targeting opsin4/melanopsin with a novel small molecule ... · the identification of oncogenic...
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Targeting Opsin4/Melanopsin with a novel small molecule suppresses
PKC/RAF/MEK/ERK signaling and inhibits lung adenocarcinoma progression
Qiushi Wang1*
, Tianshun Zhang1*
, Xiaoyu Chang1, Keke Wang
1, 2, Mee-Hyun Lee
2, Wei-Ya
Ma1, Kangdong Liu
2, Zigang Dong
1, 3+
1The Hormel Institute, University of Minnesota, 801 16th Ave NE, Austin, MN 55912
2The
China-US (Henan) Hormel Cancer Institute, No.127 Dongming Road, Zhengzhou, Henan,
China, 450000
3Department of Pathophysiology, School of Basic Medical Sciences. College of Medicine.
Zhengzhou University, Henan, 450001, China
*Qiushi Wang and Tianshun Zhang contributed equally to this work
Correspondence Author:
+ Address correspondence to Zigang Dong, No.100 Science Avenue, Zhengzhou City, Henan
Province, China. Postcode: 450001. Telephone: +86-371-66658803; Email: [email protected]
Running title: AE 51310 suppresses oncogenic signaling in lung cancer
Financial Support: This work was supported by the Hormel Foundation (Z. Dong).
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Manuscript Information:
Word count: Abstract = 201; Main text =3654 (Introduction, Materials and Methods, Results,
Discussion)
6 Figures, 5 Supplementary figures with associated legends.
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Abstract
The identification of oncogenic biomolecules as drug targets is an unmet need for the
development of clinically effective novel anticancer therapies. In the present study, we report for
the first time that opsin 4/melanopsin (OPN4) plays a critical role in the pathogenesis of
non-small cell lung cancer and is a potential drug target. Our study has revealed that OPN4 is
overexpressed in human lung cancer tissues and cells, and is inversely correlated with patient
survival probability. Knocking down expression of OPN4 suppressed cells growth and induced
apoptosis in lung cancer cells. We have also found that OPN4, a G protein couple receptor,
interacted with Gα11 and triggered the PKC/BRAF/MEK/ERKs signaling pathway in lung
adenocarcinoma cells. Genetic ablation of OPN4 attenuated the multiplicity and the volume of
urethane-induced lung tumors in mice. Importantly, our study provides the first report of AE
51310 (1-[(2,5-dichloro-4-methoxyphenyl)sulfonyl] -3-methylpiperidine) as a small molecule
inhibitor of OPN4, suppressed the anchorage-independent growth of lung cancer cells and the
growth of patient-derived xenograft (PDX) tumors in mice. Implications: Overall, this study
unveils the role of OPN4 in NSCLC and suggests that targeting OPN4 with small molecules,
such as AE 51310 would be interesting to develop novel anticancer therapies for lung
adenocarcinoma.
Keywords: OPN4; lung adenocarcinoma; patient-derived xenograft; Gα11; urethane-induced
lung cancer
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Introduction
Lung cancer is the leading cause of cancer-related death worldwide. In 2019, approximately
228,150 new cases and 142,670 deaths from lung and bronchial cancer are estimated in the
United State (1). Non-small cell lung cancer (NSCLC) is the most common malignancy,
occurring in up to 85% of all lung cancers, and is considered to be an insidious disease and has a
poor prognosis (2,3). The aggressiveness of NSCLC and its resistance to common therapies still
intractable issues. Therefore, the elucidation of the pathophysiological mechanisms to identify
biomolecules as drug targets and developing novel therapeutic agents are urgently needed and
clinically very important.
Opsin 4/Melanopsin (OPN4), a G protein coupled receptor (GPCR) commonly present in a
small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs), regulates circadian
rhythms, pupil functions, melatonin expression, cognition and sleep (4-7). GPCRs, which
transduce extracellular signals to the intracellular effector pathways through activation of
heterotrimeric G proteins, include approximately 900 members (8,9). Most GPCRs are
overexpressed in primary and metastatic tumor cells of head and neck, NSCLC, breast, prostate
and gastric tumor and melanoma (10-15). Many of this family of cell membrane receptors are
involved in aberrant intracellular signal transmission often associated with tumor growth and
metastasis. Therefore, targeting the GPCRs contributing to oncogenic signaling may be a rational
approach to develop novel anticancer therapies for NSCLC (16).
GPCRs transmit signals from extracellular into intracellular by interacting with different
signaling protein, termed G proteins (Gγ, families Gi, Gs, Gq/11, G12/13) or arrestin (17).
