A Crosstalk between the Receptor Tyrosine Kinase-Like Orphan Receptors ROR1/2 and S1P Signaling Pathways in Lung Cancer (2024)

Abstract

Key Words: SPHK1- ROR1- ROR2- Lung Cancer- NSCLC

Introduction

Lung cancer is the most common type of cancer globally. Owing to tumor heterogeneity and drug resistance, patients with non-small cell lung cancer (NSCLC) have poor prognosis [1, 2]. Treatment modalities for NSCLCs include chemotherapy, radiation, molecular targeted therapy, and immunotherapy. Furthermore, several drugs targeting various oncogenic pathways in NSCLC have been introduced in the last decade [3]. Oncogenic pathways targeted by such pharmacological molecules include, epidermal growth factor receptor (EGFR), ROS proto-oncogene 1 (ROS1), cellular mesenchymal epithelial transition (c-MET), fibroblast growth factor receptor (FGFR), mammalian target of rapamycin (mTOR), insulin like growth factor 1 receptor (IGFR), rearranged during transfection (RET), proto-oncogene B-Raf (BRAF), and anaplastic lymphoma kinase (ALK). However, most of the patients with NSCLC are diagnosed in the advanced stages (III/IV) and the median overall survival (OS) for patients with metastatic NSCLC is only 4–5 months [4, 5]. Thus, further research identifying newer drug targets for NSCLC is required.

Sphingosine-1-phosphate (S1P), a potent signaling molecule, is involved in various aspects of carcinogenesis. Sphingosine Kinases (SphK) are two enzymes that catalyze the synthesis of S1P. Overexpression of sphingosine kinase (SphK1) in several malignancies, including lung cancer, correlates with metastasis and poor prognosis (Pyne and Pyne, 2020). S1P is a natural ligand for five G-protein coupled receptors (GPCR) known as S1P receptor 1-5 (S1PR1-5) [6]. It activates downstream signaling pathways, regulating various cellular and biological functions such as cell proliferation, lymphocyte trafficking, inflammation and neovascularization [6]. On the other hand, receptor tyrosine kinase (RTK)-like orphan receptors (RORs) are also overexpressed in various forms of cancer, including NSCLC [7].

Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signalling pathways and is constitutively activated in many types of cancers [8]. S1P-S1PR1 signalling induces persistent activation of STAT3 and leads to chronic intestinal inflammation and development of colitis-associated cancer [8]. Interestingly, ROR1 expression is induced by STAT3 [9], which is in turn being activated by S1P. Additionally, S1PR1 trans-activates receptor tyrosine kinases [10]. Thus, we hypothesized that the two pathways may crosstalk to regulate cell growth, survival, and carcinogenesis.

In this study, we examined the crosstalk of S1P and ROR1/2 pathways in lung cancer cells and found an association between them. S1P treatment decreased ROR1 and ROR2 transcript levels in lung cancer cells, while treatment with PF-543, a pharmacological SphK1 inhibitor or genetic knockdown of SPHK1 by shRNA, raised ROR1 and ROR2 levels. These outcomes demonstrate the reciprocal regulation of both pathways, suggesting that both pathways have an inverse relation i.e. in the absence of one pathway, another pathway may compensate the other pathway. Therefore, simultaneously targeting both pathways could serve as a potential therapeutic target for lung cancer treatment.

Materials and Methods

Establishment of cell lines and cell culture

The lung cancer cell lines A549 and L132 were purchased from the National Center for Cell Science (NCCS), Pune India, while bronchial epithelial cells BEAS2B (ATCC# CRL-9609) was procured from the American Type Culture Collection (ATCC). Dulbecco’s modified Eagle Medium (DMEM) was used to culture the cells (A549 and L132). Furthermore, BEAS-2B cells were grown in a BEGM medium (Lonza) containing 10% fetal bovine serum (FBS). All experiments were carried out on BEAS2B cells between passages 10 and 30, with passage 1 defining the frozen cells from the supplier. Cells were trypsinized using trypsin-EDTA (0.25%). Cell cultures were incubated in a humidified 95% air: 5% CO2 environment. In 75-cm culture flasks, 2 million cells were plated and sub-cultured at 90% confluence.

