancer cell proliferation.

• Avoiding Immune Destruction: Na⁺ channels and transport proteins regulate intracellular Na⁺ levels, influencing immune evasion.

• Activating Metastasis and Invasion: Na⁺ channels and regulation of intracellular Na⁺ play a role in cancer cell invasion and metastasis.

How Do Na⁺ Channels and Transporters Contribute to Cancer Progression

• Na_v channels are expressed in strong metastatic cancer

• Intracellular Na⁺ levels are critical for cancer invasion and progression

• Increased [Na⁺]_i is common in cancer cells and the tumour microenvironment

• Changes in the expression of Na⁺ transporters (e.g., VGSC, NHE1, Na/K-ATPase) at the plasma membrane alter Na⁺ homeostasis, leading to

  • Pro-tumorigenic acidity in the TME, promoting invasion

  • Dysregulated growth signalling, invasion/metastasis, metabolic changes, and immune evasion (cancer hallmarks)

• This altered Na⁺ transporter activity is key in regulating Na⁺ levels and driving cancer progression.

How do Na⁺ channels contribute to cancer progression, and where are they expressed?

• Na⁺ channels, typically found in excitable cells (nerves, muscles), can be abnormally expressed elsewhere, e.g. in cancer cells, promoting invasion and metastasis.

• Nav channels are functionally expressed in strongly metastatic cancer cells, where they:

  • Promote Na⁺ influx, leading to increased intracellular Na⁺ ([Na⁺]i).

  • Promote invasive behaviours like migration and the formation of metastases in distal sites (away from the parent tumour).

• Na⁺ channels are involved in NSCLC, breast cancer, mesothelioma, melanoma, and neuroblastoma.

• Other Na⁺ channels (e.g., ENaC, ASIC) also increase [Na⁺]i and are linked to several cancers

Where Can Voltage Gated Ion Channels Be Found?

• Typically found in excitable cells (nerves, muscles), where they contribute to AP generation

• They can also be found in non-excitable cancer cells of epithelial origin; unusual

What are the current challenges in treating metastatic cancer, and why is survival low?

• Metastatic cancer survival remains low despite advances in education, diagnostic screening, and biomarker identification.

• ~90% of metastatic cancers are incurable, and current treatments need to focus on preventing metastasis (e.g., breast cancer surgery aims to stop cancer cells from spreading and invading later).

• Current palliative therapies (chemotherapy, radiotherapy) have poor survival rates and create a significant burden on patients.

Which Na_v Subtypes are Typically Expressed in Cancer

• Na_v1.5 – breast, ovarian, colon

  • Normally found in cardiac tissue

• Na_v1.6 – cervical

  • Normally located in CNS/PNS

• Na_v1.7 – prostate, colon, non-small cell lung cancer

  • Influence nociception

• In some cases, there are particular variants of channel subtypes expressed

How do specific ion channel variants contribute to cancer progression?

• Certain channel variants are expressed in cells, including variants that are normally not present

• This can include variants that are not normally expressed in adults.

• In prostate cancer, the SkM1 variant of Nav1.7 is expressed in metastatic cancer cells

• These channel variants can act as biomarkers for prognosis or be selectively targeted in treatments alongside existing therapies

How does the nNav1.5 variant of Nav1.5 contribute to breast cancer progression?

• The nNav1.5 neonatal variant (TMD1:S3/4) is expressed in strongly metastatic breast cancer cells

  • nNav1.5 is an early development variant not normally expressed in adults.

• nNav1.5 correlates with poor prognosis in breast cancer (biomarker)

• Blocking nNav1.5 function using antibodies reduces metastasis and invasion in cell lines → can be selectively targeted for treatment

How Was the Functional Expression of Nav Channels Shown to Affect Cell Invasion?

• Use of the patch clamp experiment comparing H460 (strongly metastatic) vs A549 (weakly metastatic) cells showed:

  • H460 cells had a strong, robust inward Na⁺ current.

  • A549 cells had weak/no Na⁺ current

• In weakly metastatic cells (A549), which lack functional Nav channels, there is a reduction in invasive potential.

• When Na⁺ influx through Nav channels is blocked using high [TTX], the inward Na⁺ current is abolished, and invasion is reduced by 40% in strongly metastatic cells (H460).

• Functional expression of Nav channels and Na⁺ influx promotes invasion in strongly metastatic cells.

• This phenomenon is observed in breast, prostate, and other cancers.

• 30-50% of cancer cell invasion is directly related to the Nav channel expressed in the cancer.

How is Nav1.7 expressed in non-small cell lung cancer (NSCLC)?

• Nav1.7 is highly expressed in NSCLC tissue.

• Detected in patient biopsy using antibodies (Ab) showing brown staining, indicating high levels of the Nav1.7l in cancer tissue.

• In contrast, normal tissue showed no staining, indicating low or no Nav1.7 expression.