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Theses G proteins are linked to multiple signaling pathways: Gq/11 plays a role in activating the
phospholipase C (PLC) family; Gs stimulates the adenylylcyclase pathway; while Gi/o
suppresses adenylylcyclase pathway; and G12/13 makes activation of small GTPase (18). Gq,
G11, G14 and G15/16 share similar structure, and the activated subunit each protein complex
can lead to PLCactivation(17-19). Furthermore, these subunits regulate both overlapping and
different signaling pathways, subsequently activating inositol lipid (e.g. calcium/protein kinase C
(PKC)) signaling PLC isoforms (20,21).
While an earlier study has demonstrated that OPN4 is highly expressed in tumors originated
from the pineal region especially in pineocytomas (22), there have no further study to investigate
the role of OPN4 in tumorigenesis. Here we provide the proof of principle to suggest that
elevated expression of OPN4 plays a contributing role in the development of lung cancer. We
studied the function of OPN4 in lung cancer development and demonstrated the effect of OPN4
in lung adenocarcinoma proliferation and apoptosis. This study aimed to ascertain the
mechanisms of OPN4 in lung carcinogenesis. The opsin 4 knockout (OPN4 KO) mice were used
to identify an oncogenic role of OPN4 in a urethane-induced lung cancer model. We also
introduce AE 51310 as a small molecule inhibitor of OPN4, and demonstrate the antitumor
potential of this OPN4 antagonist in patient-derived xenograft (PDX) tumors in mice. Our
finding suggests that OPN4 could be a potential target in lung cancer development. Targeting
OPN4 might be a potential approach against lung tumorigenesis.
Materials and Methods
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Reagents and antibodies
Cell culture media, gentamicin, penicillin, and L-glutamine were all obtained from
Invitrogen (Grand Island, NY), nonessential amino acids (Corning, NY), insulin (Gibco
Gaithersburg, MD). Fetal bovine serum (FBS) was from Gemini Bio-Products (West Sacramento,
CA). Tris, NaCl, Sodium bicarbonate, hydrocortisone, glucose, transferrin, epidermal growth
factor (EGF) and SDS for molecular biology and buffer preparation were purchased from
Sigma-Aldrich (St. Louis, MO). OPN4 antagonist AE 51310
(1-[(2,5-dichloro-4-methoxyphenyl)sulfonyl]-3-methylpiperidine) (Catalogue Number 1115306)
(23) were purchased from Otava chemical (Vaughan, Ontatio, Canada). Antibodies to detect
BRAF (sc-166), melanopsin (sc-32879), PLC4 (sc-166131), Orexin R-1/2 (sc-166111), GRK2
(sc-13143), PKC (sc-208), Bcl-2 (sc-7382), Gq (sc-136181) and G11 (sc-390382), -actin
(sc-47778) and GAPDH (sc-25778) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
p-PKC (#9371S), p-BRAF (#2696), p-MEK (#9121), MEK (#9122), p-ERKs (#9101), ERKs
(#9102), caspase-3 (#9662), c-caspase-3 (#9661), PARP (#9542), c-PARP (#9541), Bax (#2772)
and PCNA (#D3H8P) antibodies were purchased from Cell Signaling Technology (Danvers,
MA). Rabbit true blot ultra: anti-mouse Ig HRP (18-8816-31) and Mouse true blot ultra:
anti-mouse Ig HRP (18-8817-31) antibody was purchase from Rockland (Rockland, ME).
Cell culture and transfection
The lung cells were obtained from American Type Culture Collection (ATCC). Cells were
cultured at 37°C in a 5% CO2 humidified incubator according to the ATCC protocols. The cells
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were routinely screened to confirm mycoplasma negative status and to verify the identity of the
cells by Short Tandem Repeat (STR) profiling before being frozen. Enough frozen vials of each
cell line were available to ensure that all cell-based experiments were conducted on cells tested
and in culture for 8 weeks or less. NL-20 (immortalized bronchial epithelial) cells were cultured
in Ham F12 medium containing 4% fetal bovine serum (FBS), 15 000 U penicillin, 15 000 U
streptomycin, 2 mmol/L l-glutamine, 0.1 mmol/L nonessential amino acids, 10 ng/mL human
recombinant epidermal growth factor (EGF), 0.005 mg/mL insulin, 500 ng/mL hydrocortisone,
and 0.001 mg/mL transferrin. A549 human lung cancer cells were grown with F-12K medium
with 10% FBS and 1% antibiotics. All other human lung cancer cells were cultured in
RPMI-1640 medium with supplement of 10% FBS and 1% antibiotics.