Gene knockdown by short hairpin RNA (shRNA)

Transfected knockdown cells were harvested for RNA extraction. The clones (shRNA_SPHK1, shRNA_SPHK2, shRNA_ROR1, and shRNA_ROR2) were generated by transfection of cells and protocol was used according to the manufacturer. Table 1 shows the shRNA target sequences.

Lentiviral Particle Preparation

To produce a lentivirus containing shRNA, HEK293T cells were seeded in a 10-cm culture dish and co-transfected with plasmids at the concentrations shown below using the calcium phosphate technique once they reached 60-70% confluency. The following plasmids were used: pMD2.G of 1.5 µg, ps PAX2 of 3.75µg, and shRNA plasmid of 5 µg. Spent media was collected 48 hours after the transfection and filtered using a 0.45 µm syringe filter before being preserved at -70°C for future use. pMD2.G and psPAX2 plasmids were obtained from Addgene, whereas shRNA-SPHK1 were purchased from Sigma Aldrich.

The preparation of a Puromycin Kill Curve and selection of stable clones

For the preparation of kill curve, cells were seeded in 24-well plates and treated with different concentrations of puromycin for seven days, and the minimum concentration at which all the cells died, was used for the selection of transformed clones. A549 and BEAS2B were infected with lentivirus containing small hairpin RNA (shRNA) specific to ROR1 (shROR1), ROR2 (shROR2), SPHK1 (shSPHK1), and pLKO (shControl) with 8 µg/ml polybrene containing media. Cells were selected using 2µg/ml puromycin for 7 days. After selection, cells were used for downstream experiments.

RNA Extraction

Lung cancer cell lines L132, A549, and BEAS2B were cultured in 100-mm² cell culture plates in a complete DMEM medium. The confluency of cells was assessed under an inverted microscope (Model: Ti-Eclipse, make Nikon) and after washing with sterile PBS, cells were harvested and pelleted down by centrifugation. Cells were lysed in cell lysis buffer and cells were passed 8-10 times through a syringe attached with 20-gauge needle.RNA extraction was performed using PureLink RNA mini kit (Ambion) and PureLink DNase (Ambion) as per manufacturer’s protocol. Total RNA was quantified spectrophotometrically at 260 nm and 280nm using the Nanodrop (Thermo Fisher). cDNA synthesis was performed using BioRad iScript cDNA synthesis kit (Catalogue No. 17188), as per manufacturer’s protocol.

Real-time polymerase chain reaction (qRT-PCR)

To quantitate SPHK1, SPHK2, ROR1, ROR2, and GAPDH mRNA levels, reverse transcription (RT) reactions were performed using the iScript cDNA synthesis kit (Biorad, Hercules, CA) on Nexus Gradient Thermal Cycler (Eppendorf). Real-time PCR reactions were carried out using iTaq Universal SYBR-Green mix (Bio-Rad Laboratories) on CFX-96 thermal cycler (Bio-Rad, Hercules, CA) by using following primer sets SPHK1 Forward 5´-GGGAACTGGGCCACTTGT-3´Reverse 5’-CAAAGCCAAGCCCGAACC-3’; SPHK2 Forward 5’-CCGGAAGAAAGGGATCTGGG-3’, Reverse 5’-TTCAGCTCTCCAACACTGGG-3’; ROR1 Forward 5’-CAACAAGAAGCCTCCCTAATGG-3’, Reverse 5’-CCTGAGTGACGGCACCTAGAA-3’ (reverse); ROR2 Forward 5’–GGCAGAACCCATCCTCGTG-3’, reverse 5’-CGACTGCGAATCCAGGACC-3’; GAPDH Forward 5’-AATCCCATCACCATCTTCCAG- 3’; Reverse 5’-AAATGAGCCCCAGCCTTC-3’. GAPDH was used as a reference gene.

MTT Assay

Post-puromycin selection, cells were seeded in 96-well culture plates (8 × 10³ /well) for 12, 24, 36, 48, and 72 h (in triplicate for each condition). MTT (Sigma, Saint Louis, USA) solution 10 µl (50 mg/ml) was added to each well and incubated for 4h. After the incubation, formazan crystals formed in the cells were solubilized using dimethyl sulfoxide, and the optical density was recorded at 570 and 600 nm using plate reader BioTek Eon (BioTek, Winooski, USA).

