Biological insights in non-small cell lung cancer

Abstract

Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress. Several tumor types, including non-small cell lung cancer (NSCLC), upregulate the system x_c^- cystine/glutamate antiporter (xCT) through overexpression of the cystine transporter SLC7A11, thus sustaining intracellular cysteine levels to support glutathione synthesis. Nuclear factor erythroid 2-related factor 2 (NRF2) serves as a master regulator of oxidative stress resistance by regulating SLC7A11, whereas Kelch-like ECH-associated protein (KEAP1) acts as a cytoplasmic repressor of the oxidative responsive transcription factor NRF2. Mutations in KEAP1/NRF2 and p53 induce SLC7A11 activation in NSCLC. Extracellular cystine is crucial in supplying the intracellular cysteine levels necessary to combat oxidative stress. Disruptions in cystine availability lead to iron-dependent lipid peroxidation, thus resulting in a type of cell death called ferroptosis. Pharmacologic inhibitors of xCT (either SLC7A11 or GPX4) induce ferroptosis of NSCLC cells and other tumor types. When cystine uptake is impaired, the intracellular cysteine pool can be sustained by the transsulfuration pathway, which is catalyzed by cystathionine-B-synthase (CBS) and cystathionine g-lyase (CSE). The involvement of exogenous cysteine/cystine and the transsulfuration pathway in the cysteine pool and downstream metabolites results in compromised CD8⁺ T cell function and evasion of immunotherapy, diminishing immune response and potentially reducing the effectiveness of immunotherapeutic interventions. Pyroptosis is a previously unrecognized form of regulated cell death. In NSCLCs driven by EGFR, ALK, or KRAS, selective inhibitors induce pyroptotic cell death as well as apoptosis. After targeted therapy, the mitochondrial intrinsic apoptotic pathway is activated, thus leading to the cleavage and activation of caspase-3. Consequently, gasdermin E is activated, thus leading to permeabilization of the cytoplasmic membrane and cell-lytic pyroptosis (indicated by characteristic cell membrane ballooning). Breakthroughs in KRAS G12C allele-specific inhibitors and potential mechanisms of resistance are also discussed herein.

Keywords: KRAS G12C allele-specific inhibitors; Solute carrier family 7 member 11 (SLC7A11); ferroptosis; non-small cell lung cancer (NSCLC); nuclear factor erythroid 2-related factor 2 (NRF2); pyroptosis.

Figure 1

Solute Carrier family 7 member 11 (SLC7A11) mediates cystine uptake and is a central regulator of ferroptosis, together with downstream glutathione peroxidase-4 (GPX4). Membrane phospholipids (PL) comprise polyunsaturated fatty acids (PUFAs) that are sensitive to lipid peroxidation and ferroptosis. Sulfasalazine and erastin are SLC7A11 and GPX4 inhibitors, respectively. Alternative ferroptosis scavenger pathways are compromised by: ferroptosis suppressor protein 1 (FSP1), dihydroorotate dehydrogenase (DHODH), and the tetrahydrobiopterin (BH4) biosynthesis pathway. TP53 mediates ferroptosis and irradiation sensitivity by decreasing SLC7A11. Nuclear factor E2-related factor 2 (NRF2), interferon gamma (IFN-γ), and OUT domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) repress SLC7A11 expression. Yes-associated protein1 (YAP1) upregulates several ferroptosis modulators, such as transferrin receptor (TFRC) and acyl-CoA synthetase long chain 4 (ASCL4), thus favoring ferroptosis. TFRC permits access to ferritin-bound iron for cellular use (ferritinophagy), in a process that allows iron release from ferritin and conversion of ferric iron (Fe³⁺) to bioactive ferrous iron (Fe²⁺). Zinc finger E-box binding homeobox 1 (ZEB1) is a mesenchymal marker in drug-tolerant persister cells, which may indicate inactivation of Hippo signaling and subsequent derepression of YAP1. YAP1 subsequently translocates to the nucleus and initiates its transcriptional program. Both YAP1 and ZEB1 are potential predictors of sensitivity to ferroptosis inducers GTP cyclohydrolase (GCH1).

