Abstract

Ferroptosis is a recently discovered form of iron-dependent programmed cell death, characterized by the toxic accumulation of lipid peroxides that disrupt cell membrane function. Cancer cells are particularly susceptible to ferroptosis due to their high iron requirements for growth and a metabolism that creates an intracellular environment conducive to triggering this death mechanism. However, cancer cells counteract this vulnerability through carbonic anhydrase 9 (CA9), a protein that regulates intracellular pH, ensuring conditions unfavorable for ferroptosis.\\\\nThis minireview explores the factors necessary to induce ferroptosis and examines how CA9 not only protects cancer cells but also facilitates their migration. The aim is to highlight potential therapeutic opportunities arising from disrupting the functionality of this protein.\\\\n

Content

1.    Introduction

One of the hallmarks of cancer is the ability to evade programmed forms of cell death. As a result, strategies to induce such pre-planned death processes, which naturally occur within cells, are emerging as promising approaches to cancer treatment. Apoptosis has long been regarded as the traditional form of programmed cell death; however, many cancer cells develop resistance to apoptosis-inducing therapies, limiting their efficacy. This resistance has sparked interest in alternative non-apoptotic cell death mechanisms as a novel strategy to overcome therapy resistance and enhance treatment outcomes¹.

2.    Ferroptosis

Ferroptosis, first identified in 2012 by Dr. Brent R. Stockwell, is a regulated, iron-dependent form of non-apoptotic cell death.² The term "ferroptosis" reflects its reliance on iron, as shown by the ability of deferoxamine (an iron chelator) to inhibit this process. Unlike apoptosis, ferroptosis does not depend on caspase or receptor-interacting protein kinase 1 RIPK1 activity. Instead, it is triggered by iron-dependent lipid peroxidation, which can be mitigated by glutathione peroxidase 4 GPX4, a selenoenzyme that uses glutathione as a cofactor.³
Studies have demonstrated that ferroptosis can be initiated by glutathione depletion or GPX4inactivation, while iron chelators effectively prevent it.²¹ Recent research has also identified carbonic anhydrase 9 CA9, as a critical defense mechanism that cancer cells use to evade ferroptosis. CA9 regulates intracellular pH and influences other aspects of cancer cell metabolism. Inhibiting this mechanism could represent a novel therapeutic strategy to block cancer progression.⁴
Figure 1 illustrates the core aspects of ferroptosis, emphasizing how a combination of factors—altered pH balance, iron metabolism, and reactive oxygen species (ROS) production—leads to membrane lipid peroxidation.

Figure 1: Mechanism of ferroptosis. Ferroptosis results from lipid peroxidation due to the accumulation of ROS produced by cell metabolisms, as well as the accumulation of Fe2+ (which can trigger the Fenton reaction) and cellular responses induced by extracellular hypoxia. On the other hand, it is inhibited by the Fe2+ storage activity of ferritin, by the phospholipids reduction of GPX4 and by the intracellular pH control of CA9.

2.1. Tumor microenvironment triggers ferroptosis
The tumor microenvironment is often characterized by uneven oxygen availability, with many regions existing under hypoxic conditions. Hypoxia triggers an adaptive response mediated by hypoxia-inducible factors HIFs, which shift cellular metabolism toward anaerobic glycolysis and lactic acid fermentation to meet energy demands. While this limits ROS production, the high glucose requirement and metabolic activity of cancer cells still expose them to oxidative stress.⁵
Hypoxia-induced metabolic shifts disrupt the acid/base balance, threatening tumor survival and proliferation. Enzymes such as CA9, which regulate pH, play a pivotal role in maintaining intracellular and extracellular conditions that support tumor growth and invasiveness.⁵

2.2. Iron as a double-edged sword
Iron is essential for ferroptosis, as demonstrated by the inhibitory effect of deferoxamine on this process. Transferrin, responsible for transporting ferric ions (Fe³⁺) into cells, emerges as a key regulator of ferroptosis.⁵ In cancer cells, iron homeostasis is tightly controlled to support proliferation, with increased iron uptake and reduced iron efflux compared to non-cancerous cells.⁴
However, iron is a double-edged sword: while it supports biological processes, it also catalyzes oxidative stress through the Fenton reaction, contributing to lipid peroxidation and ferroptosis.⁴

2.3. Lipid peroxidation
The metabolic activity of cancer cells generates oxidative stress, leading to the formation of hydroxyl radicals that interact with polyunsaturated fatty acids (PUFAs) in membrane phospholipids. This interaction triggers a chain reaction of lipid ROS production, targeting nearby PUFAs and amplifying the damage. The Fenton reaction drives this process.¹
Ferroptosis arises not from a specific agonist but as a consequence of inadequate enzymatic control of lipid peroxidation. Dysfunctional GPX4 activity, which ordinarily converts lipid peroxides into alcohols, exacerbates this imbalance.³

3. The Role of CA9 in Cancer Cell Metabolism

The role of carbonic anhydrase 9 (CA9) in cancer cell metabolism has been extensively studied through methods such as silencing or suppressing its expression, as well as using molecular inhibitors and specific antibodies. These preclinical studies are paving the way for novel anticancer strategies, some of which have already progressed to clinical trials (Strapcova et al., 2020).⁶ However, before delving into the connection between CA9 and tumorigenesis, it is essential to understand the enzyme's biological role.