How Do Nav Channels Function in Non-Excitable Cancer Cells of Epithelial Origin?

• Nav channels are aberrantly expressed in highly metastatic cancer cells, despite these cells being non-excitable.

• Unlike neurons or muscle cells, these cancer cells do not fire action potentials (no dynamic changes in resting potential)

• Instead, Nav channels contribute to a depolarised resting membrane potential and elevated intracellular Na⁺ levels in strongly metastatic cells (e.g. H460) compared to weakly metastatic cells (e.g. A549).

• Pharmacological inhibition of H460 cells with TTX causes membrane hyperpolarisation and reduces intracellular Na⁺ to levels seen in weakly metastatic cells.

• These Na channels regulate resting membrane potential and Na⁺ homeostasis, rather than excitability, in metastatic cancer cells

What is the Nav “window current” and how does it explain Nav function in non-excitable cancer cells?

• Nav channels have:

  • An activation curve (conductance vs voltage).

  • An inactivation (availability) curve (channels available for opening)

• A “window current” occurs where these curves overlap.

• In strongly metastatic cells:

  • This overlap occurs near the resting membrane potential (≈ −40 to −20 mV).

  • The RMP of H460 cells (~ −27 mV) lies within this window.

• This results in a small but persistent Na⁺ influx at resting membrane potential in SCLC.

  • Sustained depolarisation and increased intracellular Na⁺.

• This persistent Na⁺ entry supports metastatic behaviours

Why are Nav Channels Not Predominantly in the Inactive State, Especially Metastatic Cancer Cells Have Depolarised Membrane Potentials?

• In MDA-MB-231 metastatic breast cancer cells, depolarisation opens the nNav1.5 cells

• These nNav1.5 channels rapidly enter an inactive state after a few ms (patch clamp current recordings)

• This leads to the generation of transient (I_T) and persistent (I_P) currents

• The persistent current is resistant to inactivation even at depolarised membrane potentials, resulting in a small persistent inward I_Na, leading to significant Na⁺ influx at RMP

  • I_P is primarily responsible for the increased Na⁺ influx via nNa_v1.5 in strongly metastatic (BCa) cells

• Thus, Nav channels are partially open, not fully inactivated, enabling enhanced Na⁺ influx despite depolarised Vm

What Determines the Invasive Potential of Malignant Cells?

• Invasive potential is determined by the cells’ ability to degrade the basement membrane and extracellular matrices via invadopodia formation and using proteases to invade the circulation and lymphatic systems to reach distal sites and form secondary tumours

• Key proteases involved:

  • Metalloproteases (MMPs, ADAMs)

  • Cysteine cathepsins (CATs) → important for migration and invasion

• CATs & MMPs facilitate apoptosis, angiogenesis, proliferation, migration, and invasion and have optimal activity at acidic pH, supporting function in the tumour microenvironment

What evidence demonstrates the functional relationship between cathepsins (CATs) and Naᵥ1.5 channels in breast cancer invasion?

• Investigated using selective CAT inhibitions in invasion assays:

• Inhibition of CAT-B or CAT-S alone → ~40-60% reduction in invasion

• Inhibition of Naᵥ channels with TTX + CAT-B+S inhibition → no additive effect
  → suggests CATs and Naᵥ1.5 act in the same pathway to promote invasion

• Inhibition of CAT-L + Na_v1.5 with TTX shows an additive effect, indicating that they act via separate pathways; blocking both further reduced invasion

• CAT inhibitors can reduce invasion in MDA-MB-231 cells

  • Some CAT inhibitors + TTX have no additive effect

• Therefore, CAT-B/S likely promote invasion downstream of or alongside Naᵥ1.5 activity

How was nNav1.5 and NHE1 shown to Promote Acidification?

• Inhibitors and siRNA were used to reduce Nav1.5 and NHE1 activity and expression

• Resulted in reduced H⁺ efflux

• TTX (Nav1.5 inhibitor) reduced proton efflux by ~45–50%

• EIPA (NHE1 inhibitor) reduced exchanger activity by ~80%

  • NHE1 regulates pH and is involved in extracellular protein extrusion

• No additive effect when both were inhibited → indicates Nav1.5 and NHE1 work together to promote extracellular extrusion of protons and acidification

How was it shown that Nav1.5 and NHE1 work together to promote invasion and matrix degradation?

• siRNA or inhibitors were used to inhibit Nav1.5 or NHE1

• Inhibition of either protein caused the same reduction in invasion

• No additive effect when both channel and transporter were inhibited, suggesting they act together to promote invasion

• Their contribution to matrix degradation was measured by fluorescence

  • Inhibition of each reduced fluorescence to the same extent for Nav1.5 and NHE1 (no additive effect)

• This suggested that Nav1.5 and NHE1 work in concert to promote MDA-MB-231 cell invasion and matrix degradation

What evidence shows that nNav1.5 and NHE1 are functionally coupled to promote invasion?

