
==== Front
Neoplasia
Neoplasia
Neoplasia (New York, N.Y.)
1522-8002
1476-5586
Neoplasia Press

S1476-5586(24)00086-1
10.1016/j.neo.2024.101044
101044
Original Research
Non-canonical olfactory pathway activation induces cell fusion of cervical cancer cells
Araki Keigo k-araki@den.ohu-u.ac.jp
a⁎
Torii Takeru b
Takeuchi Kohei b
Kinoshita Natsuki b
Urano Ryoto b
Nakajima Rinka c
Zhou Yaxuan c
Kobayashi Tokuo a
Hanyu Tadayoshi d
Ohtani Kiyoshi c
Ambe Kimiharu a
Kawauchi Keiko b
a Department of Morphological Biology, School of Dentistry, Ohu University, Koriyama, Fukushima 963-8611, Japan
b Frontiers of Innovative Research in Science and Technology, Konan University, Kobe, Hyogo 650-0047, Japan
c Department of Biomedical Sciences, School of Biological and Environmental Sciences, Kwansei Gakuin University, Sanda, Hyogo 669-1330, Japan
d Department of Gynecology, Tsuboi Cancer Center Hospital, Koriyama, Fukushima 963-0197, Japan
⁎ Corresponding author. k-araki@den.ohu-u.ac.jp
01 9 2024
11 2024
01 9 2024
57 10104425 7 2024
26 8 2024
26 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• The olfactory receptor OR1N2 gene is mutated in multinucleated cervical cancer cells.

• Mutation in OR1N2 activates the non-canonical olfactory pathway mediated by PKA.

• Furin protease is phosphorylated and activated by PKA.

• Furin inhibitor treatment prevents multinucleation.

Multinucleation occurs in various types of advanced cancers and contributes to their malignant characteristics, including anticancer drug resistance. Therefore, inhibiting multinucleation can improve cancer prognosis; however, the molecular mechanisms underlying multinucleation remain elusive. Here, we introduced a genetic mutation in cervical cancer cells to induce cell fusion-mediated multinucleation. The olfactory receptor OR1N2 was heterozygously mutated in these fused cells; the same OR1N2 mutation was detected in multinucleated cells from clinical cervical cancer specimens. The mutation-induced structural change in the OR1N2 protein activated protein kinase A (PKA), which, in turn, mediated the non-canonical olfactory pathway. PKA phosphorylated and activated furin protease, resulting in the cleavage of the fusogenic protein syncytin-1. Because this cleaved form of syncytin-1, processed by furin, participates in cell fusion, furin inhibitors could suppress multinucleation and reduce surviving cell numbers after anticancer drug treatment. The improved anticancer drug efficacy indicates a promising therapeutic approach for advanced cervical cancers.

Graphical abstract

Image, graphical abstract

Keywords

Cervical cancer
Cell fusion
Olfactory pathway
Anticancer drug resistance
PKA
Furin
Abbreviations

PKA protein kinase A

ORs olfactory receptors

OSNs olfactory sensory neurons

cAMP cyclic adenosine monophosphate

CBEs cytosine base editors

SNVs single nucleotide variants

WES whole exome sequencing

GPCRs G protein-coupled receptors

CREB cAMP response element-binding protein

CDS coding sequence

TM transmembrane

ECL extracellular loop

ICL intracellular loop
==== Body
pmcIntroduction

Cancer is a group of diseases that exhibits a variety of undesirable cellular characteristics, most typically uncontrolled cellular proliferation [1,2]. Given that cancer cells can uncontrollably invade and metastasize to other parts of the human body, appropriate cancer management is crucial for improving treatment outcomes. Cancer is a fundamental consequence of genetic mutations [3] that can occur spontaneously or be triggered by environmental factors, including synthetic chemicals, non-ionizing radiation, and infectious agents such as oncoviruses [4,5]. Various types of genetic mutations, such as chromosomal deletions, duplications, and translocations, can be related to cancer initiation [6]; however, point mutations are the most frequent genetic mutations and alter protein functions. Cancer initiation is a complex biological process, and a combination of mutations in specific genes, such as oncogenes and tumor suppressor genes, often leads to cancer onset [3]. Genetic mutations play crucial roles in cancer progression as well as initiation [[7], [8], [9]]. Cancers are heterogeneous, with various subpopulations of cells and additional mutations driving clonal evolution, and aggressive clones can outcompete others and develop into advanced cancers. The accumulation of more mutations during cancer progression enhances malignant characteristics, such as metastasis, invasion, and angiogenesis [[7], [8], [9]]. These mutations can render cancer cells resistant to chemotherapy and targeted therapies [8]. Intracellular signaling pathways induced by these mutations contribute to the malignant characteristics of cancers [10]. Because these mutations and subsequent intracellular signaling pathways are potential therapeutic targets, identifying these cancer-related mutations and signaling pathways by comprehensive analysis of cancer genomes will help develop better treatments and preventive strategies [[8], [9], [10]].