3.1. Structure and Functions of CA9
Carbonic anhydrases are zinc-dependent metalloenzymes primarily responsible for regulating pH through the reversible hydration of CO₂ into H⁺ and HCO₃⁻.⁴ Tumor cell survival in hypoxic environments depends on pH control mechanisms, with CA9 playing a critical role. Responding to hypoxia via activation by HIF and PKA-mediated phosphorylation, CA9 helps maintain a neutral or alkaline intracellular pH. This environment promotes cell proliferation and inhibits apoptosis by suppressing caspase activity.⁶
CA9 activation under hypoxic conditions has been demonstrated in pancreatic ductal adenocarcinoma cells, where HIF1A stabilization increases CA9 expression and glycolysis. Conversely, KRAS inhibition reduces HIF1A stabilization, CA9 production, and glycolytic activity.^7,8
CA9 is overexpressed in tumor tissues, regulating both intracellular and extracellular pH. It modulates extracellular acidity to support tumor progression via pH-dependent mechanisms, including integrin-mediated cell-matrix adhesion, extracellular matrix degradation by cathepsins, and activation of matrix metalloproteases.
Structurally, CA9 consists of an extracellular domain (ECD), a single-pass transmembrane portion, and a small intracellular tail. The ECD includes an N-terminal proteoglycan-like (PG) region and a central catalytic domain. The PG region interacts with the extracellular matrix and aids tumor cell adhesion during metastasis. This region, absent in other carbonic anhydrases, also binds the monoclonal antibody M75, commonly used for CA9detection in tumor tissues.
Catalytic activity occurs on the extracellular surface of the plasma membrane, where CA9converts CO₂ into HCO₃⁻ and H⁺, as shown in Figure 2.^9,10

Figure 2: CA9 structure. ECD: large extracellular portion, TM: single-pass transmembrane portion, IC: intracellular tail, PG: N-terminal proteoglycan-like region. It can be seen that the catalytic domain is located in the extracellular space.

3.2. The Bicarbonate Transport Metabolon
A metabolon is a transient, functional complex of sequential metabolic enzymes and structural elements, facilitating efficient metabolite transfer without bulk diffusion.⁹ The bicarbonate transport metabolon, comprising CA9and bicarbonate transporters such as NBC1SLC4A4 and AE2 SLC4A2, exemplifies this concept.
CA9-catalyzed reactions in the extracellular space generate bicarbonate ions, which are taken up by transporters and combined with intracellular protons to form CO₂. This process neutralizes intracellular pH while acidifying the pericellular space, fostering tumor invasion and progression (Figure 3).^10,11

Figure 3: Transport metabolon. CA9 converts CO2 into H+ and HCO3- in the extracellular space. HCO3- enters the cell via AE2 and NBC1, where it reacts with intracellular H+ and forms CO2, which leaves the cell by diffusion. Thus, a basic intracellular pH and an acidic extracellular pH are maintained.

3.3. CA9as a Suppressor of Ferroptosis
Numerous studies have explored the role of CA9in suppressing ferroptosis. Four key investigations are summarized below, with results detailed in Table 1.

Table 1: Experimental data on CA9 functions. CA9 protects the cell from ferroptosis because it mainly interferes with iron metabolism and ROS production. CA9 is also correlated with cancer cells migration.

3.3.1. Human Mesothelioma Cells Under Hypoxia
In mesothelioma cells, CA9 inhibition reduced cell migration, increased intracellular iron and lipid peroxidation, decreased ferritin, and elevated transferrin receptor TFRC and iron regulatory proteins ACO1, and IREB2. By stabilizing ferritin and maintaining cytoskeletal integrity, CA9 prevents iron toxicity and ferroptosis.¹²

3.3.2. Non-Small Cell Lung Cancer (NSCLC)
In NSCLC, CA9 provided protection against ferroptosis by stabilizing transferrin and ferritin, preventing transferrin endocytosis, and reducing ROS and labile iron levels. Blocking CA9 heightened cancer cell sensitivity to ferroptosis inducers like erastin, while overexpression conferred resistance even in non-tumor cells.¹³

3.3.3. Hypoxic Triple-Negative Breast Cancer
CA9 interacts with cysteine desulfurase (NFS1), which supports iron-sulfur cluster synthesis critical for cellular metabolism. Dual suppression of CA9 and NFS1 increased iron levels, lipid peroxidation, and ferroptosis. Furthermore, CA9 inhibition amplified glutamine uptake and glutathione synthesis, intensifying stress responses.^14,15
These findings underscore CA9’s multifaceted role in regulating iron metabolism, ROS production, and lipid peroxidation, making it a critical target for disrupting cancer cell survival mechanisms.

