Novel insights into the RTK-dependent metastatic phenotype of KRAS-mutant lung adenocarcinoma

ABSTRACT

In a recent study, our group identified RAC guanine nucleotide exchange factors (RAC-GEFs) driving motility signaling in KRAS mutant lung adenocarcinoma cells. The RAC-GEFs FARP1, ARHGEF39 and TIAM2 play fundamental roles in the formation of membrane ruffles in response to growth factor receptor stimulation.

Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) representing ∼85% of new diagnoses. NSCLC encompasses three main histological subtypes: adenocarcinoma (∼40-50%), squamous cell carcinoma (∼25-30%) and large cell carcinoma (∼10-15%).^1–3 The disease is often detected at locally advanced or advanced metastatic stages, with poor prognosis and clinical outcome. According to comprehensive molecular profiling of lung adenocarcinomas, the most common somatic mutations occur in TP53 (46%), KRAS (33%), KEAP1 (17%), STK11 (17%), EGFR (14%), NF1 (11%), and BRAF (10%), and less frequently in MET, PIK3CA, RB1, and CDKN2A, among other genes.³

In order to escape from the primary tumor, cancer cells attain traits associated with high motility and invasiveness, rendering cellular phenotypes that comprise changes in the dynamics of actin cytoskeleton reorganization. These pro-metastatic processes are firmly regulated by RAC1, a small G-protein member of the RHO family of small guanosine triphosphatases (GTPases). RAC1 cycles between active GTP-bound and inactive GDP-bound states, and it is prominently expressed in lung cancer. Activation of RAC1 is closely associated with tumor migration, invasion, metastasis, and poor prognosis in carcinomas.⁴ This small GTPase participates in the formation of actin-rich membrane protrusions required for tumor cell motility and for the secretion of extracellular matrix-degrading proteases.⁵ The biological processes leading to RAC1 activation in NSCLC progression and metastasis involve the activation of RAC Guanine nucleotide Exchange Factors (GEFs), proteins that enable the displacement of bound GDP. Consequently, nucleotide exchange occurs due to the excess of GTP in the cytosol. A well-established paradigm of RAC1 signaling is the binding of RAC1-GTP to effectors such as p21 Activated Kinase-1 (PAK1), activates downstream mechanisms for actin cytoskeletal reorganization.^6,7 The majority of RAC-GEFs belong to the Dbl-like family and have characteristic Dbl Homology-Pleckstrin Homology (DH-PH) domains that are crucial for nucleotide exchange activity and regulation.⁸ Although poorly understood, the prediction is that an intricate network of receptor-activated RAC-GEFs exists, thus illustrating the complexity of RAC1-mediated cell biological functions. Conceivably, specific RAC-GEFs activate discrete intracellular pools of RAC1, leading to unique functional responses in the context of specific oncogenic drivers.^4,6

