Targeting Opsin4/Melanopsin with a novel small molecule ... · The identification of oncogenic biomolecules as drug targets is an unmet need for the development of clinically effective

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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).

on July 8, 2020. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 8, 2020; DOI: 10.1158/1541-7786.MCR-19-1120

mailto:[email protected]

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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.



https://string-db.org/


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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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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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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,




28

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




29

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).




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.






















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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