Wound healing assay

Post puromycin selection, 5× 10⁴ cells/well were seeded in 24-well plate, and at approximately 100% confluency, a wound (scratch) was created using a 200 μl pipette tip and washed with 1× PBS for two times to remove cellular debris. Wounds were visualized at 10× with an inverted microscope Eclipse Ti (Nikon India Pvt Ltd), and three random images were captured at various time points. Wound width was measured using Image J software, and the graph was plotted with GraphPad Prism9 (La Jolla, CA, USA).

Bioinformatics analysis

Correlation between the two pathways in patient samples was determined using the available online tool GePIA (http://gepia.cancer-pku.cn/ ) [11]. Pearson’s correlation was used to assess the correlation between expressions of two genes in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumor and normal.

Statistical analysis

The statistical analysis was performed with GraphPad Prism 9 (La Jolla, CA, USA). The difference in the mean between the two groups was compared using student’s t-test. Differences among multiple groups were compared using two-way-ANOVA with Sidak’s multiple comparison test. The differences were considered statistically significant with P < 0.05.

Results

Treatment with S1P inhibits the expression of ROR1/ROR2

Lung cancer cells were treated with vehicle, 10 nM, 100 nM, and 1 µM concentrations of S1P for 24 hours. mRNA expression of ROR1 and ROR2 was measured using qRT-PCR. S1P decreased the mRNA expression in a dose-dependent manner, with a substantial reduction in mRNA transcript at 100 nM concentration (Figure 1A and B). However, the decrease in ROR2 mRNA levels was more significant at 1 µM concentration (Figure 1A and B). To strengthen our findings, we employed an alternative approach in which we controlled intracellular S1P levels in lung cancer cells using PF-543, an SphK1 inhibitor. Lung cancer cells, A549 were treated with either a vehicle or a 1 µm dose of PF-543, and gene expression of ROR1 and ROR2 was evaluated using qRT-PCR. As shown in Figure 1C, we noticed that ROR1 and ROR2 expression elevated approximately twofold in PF-543-treated cells.

The preliminary results suggested that increasing intracellular S1P levels inhibits ROR1 and ROR2 transcription, whereas blocking S1P production using an SphK1 inhibitor, PF-543, induces ROR1 and ROR2 transcription. To confirm these findings, an shRNA was used to knockdown SPHK1 in HEK293T and BEAS2B cells, showing greater than 95% gene silencing efficiency (Figure 2A). Transient knockdown of SPHK1 in BEAS2B increased ROR1 and ROR2 expression, suggesting SPHK1 can alter ROR1/ROR2 expression at the transcription level (Figure 2B).

ROR1/ROR2 knockdown promotes SPHK1 expression

Then, we asked whether ROR1 and ROR2 influence the expression of SPHK1 in lung cancer cells. ROR1 and ROR2 expression was silenced using siRNAs against ROR1 and ROR2, and a 2-fold increase in SPHK1 expression was observed in ROR1-knocked down cells. However, SPHK1 expression was not changed in ROR2-knocked down cells (Figure 2C and 2D). Two shRNAs against the ROR1 gene were used to knock down ROR1 expression, with shROR1-A showing better efficacy than shROR1-B as shown in Figure 3A. Therefore, shROR1-A was employed for further experimental procedures. ROR1 expression was reduced by approximately 65% in A549 cells with stable knockdown and by approximately 50% in BEAS2B cells (Figure 3B and 3C). However, decreased ROR1 expression had no influence on SPHK1 mRNA expression, and a 50% decrease in SGPL1 mRNA expression in A549 cells as mentioned in Figure 3B. This suggests that ROR1 and ROR2 work to maintain intracellular S1P homeostasis, as ROR1 lowers SGPL1 and thus increases S1P levels in the cells.

SPHK1 and ROR1 deficiency reduces the proliferation of lung cancer cells

To assess the role of two pathways individually on cell proliferation, SPHK1 and ROR1 knocked down cells were seeded on a 96-well plate, and cell proliferation at various time points was determined using an MTT assay. As shown in Figure 4A, SPHK1 and ROR1-knockdown cells respectively exhibited decreased cell growth compared to shControl cells.