3.4. CA9 and Tumor Invasiveness
In agreement with the above, CA9 not only neutralizes intracellular pH but also influences extracellular acidity. The acidic extracellular environment facilitates the remodeling of the extracellular matrix by upregulating the expression and activation of specific proteases, such as metalloproteases. This activity enhances the migration and invasion of cancer cells by promoting collagen degradation, as demonstrated in breast cancer cells.⁴
Emerging evidence suggests that CA9 might function similarly to a chaperone, akin to heat shock proteins (HSPs). It has been proposed that the suppression of TF/TFR1-mediated endocytosis by HSPs is linked to increased actin polymerization, a feature commonly observed in cancer cells with high CA9 levels. Furthermore, blocking HSPB1 has been found to induce ferroptosis, a form of cell death driven by lipid peroxidation, while elevated HSPB1 levels confer resistance to this process. Similarly, CA9 appears to regulate the cytoskeletal structures of cancer cells by controlling cytoplasmic filaments, contributing to their migratory and invasive properties.⁴
CA9 also plays a pivotal role in tumor invasiveness. Studies have identified CA9 as a marker for circulating tumor cells (CTCs), and its inhibition has been shown to reduce CTC levels significantly.^14,16,17

3.5. LINC02086/miR-342-3p/CA9 Axis
A recent investigation into pancreatic cancer cells, which exhibit high expression of the long noncoding RNA LINC02086, explored its potential involvement in the miR-342-3p/CA9 axis. The study found that knockdown of LINC02086 promotes ferroptosis by functioning as a competing endogenous RNA (ceRNA) for miR-342-3p. This interaction leads to the overexpression of CA9 and GPX4, both suppressors of ferroptosis.
LINC02086 contains a binding site for miR-342-3p, which is notably less expressed in pancreatic cancer cells compared to normal tissue. Increased expression of miR-342-3p elevates intracellular Fe²⁺ and ROS levels while reducing GPX4 and CA9. By inhibiting miR-342-3p, LINC02086 prevents these effects, thereby promoting cancer cell survival. These findings suggest that CA9 activity is regulated upstream by a long noncoding RNA (Figure 4).¹⁸

Figure 4: LINC02086 control of CA9. LINC02086 controls CA9 expression by inhibiting the function of miR-342-3p, which decreases the levels of the anhydrase.

4. CA9 Beyond Ferroptosis: Promising Findings on Its Other Roles in Tumorigenesis
Another hallmark of tumor behavior, alongside evading cell death, is the ability to escape immune system regulation. It is, therefore, unsurprising that CA9 also functions as an immune suppressor in addition to its role in ferroptosis suppression.
The pH-regulating activity of CA9 has been linked to the inhibition of CD8⁺ T cell infiltration. Specifically, CA9 promotes the expression of SIGLEC15 (Sialic Acid Binding Ig Like Lectin 15) on cancer cell surfaces. SIGLEC15 binds to receptors on CD8⁺ T cells, antagonizing their cytotoxic activity.¹⁹ Moreover, CA9 has been shown to confer resistance to immune checkpoint inhibitors and impair CD8⁺ T cell functionality in acidic tumor microenvironments.²⁰
Intriguingly, CA9 appears to suppress immune responses through mechanisms that extend beyond its pH-regulating functions.^19,20 These findings establish CA9 as a central player in various processes that enable cancer cells to adapt and thrive within the host tissue (Figure 5).

Figure 5:  Overview of CA9 functions in cancer cells. Here are summarized the roles of CA9 discussed in this minireview in the tumour phenotype acquisition process. The main one is its activity as a pH regulator, but there are certainly other mechanisms that we do not yet know about.

Conclusion
Cancer cells have evolved adaptive mechanisms to survive within hostile tumor microenvironments. The CA9 protein plays a crucial role in these adaptations, acting as a safeguard against intracellular acidity—a direct consequence of altered cancer cell metabolism. By mitigating environmental stress that would otherwise trigger programmed cell death, CA9 enables cancer cell survival and progression.
However, this essential role also presents a potential vulnerability. As demonstrated in this mini-review, loss of CA9 functionality exposes cancer cells to a high risk of death, suggesting that CA9 could be a key target for anticancer therapies.
Thus, CA9 represents a true Achilles' heel in cancer survival and proliferation mechanisms, paving the way for innovative therapeutic strategies.