Lentiviral Infection
Lentivirus plasmids shOPN4 (#1, TRCN0000009255; #2 TRCN0000009256; #3
TRCN0000009257) were purchased from University of Minnesota Genomics Center (University
of Minnesota, MN). pLKO.1-puro Non-Target shRNA Control Plasmid DNA (shNT) was
purchased from Sigma-Aldrich Co. LLC (St. Louis, MO). Another non-target control plasmid
(shLuc, 19125) was purchased from Addgene (Cambridge, MA). As previously described (24),
to generate knockdown OPN4 cells, the lentiviral expression vector of OPN4 (shOPN4) or
pLKO.1-puro Non-Target shRNA Control Plasmid DNA (shNT) and packaging vectors
(pMD2.0G and psPAX) were transfected into HEK293T cells using iMfectin Poly DNA
transfection reagent (GenDEPOT, Barker, TX) following the manufacturer's suggested protocols.
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MTS assay
H441 and A549 cells (1 103 cells/well) expressing Control (shNT or shLuc) and shOPN4
were seeded into 96-well plates. NL-20 Cells (1 104 cells/well) were seeded into 96-well plates
for evaluating cytotoxicity. After an overnight incubation, the different concentrations of OPN4
antagonist were used to treat cells. After incubation for 24, 48 or 72 h, 20 L of the CellTiter 96
Aqueous One Solution (Promega, Madison, WI) were added to each well and cells were then
incubated for an additional 1 h at 37°C, 5% CO2. Absorbance was measured at 492 nm using the
Thermo Multiskan plate-reader (Thermo Fisher Scientific, Waltham, MA).
Anchorage-independent Cell Growth Assay
Control (shNT or shLuc) and shOPN4 Cells (8 103/well) were suspended in 1mL BME,
0.3% Basal Medium Eagle agar with 10% FBS and plated on 3mL of solidified BME containing
10% FBS and 0.5% agar. The different concentrations of OPN4 antagonist with cells (8 × 103)
were mixed in 1 ml BME/10% FBS/0.33% agar. The mixture was plated on 3 ml of solidified
BME/10% FBS/0.5% agar with the same concentration of OPN4 antagonist in each well of
6-well plates. After 14 days, Colonies were scored under a microscope using the Image-Pro
PLUS (v6.) computer software program (Media Cybernetics. Rockville, MD).
Flow cytometry for analysis of apoptosis
As previously described (25), briefly Control (shNT or shLuc) and shOPN4 cells (2
105/well) were seeded into 60-mm dishes and cultured for 48 h. Cells were trypsinized and
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washed twice with cold PBS and then resuspended with phosphate-buffered saline and incubated
for 5 min at room temperature (RT) with annexin V-FITC plus propidium iodide. Cells were
analyzed using a FACSCalibur flow cytometer (BD Bioscienes, San Jose, CA).
Immunoprecipitation and Western blotting analysis
Nonidet P-40 lysis buffer (50 mmol/L Tris-HCl, pH8.0, 150 mmol/L NaCl, 0.5% Nonidet
P-40 and protease inhibitor mixture) was used to extract protein. For immunoblotting, 30 g
proteins were used to detect with specific antibodies. Proteins were visualized by
chemiluminescence (Amersham Biosciences). For immunoprecipitation (IP) assay was
performed as described previously (26). The extractions were precleared with 10L protein G
agarose beads (GenDEPO; Barker, TX) by rocking for 30 min at 4°C. The precleared supernatant
fractions were combined with fresh protein A/G agarose beads (Santa Cruz) and appropriate 2g
antibodies by rocking for overnight at 4°C. The immunoprecipitates were washed four times with
the above lysis buffer. Immunoprecipitates were suspended in SDS sample buffer and subjected
to SDS-PAGE and Western blotting. For IP under denaturing conditions, protein was extracted
using regular IP lysis buffer plus 1% SDS and heated at 95°C for 5min. Samples were diluted ten
times by using regular IP lysis buffer before IP. The beads were washed, mixed with SDS sample
buffer, boiled then resolved by SDS-PAGE. Signals were visualized by immunoblotting, which
was previously described (27).