We developed an unbiased and unambiguous RT-PCR array for the simultaneous determination of the expression of 32 RAC-GEFs, coupled to a systematic RNAi screening for ruffle formation analysis. Using 14 lung adenocarcinoma cell lines with different oncogenic alterations (i.e., KRAS mutation, EGFR mutation, EML4-ALK fusion), we identified FARP1, ARHGEF39 and TIAM2 as the key RAC1 signaling regulators responsible for the formation of actin-rich ruffles upon receptor tyrosine kinase (RTK) stimulation in KRAS lung adenocarcinoma cells. Contrariwise, well-studied pro-motility/invasive RAC-GEFs (namely P-REX1 and VAV3, among others) turned out to be expendable. Intriguingly, FARP1, ARHGEF39 and TIAM2 act as downstream effectors of multiple RTKs such as Epidermal Growth Factor Receptor (EGFR), Platelet-Derived Growth Factor Receptor (PDGFR) and Tyrosine Protein Kinase MET/Hepatocyte Growth Factor Receptor (c-MET) (Figure 1). It is worth to note that these RAC-GEFs are also effectors for AXL Receptor Tyrosine Kinase (AXL), an RTK associated with metastatic disease, poor clinical outcome, and targeted therapy resistance in lung adenocarcinoma patients. We found that the AXL cognate ligand Growth Arrest Specific-6 (GAS6) induces a prominent relocalization of FARP1 to ruffle protrusions. The involvement of AXL in EGFR-driven ruffle formation in lung adenocarcinoma cells also attests to its paramount relevance in RAC-GEF/RAC1-mediated motility signaling via a receptor transactivation mechanism. Moreover, our results identified PI3K as a common connection for RAC-GEF activation downstream of RTKs, consistent with the presence of DH-PH domains in FARP1, ARHGEF39, and TIAM2. We also found a major dependence on the adaptor GAB1 for ruffle formation for all RTKs analyzed, including AXL. GRB2 Associated Protein 1 (GAB1), which recruits the p85 PI3K subunit to ensue activation of the PI3K/AKT pathway upon growth factor stimulation, can arbitrate motility signaling upstream of RAC1. Hereafter, we theorize that GAB1/PI3K embodies a fundamental node for RAC-GEF activation in lung adenocarcinoma cells. An additional important conclusion is that FARP1, ARHGEF39 and TIAM2 operate in a non-redundant fashion, since specific silencing of each RAC-GEF is sufficient to prevent ruffle formation, motility and invasion in lung adenocarcinoma cells.⁹

Figure 1.

RAC-GEFs as RTK effectors. FARP1, ARHGEF39 and TIAM2 mediate Rac1-dependent motility signaling in human KRAS mutant lung adenocarcinoma cells. These exchange factors facilitate GTP loading onto the small GTPase Rac1 leading to its activation. Rac1-GTP promotes actin cytoskeleton reorganization and the formation of membrane ruffles required for cell motility. These RAC-GEFs are important players in the metastatic cascade. c-MET, Tyrosine Protein Kinase MET/Hepatocyte Growth Factor Receptor; EGFR, Epidermal Growth Factor Receptor; PDGFR, Platelet-Derived Growth Factor Receptor; AXL, AXL Receptor Tyrosine Kinase; HGF, Hepatocyte Growth Factor; EGF, Epidermal Growth Factor; PDGF, Platelet-Derived Growth Factor; GAS6, Growth Arrest Specific 6; RAC-GEF, RAC Guanine nucleotide Exchange Factors; RAC-GAP, RAC GTPase Activating Protein.

In silico analysis revealed that 13 of the 32 Dbl-like RAC-GEFs display statistically significant up-regulation in tumors vs. normal tissue, and this includes FARP1, ARHGEF39, and TIAM2. We determined the expression of FARP1, ARHGEF39, and TIAM2 in tumor cells isolated from fresh surgically resected human lung adenocarcinomas using a methodology that involves fluorescence-activated cell sorting (FACS). Epithelial Cellular Adhesion Molecule positive (EpCAM⁺) cells were isolated with >95% purity from the tumors as well as from the corresponding normal adjacent tissue. RT-PCR analysis of RAC-GEF expression unveiled a marked up-regulation of FARP1 and ARHGEF39 in a significant proportion of EpCAM⁺ tumor samples relative to normal. As expected from database analysis, the RAC-GEF P-REX1 is not upregulated in tumor EpCAM⁺ cells, thus underlining the up-regulation of selected RAC-GEFs in primary human lung adenocarcinoma.⁹ It is also notable that some of these RAC-GEFs, such as ARHGEF39, are strong predictors of poor outcome in lung adenocarcinoma patients.¹⁰ Hence, the upregulation of selected RAC-GEFs and their associations with poor clinical outcome in lung adenocarcinoma highlights their likely relevance in disease progression.

The systematic approach described in our study could be straightforwardly utilized in different solid tumor types toward identifying specific motility and invasive effectors in a particular malignancy. Furthermore, it should provide solid rationale for the development of actionable target antineoplastic agents against RAC-GEF/RAC signaling players. In addition, our unique approach may serve to predict critical biomarkers for advanced stage in lung cancer patients, eventually assisting in upgrading patient prognosis and decision-making in the clinical setting.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).