• nNav1.5 and NHE1 are co-localised at the membrane, shown by yellow fluorescence

• This close proximity suggests functional coupling

• Sucrose gradient fractionation was used to identify lipid raft localisation of NHE1 and nNav1.5

  • Both proteins were found in buoyant lipid raft fractions

• Nav1.5 was detected using a Cav1 (caveolin-1) antibody, a lipid raft marker

• This demonstrated that nNav1.5 and NHE1 are co-localised in lipid rafts and are in close proximity, supporting functional coupling in invasion

How was interaction between Nav1.5, NHE1, and Caveolin-1 demonstrated in MDA-MB-231 cells?

• Nav1.5, NHE1, and Caveolin-1 are all expressed in MDA-MB-231 cells

• Co-immunoprecipitation (co-IP) was used to test protein interactions, identifying strongly associated proteins

• Nav1.5 and Caveolin-1 co-immunoprecipitated with NHE1, but not with control IgG

  • Caveolin-1 was used as a lipid raft marker

• Strong bands for Nav1.5 and Cav-1 when NHE1 was immunoprecipitated, indicating a close association (direct or indirect)

• This demonstrated that Nav1.5, NHE1, and Caveolin-1 form a strongly associated complex in lipid rafts

How does Nav1.5 modulate NHE1 activity to increase H⁺ efflux?

• Nav1.5 interacts with and allosterically modulates NHE1

  • NHE1 activity was measured by Li⁺ uptake

• Nav1.5 increases NHE1 activity at intracellular pH ~6.4–7.0

• Knockdown of Nav1.5 reduced NHE1 activity at pHi ~6.5

• This suggests that Nav1.5 allosterically enhances NHE1 function, increasing H⁺ efflux at acidic pH

How Does nNav1.5 Contribute to Invasion in Breast Cancer

• nNav1.5 contributes to invasion via:

  • pH regulation → association and allosteric modulation of NHE-1 → H+ efflux and cystine cathepsin activity → ECM degradation

  • Promotion of Src medated actin polymerisation → invadipodia formation and acquistion of invasive phenotype

• pH dependent mechanism between NHE-1 and Na⁺ channel

What preliminary evidence links Nav1.7 to cancer invasion in NSCLC?

• In NSCLC, Nav1.7 works in concert with NHE1 to promote H⁺ efflux

• This increased H⁺ efflux contributes to cancer cell invasion

• EGF regulates the expression of Nav1.7

What is Ranolazine and What is its Effect in Breast Cancer ?

• An anti-arrythmic → approved for chronic angina in 2006

  • Na_v1.5 selective inhibitor of late (persistent) I_Na current in the heart

• In breast cancer cells, it:

  • Reduced I_Na amplitude (persistent I_Na current response for Na+ influx and invasion in breast cancer )

  • Leftward shift in availability curve → fewer channels available for opening at depolarised potentials → reduces Na+ influx → reduces breast cancer invasion

  • Reduced Na_v1.5 activty

How was the role of Nav1.5 in metastatic lung colonisation assessed in vivo?

• Breast cancer cells were treated with (and without) ranolazine or Nav1.5 siRNA/shRNA

• Control cells had normal Nav1.5 expression

• Cells were implanted into mice

• Lung colonisation was monitored over 8 weeks

What effect does Nav1.5 inhibition have on lung colonisation by breast cancer cells?

• Control cells showed significant lung colonisation

• Knockdown of Nav1.5 abolished lung colonisation after 8 weeks

• Ranolazine reduced lung colonisation, though some metastasis remained

• This demonstrated that Nav1.5 activity promotes metastatic lung colonisation

Why is ranolazine considered for drug repurposing, and what is a limitation?

• It has a known safety and toxicity profile (drug repurposing)

• It reduces invasion and lung colonisation

• Limitation: Nav blockers (e.g. ranolazine, phenytoin) do not distinguish between adult Nav1.5 and nNav1.5 → potential side-effect concerns

How Was 3D QSAR Used to Develop Neonatal nNav1.5 Specific Variant Blockers?

• Aim to selectively target the neonatal Nav1.5 variant theraputically

• A 3D QSAR model was used to develop 5 small-molecule nNav1.5 blockers

• Compound 1 shows concentration-dependent inhibition of Nav1.5 current

  • slowed channel activation

  • left shift in the availability curve, reducing n.o channel opening during depolarisation

• At 1 µM, compounds inhibited invasion as effectively as 30 µM TTX

• No further development reported since 2018

What drug classes are being explored for repurposing to target voltage-gated sodium channels in cancer?