Animals and carcinogen treatment
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All animal studies were performed and approved by the University of Minnesota
Institutional Animal Care and Use Committee (Protocol ID: 1709-35106A). OPN4 deletion mice
(021153) were purchased from the Jackson Laboratory and purified to the background to
C57BL/6. The OPN4 +/+ (WT) and OPN4 -/- (OPN4 KO) mice were subject to a
urethane-induced lung cancer mouse model, which was described in the previous study (24).
Briefly, the mice were housed and bred under virus- and antigen- free conditions. Mice were
genotyped by standard PCR analysis according to the Jackson Laboratory genotyping protocol
with 5’- AGGCTGGATGGATGAGAG C-3’, 5’-GTTGTGAAGCTGGGATCCTG-3’, and
5’-GGTCTTCCAGGTTGGATGTG-3’ as the primers. Mice (6 weeks old) were divided into four
groups: (1) WT- vehicle–treated; (2) OPN4 KO-vehicle–treated (for vehicle treatment, 5 males
and 5 females each group); (3) WT-urethane–treated; (4) OPN4 KO- urethane–treated (for
urethane treatment, 12 males and 12 females each group).
Patient-derived xenograft (PDX) mouse model
The lung tumor LG17 (Adenocarcinoma, Grade 2; Stage II) and LG55 (Adenocarcinoma,
Grade 2; Stage I) was obtained from First Affiliated Hospital of Zhengzhou University. All
patients neither received chemotherapy nor radiotherapy before the surgery. The lung tumor
tissue fragments (2-3 mm) were implanted into severe compromised immune deficient (SCID)
mice. This study followed a protocol that was approved by the Zhengzhou University
Institutional Animal Care and Use Committee (Zhengzhou, Henan, China). After tumor
implantation, when the tumors reached around 100 mm3, mice were randomly divided into 3
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groups (n = 7 mice per group). The groups were: 1) vehicle (PBS with 10% PGE400 and 1%
DMSO) control; 2) 10 mg/kg OPN4 antagonist; and 3) 50 mg/kg OPN4 antagonist. Mice were
administered drug or vehicle by oral gavage daily. Body weight and tumor volume were
measured once a week and tumor volume was calculated based on the formula: length × width ×
width ×0.52. At the end of the experiment, mice were euthanized prior to removal of tumors for
further analysis.
Immunohistochemical analysis of tissue array and mice lung tissues
A human lung tissue array (BC041115a and LC483) was purchased from US Biomax Inc
cancer tissue bank collection (US Biomax Inc, MD). A Vectastain Elite ABC Kit obtained from
Vector Laboratories was used for immunohistochemical staining according to the protocol
recommended by the manufacturer. Mice lung tissues were embedded in paraffin for examination.
Sections were stained with hematoxylin and eosin (H&E) and analyzed by
immunohistochemistry, which was described in previous study (24).
Statistical analysis
All quantitative data are expressed as mean values standard deviation (S.D.) or standard
error (S.E.) of at least three independent experiments or samples. Significant differences were
determined by a Student’s t test or one-way ANOVA. A probability value of p<0.05 was used as
the criterion for statistical significance.
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Results
OPN4 is overexpressed in human lung adenocarcinoma and associated with
adenocarcinoma lung cancer patient survival probability
Lung adenocarcinoma is a common primary lung cancer. Initially, we evaluated protein
expression level of OPN4 in human lung cancer tissue arrays and lung cancer cell lines. Results
showed OPN4 is overexpressed in human lung adenocarcinoma as compared to the normal tissue
(Fig. 1A; Supplementary Fig. S1). The cell lines results confirmed that the elevated expression
of OPN4 in NSCLC (Fig. 1B). We analyzed the association between elevated OPN4 expression
and patient survival probability using Kaplan-Meier plotter (Fig. 1C). Results clearly showed
that the survival probability of patients with high OPN4 expression is significantly lower than
patients with low OPN4 expression (p = 0.0014).
Knockdown of OPN4 inhibits proliferation of lung adenocarcinoma cells
OPN4 knockdown H441 and A549 lung cancer cells was generated with three different
shOPN4 sequences (Fig. 2A; Supplementary Fig. S2A). The MTS and anchorage-independent
cell growth assays showed that knockdown of OPN4 attenuates the absorbance reading at 492
nm, indicating the attenuation of cell proliferation (Fig. 2B; Supplementary Fig. S2B).
Similarly, colony number was reduced in OPN4 knockdown cells (Fig. 2C; Supplementary Fig.
S2C), which indicated that blocking OPN4 expression decreased anchorage-independent cell
growth ability.