Combinatorial inhibition of SphK1 and ROR1 significantly reduces cell proliferation

Since previous findings established the reciprocal role, the deficiency of one pathway can be compensated by the other. Therefore, to understand the effect of combined inhibition of both pathways, ROR1 knocked-down cells or shControl cells were treated with PF543 at various doses. ROR1 knockdown cells showed reduced viability than the shControl as well as untreated cells (Figure 4B-C). In addition to this, wound healing assay suggests that inhibiting both pathways individually decreases the migration efficiency of the cells (Figure 5A-B), whereas simultaneous inhibition of SPHK1 along with ROR1 but not with ROR2, reduced the migration of lung cancer cells (Figure 5C). This finding suggests that simultaneous inhibition of both pathways might inhibit metastasis.

Furthermore, in order to assess the correlation between the two pathways in the patient samples, publicly available online tool GePIA was utilized. Correlation between the expression of two genes in lung adenocarcinoma and lung squamous cell carcinoma tumor was analyzed. SPHK1 was found to be negatively correlated with ROR1 expression and positively correlated with ROR2 expression (Figure 6A-B). Therefore, it becomes crucial to understand how the complex regulation of these pathways together can modulate the process of carcinogenesis.

Discussion

Here, we show that S1P signaling and ROR1/ROR2 signaling have reciprocal regulation in lung cancer as well as normal lung epithelial cells (Figure 7). Treatment of the cells with S1P reduces ROR1 and ROR2 transcript levels, whereas inhibition of S1P synthesis either by a pharmacological inhibitor of SPHK1 (PF543) or genetic knockdown of SPHK1 by shRNA increases ROR1 and ROR2 (prominently). Thus, these findings indicate that both pathways have reciprocal regulation. ROR1 and ROR2 try to maintain intracellular S1P homeostasis, as the knockdown of ROR1 decreases SGPL1 (an S1P catabolizing enzyme) and increases SPHK1 protein levels in the cells, thereby increasing S1P levels. Both pathways are known to promote cell growth and are involved in oncogenesis. Also, ROR1 is known to mediate its effect via Wnt signaling [12], which is also one of the downstream targets of SPHK1 [13]. Therefore, it is plausible that deficiency of one pathway may compensate for the other pathway, whereas the excess of intracellular S1P (as it occurs in the exogenous treatment) reduces the other oncogenic pathway (ROR1 and ROR2). However, simultaneous obstruction is more efficient at preventing cell growth.

To improve efficacy, minimize off-target toxicity, and provide a therapeutic benefit, targeted therapies require cellular protein expression that meets specific requirements. Lung cancer in India is a major health problem that is unfortunately diagnosed at an advanced stage, contributing to a poor prognosis. Therefore, to develop novel and efficient chemotherapeutic drugs, it is pertinent to fully understand the mechanism of lung carcinogenesis. Previous research found that ROR1 protein expression was significantly higher in lung ADC tissues than adjacent non-tumor tissues [14]. SphK1 has been shown to be overexpressed in the NSCLC tumors. SphK1 expression predicts the survival of NSCLC patients [15] and we had shown that NSCLC patients with high SPHK1 expression had a shorter OS [16]. In addition, the expression of PLPP1, PLPP3, and S1PR1 has been shown to decrease in NSCLC tumors as compared to normal tissues and serve predictive biomarkers [16].

Patients in advanced stages and those with positive lymph node metastases had greater ROR1 levels [17], and S1P signaling might be explored as targets for treatments that fulfill these requirements. Our findings revealed that when ROR1 and ROR2 knockdown cells were treated with PF-543, an inhibitor of SPHK1, cell growth was reduced, and when the knockdown cells were treated with FTY720 (fingolimod), a first-in-class S1P receptor modulator, cell growth was also reduced. Validation of our lung cancer result might provide an additional therapeutic feature. RORs and S1P signaling have recently been shown to be expressed in human tumors as part of a huge effort in target identification. Safingol, a SPHK1 inhibitor along with cisplatin, has been tested in a Phase I clinical trial for treating advanced-stage solid tumors [18]. Similarly, a ROR1 monoclonal antibody has shown promise in a variety of cancers, including lung cancer [19]; however, its efficacy in solid tumors has yet to be determined. Combinatorial targeting of both pathways might lead to better efficacy for tumor cell killing and might increase the survival outcome in lung cancer patients.

Author Contribution Statement

AK: Conception, study design, interpretation of data, and critical reading and intellectual assessment of manuscript. RN: Study design, analysis and interpretation of data, preparation of the manuscript. RP: Analysis and interpretation of data, preparation of the manuscript.

Acknowledgements

References