• Antiarrhythmics: e.g. ranolazine, lidocaine

• Analgesic inhibitors (local anaesthetics): e.g. lidocaine, ropivacaine

  • Retrospective studies suggest that regional anaesthesia during breast and prostate cancer surgery may increase disease-free survival

• Anticonvulsants/analgesics: e.g. phenytoin, carbamazepine

How has rational design of small-molecule Nav blockers become possible, and what are key examples?

• Rational drug design was difficult until structural data became available

• Recent structures of human and bacterial Navα subunits with bound ligands revealed drug-binding sites

• These structures enabled the rational design of Nav blockers

• Example: Dutta et al. (2018) developed nNav1.5 blockers

  • Clathrodin (marine alkaloid): state-dependent, non-selective Nav blocker

• Resveratrol (plant poylphenol): non-selective Nav blocker with effects in breast cancer and NSCLC

What Have Retrospective Studies Revealed About the Use of Voltage-Gated Nav Channel Inhibitors on Cancer Patient Survival?

• Fairhurst et al., (2023) Retrospective cohort study in colon, breast, prostate cancer patients found:

  • Increased cancer-specific survival in patients exposed to Class 1b & 1c antiarrhythmics (VGSC inhibitors, e.g. lidocaine)

  • No survival benefit with other local anaesthetics, Tricyclic Antidepressants, or anticonvulsants found

• Further trials needed studing a broader range of VGCS inhibitors, including agents like ranolazine

What clinical evidence supports a survival benefit of Naᵥ channel blockade in breast cancer patients?

• Open-label, multi-centre randomised trial tested impact of pre-surgical perit CT in early breast cancer surgery

• Peri-tumoral lidocaine at surgery → ↑ DFS & OS vs no lidocaine

• Risk reduction: 26% (DFS), 29% (OS)

• Benefit across all subgroups (tumour size, nodes, age, surgery type)

• Supports Nav channel block improving cancer survival

• Suggests simple, low-cost surgical changes may reduce metastasis

• Open-label, multi-centre randomised trial in breast cancer mastectomy patients

• Tested pre-surgical peri-tumoral infiltration of lidocaine (local anaesthetic and Naᵥ blocker)

• Lidocaine at surgery significantly improved disease-free survival (DFS) and overall survival (OS) in patients with early BCa compared to control

• Relative risk reduction:

  • 26% (DFS)

  • 29% (OS)

• Benefit observed across all subgroups (tumour size, lymph node status, age, surgery type - conversation/mastectomy)

Why is peri-operative lidocaine considered strong evidence for targeting Naᵥ channels in breast cancer?

• It suggests that blocking Naᵥ channels during surgery reduces metastasis formation

• This indicates that small, inexpensive surgical interventions can influence long-term cancer outcomes

• Represents the best clinical evidence to date linking the benefit of Naᵥ channel block to improved survival in BCa patients

• Provides key “Go/No-Go” evidence required for pharmaceutical development of Naᵥ channel blockers in cancer

  • It highlights a non-toxic, peri-operative therapeutic window

Would Targeting Nav and Other Channels Add Value to Current Treatment Options?

• Yes, as

  • Clinical and retrospective studies show that channel blockers produce a beneficial effect

  • No current effective treatments that specifically reduce metastasis (or against metastatic cancers) exist

  • Supporting evidence by Badwe et al (2023) lidocaine clinical trial

    • Perioperative lidocaine improved DFS and OS in breast cancer patients

  • Naᵥ channel inhibitors could be used as an adjunct to existing therapies

  • Combination therapy may increase the therapeutic window and reduce metastatic spread (more effective)

Should cancer therapies target specific Naᵥ channel subtypes or use broad-spectrum blockers?

• Subtype-selective blockers may be advantageous in some cancers

  • e.g. nNaᵥ1.5 in breast cancer to minimise cardiac side effects

• Broad-spectrum Naᵥ blockers (e.g. lidocaine, phenytoin) may be sufficient in other contexts

• Prolonged use of non-selective drugs, e.g. lidocaine, may cause off-target effects, supporting the development of selective agents

• The optimal strategy may depend on tumour type and channel expression profile

Why must ion pumps and transporters be considered alongside Naᵥ channels in cancer therapy?

• Intracellular Na⁺ levels strongly influence cancer cell behaviour

• Ion pumps and transporters (e.g. Na⁺/K⁺-ATPase) regulate:

  • Membrane potential

  • Secondary Ca²⁺ signalling

  • pH regulation

• Disrupting Na⁺ homeostasis (e.g. via ionophores) can:

  • Increase Na⁺ influx

  • Alter intracellular pH

  • Promote acidity and impact tumour progression

• Effective therapies may need to target channels, pumps, and transporters together

What is the main limitation in advancing Naᵥ channel–targeted cancer therapies?

• A need for more compelling human clinical data

• Greater awareness among clinicians and pharmaceutical developers is required

• Additional trials are needed to support drug development and regulatory approval