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Knocking down of OPN4 enhances apoptosis in lung adenocarcinoma cells
The antiproliferative effects observed upon knockdown of OPN4 may results from the
induction of tumor cell death. We, therefore, examined the alterations in cellular apoptotic
signaling after silencing of OPN4 in lung adenocarcinoma cells. The results demonstrated that
knockdown of OPN4 induced apoptosis in both H441 and A549 lung cancer cells (Fig. 3A and B;
Supplementary Fig. S2D and E). Then, we detected a special class of proteases that are
associated with apoptosis. The OPN4 partial silenced H441 and A549 lung cancer cells showed
higher levels of proapoptotic proteins including c-caspase-3, c-PARP, and Bax, while decreased
total form of caspase-3 and PARP, and reduced levels of antiapoptotic protein Bcl-2 (Fig. 3C).
Overall, silencing of OPN4 triggered apoptosis, suggesting that OPN4 participates in
intracellular signaling pathways modulating cell apoptosis.
OPN4 interacted with Gα11 and triggered the PKC/BRAF/MEK/ERKs signaling pathway
in lung cancer cells
To investigate intracellular signaling partner of OPN4, we used STRING: functional protein
association networks (https://string-db.org/) program and found top 5 potential interaction
protein candidates including PLC4, GRK2, OrexinR-1/2, Gq and G11 (Fig. 4A). Then,
verified the interaction between OPN4 and the candidates with conducting immunoprecipitation
in H441 cells. The result demonstrated that G11 is the protein-binding partner with OPN4 in
lung cancer cells (Fig. 4B and C). Intriguingly, knocking down of OPN4 expression markedly
decreased the phosphorylation level of PKC, followed by inhibition of BRAF/MEK/ERKs.
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Concurrently, knocking down of G11 expression also suppressed the phosphorylation level of
PKC and the downstream signaling pathway (Fig. 4D, E). Overall, OPN4 binding with G11
regulates PKC activation, which activates BRAF, MEK and ERKs mediation of lung cancer cells
growth, apoptosis and lung tumorigenesis (Fig. 4F).
Loss of OPN4 decelerates tumor invasion in urethane-induced lung carcinogenesis.
Urethane-induced lung cancer has been well- characterized and accepted as a model for
human lung adenocarcinoma. In lung cancer research, the urethane model of lung cancer has
been widely used (28,29). In thisstudy, the WT and OPN4 KO mice received 1g/kg urethane
once a week for 10 consecutive weekly by i.p. injections, while the control group mice were
given vehicle (1 PBS, i.p.), after then tumors were counted at 30 weeks. The results indicated
that the OPN4 KO mice exhibited significantly decreased number of lung tumors as compared to
WT mice (Fig. 5A; Supplementary Fig. S3A). Tumor multiplicity averaged 4.0 1.7 tumors in
the OPN4 KO mice, but 10.7 4.0 in WT mice (***, p < 0.001) (Fig. 5B). Consequently the
expression levels of p-BRAF, p-MEK and p-ERKs were substantially reduced in the tumor
tissues from the OPN4 KO mice compared with WT mice (Fig. 5C). Moreover, the result from
H&E staining showed that the tumors from OPN4 KO mice displayed only a few adenomas
compared with WT mice. Importantly, the lungs from OPN4 KO group retained a majority of the
normal alveolar architecture (Fig. 5D). The expression of proliferating cell nuclear antigen
(PCNA), a marker of cell proliferation (30), was markedly reduced in tumor tissues from OPN4
KO mice as compared to that of WT mice (Fig. 5D). Although there was no significant
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difference in body weight among different groups of mice (Supplementary Fig. S3B), The
survival rate of the OPN4 KO- urethane-treated group was significantly higher as compared to
the WT group (Supplementary Fig. S3C). The genotyping of all mice was determined by PCR
analysis in lung tissue (Supplementary Fig. S3D). Collectively, these results suggested that
deficient of OPN4 signaling attenuated tumor growth in urethane-induced lung carcinogenesis.
OPN4 antagonist AE 51310 suppressed the cancer cells growth and tumor growth in PDX
mouse model.
We next examined the effects of an OPN4 antagonist AE 51310 on lung cancer cells growth.