Figure 2

The transsulfuration pathway is a powerful default pathway maintaining cysteine biosynthesis when the cystine supply is depleted or SLC7A11 is pharmacologically inhibited. In the transsulfuration pathway, methionine is converted into cysteine. The cystathionine-β-synthase (CBS) enzyme condenses serine with the methionine cycle intermediate homocysteine, thus forming cystathionine, which is later cleaved by cystathionine-γ-lyase (CTH), thereby releasing cysteine. Hydrogen sulfide (H₂S), a gas transmitter, is produced mainly by CBS, and persulfidation of OTUB at cysteine 91 enhances the stability of SLC7A11. Aminooxy acetic acid (AOAA) inhibits CBS and CTH. ASCT2 and LAT1 are amino acid transporters in the plasma membrane S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH).

Figure 3

Working model of MET in KRAS-mutant NSCLC. Enolase (ENO1) is an important enzyme that may be necessary for MET activation, through hepatocyte growth factor (HGF) and transphosphorylation of low-density lipoprotein receptor-related proteins (LRP5/6) coupling with the coreceptor Frizzled, which are together bound by the prorenin receptor (PRR), which acts as an adapter between the receptor complex and the ATP-driven pump V-ATPase. In KRAS-mutant NSCLC models growing in anchorage-independent conditions, the translation of MET is enhanced. A combination of MET inhibitors with v-ATPase inhibitors may be a complementary strategy for KRAS G12C inhibitors and MEK inhibitors.

Figure 4

Working diagram indicating that NSCLC, primarily KRAS-mutant NSCLC, has several commonalities with NSCLCs with distinct driver alterations or no drivers. The diagram shows that nuclear factor E2-related factor 2 (NRF2) hyperactivation is necessary for NSCLC cells to grow in 3-D spheroid conditions. Additionally, in 3-D conditions, SHOC2, a component in noncanonical RAS pathways, and MET are factors driving KRAS-mutant cell proliferation. Mechanisms of resistance in KRAS G12C cells (H358 parental) or AMG-510 (sotorasib) involve the downregulation of the proapoptotic BIM protein and increased Bcl-x expression. Celastrol is an inhibitor of SHOC2 in KRAS and EGFR-mutant non-small cell lung cancers (NSCLCs) (see text). Treatment with v-ATPase inhibitors and MET inhibitors warrants investigation in preclinical models. Aquaporin 5 (AQP5) is elevated in NSCLC, and may potentially influence intracellular swelling and interconnect with other signaling pathways, as shown in Figure 5. PIM1 kinase is upregulated, in a mechanism of resistance to MET inhibition in cell lines with MET amplification. A major subject of interest is that PIM1 is promoted by cancer hypoxia and is a principal regulator of the master regulator NRF2, which is involved in ferroptosis and many other pathways, including positive, direct regulation of enolase 1 or though the activation of CBS. If PIM1 regulates NRF2, and persister cancer cells are dependent on glutathione 4 peroxidase (GPX4) and methionine extra-supply (transsulfuration pathway), then PIM1 inhibition might serve as a relevant therapeutic strategy. Immunohistochemical analysis of 4-hydroxy-2-nonenal (4-HNE) is indicative of ferroptosis. Long noncoding RNAs, such as LINC00857, have important functions and have been found to be upregulated in cell lines bearing TP53 mutations. In addition, these RNAs are involved in NSCLC containing many other drivers. KRAS-resistant cells exhibit an active KRAS GTP state with downregulation of the GTPase protein regulator of G protein signaling 3 (RGS3). Persister cancer cells might possibly also display high activity of NRF2 and enolase 1. Additional descriptions of the processes depicted in the figure are provided in the main text.

Figure 5

Various intracellular processes further promote chemotherapy resistance and are refractory to other anticancer modalities. Cystic transmembrane conductance regulator (CFTR) expression is diminished in NSCLC. Mechanistically, low CFTR expression impairs the expression of the chloride ion-sensitive with-no-lysine kinase 1 (WNK1) protein, thereby leading to activation of transient receptor potential cation vanilloid 4 (TRPV4). TRPV4 serves as a mediator of calcium influx, promoting increased cell contractibility and cell migration. Ivacaftor is a potentiator of CFTR and temozolomide that activates WNK1. Acetazolamide blocks aquaporin 5 (AQP5), which is overexpressed in NSCLC and contributes to intracellular swelling. MET activates HECT, UBA, and WWE domain-containing protein 1 (HUWE1) E3 ligase, thus resulting in degradation of T lymphoma invasion and metastasis inducing protein 1 (TIAM1)—a guanine nucleotide exchange factor that regulates the GTPase RAC—and ultimately causing loss of cell adhesion. RAC1 is involved in the actin-related protein 2/3 (ARP2/3) complex dependent actin network.