Initially, our data showed that OPN4 antagonist had no cytotoxicity till to 100 M in NL-20
normal cells (Supplementary Fig. S4). The anchorage-independent growth assay showed that
colony formation of H441 and A549 cells was attenuated after treatment with different
concentrations of AE 51310 (Fig. 6A, B). In addition, treatment with OPN4 antagonist AE
51310 markedly decreased the activation of PKC and the downstream BRAF/MEK/ERKs
signaling pathway (Fig. 6C). AE 51310 on the growth of lung cancer cells in vitro led us to
examine the effects of AE 51310 on PDX tumor growth in mice. Based on the expression of
OPN4 in the PDX samples (Supplementary Fig.5A), LG17 and LG55 were selected for the
further study. Our results showed that OPN4 antagonist AE 51310 at 10 or 50 mg/kg body
weight decreased the growth of both LG17 and LG55 PDX tumors without affecting mouse body
weight. Treatment with AE51310 at 50 mg/kg body weight markedly decreased the tumor
growth as compared with vehicle treatment group (Fig. 6D-G; Supplementary Fig. 5B, C, D).
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Treatment with AE 51310 also significantly decreased tumor weight (Fig. 6F; Supplementary
Fig. 5E) and the PCNA expression as compared to vehicle-treated group (Fig. 6H;
Supplementary Fig. 5F).
Discussion
Molecular target-based anticancer therapies, such as growth factor receptor antagonists,
kinase inhibitors and immune checkpoint inhibitors have gained clinical success in many solid
tumors. However, lung cancer still remains as a major cause of cancer-related mortality
worldwide (31). Because of the involvement of diverse oncogenic signaling pathways in
carcinogenesis, identification of new oncogenic biomolecules and their validation as novel drug
targets offer the opening of additional therapeutic avenue in cancer treatment.
GPCRs are a broad and diverse family of signaling receptors that play a role in the growth
and development of cancer trough mediating cell proliferation, invasion, migration, immune
cell-mediated function, angiogenesis and metastasis (32-35). GPCRs are cell surface receptors
that contain highly druggable binding sites and the largest class of drug targets, and currently
more than 30% of FDA-approved GPCR-targeted drugs (36,37). OPN4 belongs to the GPCR
family and is largely involved in the regulation of circadian rhythm (6). Considering the role of
many GPCRs in tumorigenesis process (10-13) and the initial report of the elevated expression of
OPN4 in tumors originated around pineal gland (22) led us to investigate the role of OPN4 in
lung tumorigenesis. The finding that OPN4 is overexpressed in human lung cancer cell lines and
tissues, and is inversely proportional to the survival of lung adenocarcinoma patients (Fig. 1A, B;
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supplementary Fig. 1) suggests that OPN4 contributes to lung cancer. This has been supported
by the inhibition of lung cancer cells proliferation and the activation apoptotic markers, such as
cleaved caspase 3, PARP and Bax along with the inhibition of Bcl-2 upon knocking down of
OPN4 (Fig. 2, 3). Importantly, the attenuation of anchorage-independent growth of
OPN4-deficient lung cancer cells (Fig. 2) and the reduced burden of urethane-induced lung
tumors in OPN4-deficient mice (Fig. 5A and B; supplementary Fig. 2A) provide strong
evidence to the contributing role of OPN4 in lung tumorigenesis. These results also indicated that
OPN4 is a potential target for developing therapies for lung cancer.
OPN4 protein is an opsin subgroup of GPCRs (38) linked to a chromophore containing
11-cis-retinal (specific form of vitamin A) and is highly sensitive to blue light (39-41), which
activates PKC (42,43) and plays critical roles in intracellular oncogenic signaling pathways
(44,45). In addition to its familiar photoreceptor function, all-trans-retinal can also combines
with opsin independent of light, forming activating species of the receptor (46,47). It is well
known that GPCRs undergo conformational changes and interact with G-proteins, which can
modulate downstream signaling pathways. In addition, activated GPCRs can regulate cell
function via β-arrestins, scaffolding proteins for a variety of signaling entities (17-19,48).
Interestingly, the present study found that OPN4 binds with Gα11 in lung cancer cell to modulate
activation of PKC, resulting in inhibition of BRAF/MEK/ERKs downstream signaling (Fig. 4).
Evidence indicates that Gαq and Gα11 stimulate downstream effector pathways
PKC/BRAF/MEK including ERKs activation (49), which can result in increased cell
proliferation, differentiation, or survival (50). Our results indicate that activation of
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BRAF/MEK/ERKs could be the key regulatory element for the function of OPN4 in lung cancer.
Based on the experimental findings, we suggest that OPN4 is a potential target for designing
novel therapy for lung cancer. Designing of small molecules as inhibitors of OPN4 resulted in
identifying AE 51310 (1-[(2,5-dichloro-4-methoxyphenyl)sulfonyl]-3-methylpiperidine) as an
OPN4 antagonist (23). We further examined the effect of AE51310 on lung carcinogenesis.
Results showed strong inhibition of lung cancer cells proliferation and the growth of lung cancer
PDX tumors in mice (Fig. 6; supplementary Fig. 3). Overall, OPN4 inhibitor might be a
potential new drug for lung cancer treatment. Although we have not noticed any remarkable sign
of abnormal phenomenon from the PDX mouse model, additional toxicity studies for AE 51310
or its derivatives are warranted for clinical development of OPN4 as a drug therapy of lung
cancer. Moreover, pharmacokinetics of OPN4 inhibitor should be assessed before clinical
application. In the present study, we report for the first time that OPN4 plays a critical role in the
pathogenesis of lung cancer and is a potential drug target. However, whether the OPN4 has
selective effect to lung cancer or has the role in other cancer type are still unknown. Further
experiments need to be conducted to clarify its function.
Overall, the current study suggested that OPN4 positively mediates RAF/MEK/ERKs
pathway by activating PKC, thereby contributing to lung tumorigenesis. It is noteworthy that we
found that OPN4 binds with G11 and mediates BRAF activation, triggering cellular responses,
involving growth, differentiation, and death. Thus, drug discovery approach by targeting OPN4
may lead to development of novel therapy for lung cancer.
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19
Acknowledgments
The authors thank Tara Adams for supporting animal experiments (The Hormel Institute,
University of Minnesota).
Conflicts of Interest: The authors declare no potential conflicts of interest
Author contribution
Q.W. and T.Z. contributed equally to the manuscript and designed the experiments, and
performed experiments, analyzed and interpreted data, prepared figures and wrote the manuscript.
X.C. and K.W. performed part of animal study and data analysis.
W.M. assisted in establishing experimental methods. M.H.L. and K.L. K.W. assisted with the
PDX mouse study. Z.D. contributed to study supervision, experimental design, data discussion,
and revision of the manuscript.
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20
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Figure Legends
Figure 1. OPN4 expression is upregulated in human lung cancer and is associated with lung
cancer patient survival probability. A, Immunohistochemical analysis of OPN4 protein
expression in normal and lung cancer tissues. OPN4 protein detection was accomplished using
DAB (brown) staining, and nuclei were counterstained with hematoxylin (blue). Density scores
were obtained from each sample, and statistical significance was determined by one-way
ANOVA. Tissues include normal (n=10), adenocarcinomas (n=44) and squamous cell carcinoma
(n=40); scale bar, 200 m. B, Expression of OPN4 in human normal and lung cancer cell lines. C,
Kaplan–Meier survival curves relative to OPN4 expression were generated for lung cancer
(Kaplan-Meier plotter, http://kmplot.com/ analysis). The desired Affymetrix IDs are validated
234226_at OPN4. Asterisk significant difference between normal and adenocarcinoma (***, p <
0.001).
Figure 2. Knockdown of OPN4 inhibits H441 and A549 lung cancer cell growth. A, H441and
A549 lung cancer cells with stable knockdown of OPN4 were established. The expression of
OPN4 was determined by Western blotting. The band density was measured using the Image J
(NIH) software program. The band density of OPN4/β-actin in H441 with shNT, shOPN4-1,
shOPN4-2 and shOPN4-3 was 1, 0.39, 0.50 and 0.24, respectively. And the band density of
OPN4/β-actin in A549 with shNT, shOPN4-1, shOPN4-2 and shOPN4-3 was 1, 0.54, 0.36 and
0.58, respectively. B, Knockdown of OPN4 decreases proliferation of H441and A549 lung
cancer cells. Cell growth was determined at 24, 48, and 72 h using the MTS assay. C,
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Knockdown of OPN4 reduces anchorage-independent growth of H441and A549 lung cancer
cells. H441and A549 cells stably expressing shNT or shOPN4 were incubated in 1.25% agar.
Colonies were counted using a microscope and the Image-Pro Plus (v.6) computer software
program. Data are presented as mean values ± SD from triplicate experiments. Statistical
differences were evaluated using the Student t test. The asterisks indicate a significant difference
between OPN4 knockdown and control cells (**, p < 0.01; ***, p < 0.001).
Figure 3. Knockdown of OPN4 induces apoptosis of H441 and A549 lung cancer cells. A
and B, Knockdown of OPN4 in H441 and A549 cell and then stained with annexin V. Apoptosis
was determined by flow cytometry. Data are quantified (right plots) and the asterisks (***)
indicate a significant increase of apoptosis in OPN4 knockdown cells (*, p < 0.05, **, p < 0.01;
***, p < 0.001). C, Cells with OPN4 knockdown exhibit increased expression of proapoptotic
proteins and decreased expression of antiapoptotic proteins in Western blotting anaylsis.
Figure 4. Knockdown of OPN4 inhibit PKC/BRAF/MEK/ERKs signaling pathway through
binding with G11. A, STRING network showed top 5 potential interaction protein candidates
[PLCB4 (PLCβ4), ADRBK1 (GRK2), HCRT (OrexinR-1/2), GNAQ (Gαq) and GNA11 (Gα11)]
with OPN4. B, H441 cell lysates were immunoprecipitated with anti-OPN4 or control IgG. The
immunoprecipitated complex was detected by Western blotting with anti PLCβ, anti-GRK2,
anti-OrexinR-1/2, anti-Gq, anti-G11 and anti- OPN4. C, H441 cell lysates were
immunoprecipitated with anti- G11 or control IgG. The immunoprecipitated complex was
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detected by Western blotting with anti-G11 and anti- OPN4. D, Knockdown of OPN4 inhibit
PKC/BRAF/MEK/ERKs pathway signaling. E, Knockdown of G11 inhibit
PKC/BRAF/MEK/ERKs pathway signaling as well. F, Opsin 4 binding with G11 decreases
activation of PKC, and BRAF, which activates MEK and ERKs' effects on lung cancer cell
growth, apoptosis, and NSCLC tumorigenesis.
Figure 5. Loss OPN4 inhibits urethane-induced lung carcinogenesis. A, WT and OPN4 KO
mice were used. Urethane (1 g/kg in 1 PBS) or vehicle only was i.p. administered weekly for 10
weeks. Lungs were collected at 30 weeks after first urethane treatment. B, Tumor multiplicity
averaged 4.0 1.7 tumors in the OPN4 KO group treated with urethane and 10.7 4.0 in the
urethane-treated WT group (***, p < 0.001), with no significant difference between male or
female mice. C, Protein levels of p-BRAF, BRAF, p-MEK, MEK, ERK, p-ERKs and GAPDH
were substantially decreased in the tumor tissues of the OPN4 KO mice compared with the WT
mice. The tissue lysates were prepared from pooled lung tumor nodules or normal lung tissue
from each mouse of each group. Three sets were prepared for each group, and each lane shows
one set of pooled samples subjected to Western blotting. D, Lung samples were harvested and
stained with H&E. Immunohistochemistry analysis was used to determine the levels of PCNA in
lungs from urethane-treated mice compared with those treated with vehicle. scale bar, 100 μm.
Density scores were obtained from each sample, and statistical significance was determined by
one-way ANOVA (***, p < 0.001).
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30
Figure 6. OPN4 antagonist AE 51310 inhibits lung cancer cells growth and tumor growth in
a PDX mouse model. A, B, H441 and A549 lung cancer cells were treated with different
concentration of OPN4 antagonist. C, Protein levels of OPN4, p-PKC, PKC, p-BRAF, BRAF,
p-MEK, MEK, ERKs, p-ERKs, GAPDH and -actin were substantially decreased in a dose
dependent manner. PDX mice model, the groups were: 1) vehicle (5% PGE400 + 5% Tween80
solution + 2.5% DMSO) control; 2) 10 mg/kg OPN4 antagonist; and 3) 50 mg/kg OPN4
antagonist. (LG 17 case n = 7 mice per group;). D, The picture of tumor (The scale bar, 1 cm). E,
F and G, tumor volume, tumor weight and Body weight were measured. H,
Immunohistochemistry analysis was used to determine the levels of PCNA in tumors treated with
OPN4 antagonist compared with vehicle-treated. The integrated optical density (IOD) was
evaluated using the Image-Pro Premier software offline (v.6) program. The asterisks (***)
indicate a significant (p < 0.001) decrease in compound-treated compared to vehicle-treated
samples.
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Published OnlineFirst April 8, 2020.Mol Cancer Res Qiushi Wang, Tianshun Zhang, Xiaoyu Chang, et al. adenocarcinoma progressionsuppresses PKC/RAF/MEK/ERK signaling and inhibits lung Targeting Opsin4/Melanopsin with a novel small molecule
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