Advances in genomics have identified genetic mutations in cell surface receptors [11,12] and elucidated the intriguing roles of certain intracellular signaling pathways. These findings are noteworthy as they may be implicated in a wide range of diseases, including cancer. Olfactory signaling is broadly divided into two distinct pathways: canonical and non-canonical [13,14]. The former is the conventional pathway that explains the perception of odors, principally functioning in the olfactory epithelium. Olfactory receptors (ORs) in the membranes of olfactory sensory neurons (OSNs) detect odorants and activate downstream factors such as olfactory neuron-specific G protein (Golf) and cyclic nucleotide-gated (CNG) cation channels, leading to elevated intracellular Ca2+ and Na+ concentrations and subsequent membrane depolarization [15]. The alternative pathway is driven by the spontaneous conformational transition between the active and inactive conformations of ORs in the absence of odorants, and this odorant-independent OR activity produces a specific amount of cyclic adenosine monophosphate (cAMP) using ubiquitously expressed stimulatory G protein (Gs), rather than Golf [13,14]. As the name suggests, the expression of ORs predominantly occurs in OSNs, although not confined to them [16,17]. These receptors are also expressed in multiple extranasal tissues, including the normal heart, lungs, skin, and even cancerous tissues. Ectopic ORs participate in the development of diseases in non-olfactory tissues, such as asthma and obesity, and are possibly involved in cancer progression [18,19]. The non-canonical olfactory pathway is considered the principal pathway from ectopic ORs because this pathway is capable of being transduced via Gs but not Golf. Thus, it may be a potential therapeutic target in certain cancerous tissues.

Changes in cellular morphology are known to occur during cancer progression. In particular, cellular multinucleation is a relevant feature of advanced cancers that arises from cell fusion or acytokinetic mitosis [[20], [21], [22]]. In multinucleated cells, nuclei of unconventional size and shape occasionally appear because of intracellular nuclear fusion and inappropriate nuclear division [22,23], which fuel cancer progression by promoting chromosomal abnormalities. Multinucleation in advanced cancers affects several malignant characteristics, including enhanced invasiveness and resistance to cell death [[21], [22], [23]]. Multinucleated cancer cells exhibit altered cytoskeletal dynamics and increased motility, facilitating more aggressive invasion of the surrounding tissues and dissemination to distant sites [[21], [22], [23]]. In addition, these cancer cells can evade apoptosis, and their survival characteristics contribute to tumor persistence [21,22]. Therefore, understanding the mechanisms underlying multinucleation may advance cancer treatment.

Chromosomal fragile sites are specific regions of the human genome that are normally stable but are sensitive and frequently mutated in human diseases, including cancers [24]. To explore genetic mutations related to cancer progression, we focused on these fragile chromosomal sites. We considered that off-target mutations induced by genome editing technology are indiscriminately distributed in vulnerable regions of the entire genome, with some off-target mutations possibly overlapping with the somatic mutations that arise and accumulate during cancer progression. In the current study, we developed multinucleated cervical cancer cell lines and identified a genetic mutation in the olfactory receptor OR1N2 gene that is common among them. This mutation activated the non-canonical olfactory pathway and induced multinucleation via cell fusion. Furthermore, multinucleated cells exhibited anticancer drug resistance, and the suppression of multinucleation improved anticancer drug sensitivity. Our data suggest a potential therapeutic target for advanced cervical cancer.

Materials and methods

Detailed descriptions of the materials, including antibodies, reagents, and recombinant plasmids, are provided in Supplementary materials and methods. Further information and requests for resources are available from the corresponding author upon reasonable request.

Cell culture

HeLa and 293T cells were obtained from American Type Culture Collection (ATCC CCL-2 and ATCC CRL-3216, respectively). The cells were cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM; Fujifilm Wako, #044-29765), supplemented with 10 % fetal bovine serum and antibiotic-antimycotic mixed solution (Nacalai Tesque, #09366-44) at 37 °C under 5 % CO2. Non-fusion (NF) and constitutive fusion (CF) clones were generated from HeLa cells co-transfected with CBE4-Gam and pSpCas9(BB)-2A-Puro (PX459) v2.0 plasmids (Addgene plasmids #100806 and #62988, respectively) [25,26]. Transfected cells were selected using puromycin (3 μg/mL; InvivoGen, #ant-pr-1) for 2 days. Single colonies of transfected cells were isolated using cloning cylinders and expanded for further experiments.

Human tissues

Formalin-fixed paraffin-embedded (FFPE) tissue blocks of human cancer specimens were obtained from the Tsuboi Hospital Group and REPROCELL; appropriate informed consent was acquired from patients under Institutional Review Board (IRB) approval in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects in Japan and the Health Insurance Portability and Accountability Act of 1996 (HIPAA), respectively. Characteristics of subjects with cervical squamous cell carcinoma are as follows: stage ⅠB2, 50-year-old Asian female; stage ⅡA, 61-year-old Asian female.

Live-cell imaging

Live-cell imaging experiments were performed using two different microscopes. For cell fusion and division analyses, cells were seeded on glass-bottom dishes, and culture dishes were moved into a stage incubator placed on a Nikon A1R HD25 confocal microscope, in which the living cells were maintained at 37 °C under 5 % CO2. Bright-field images were acquired at 10-min intervals using a dry objective (×40). To monitor cisplatin-induced cell death, the culture dishes were moved into an EVOS onstage incubator placed on an EVOS M7000 Imaging System (Invitrogen Thermo Fisher Scientific). Bright-field images were acquired at 1-h intervals using a dry objective (×20).

Hematoxylin and eosin staining

The FFPE tissue blocks were sectioned at the thickness of 4 μm and deparaffinized (Xylene for 5 min, 100 % ethanol for 5 min, 95 % ethanol for 3 min, 90 % ethanol for 3 min, 80 % ethanol for 3 min, 70 % ethanol for 3 min, 50 % ethanol for 3 min, and water wash for 2 min). Sections were stained with hematoxylin (Muto Pure Chemicals, #30022) for 15 min, washed with water for 2 min, and stained with eosin (Muto Pure Chemicals, #32002) for 30 s. Stained sections were washed with water for 2 min and dehydrated in a graded series of ethanol (50 %, 70 %, 80 %, 90 %, 95 %, and 100 %) and xylene.

Genomic DNA sequencing for OR1N2

Genomic DNA was extracted from NF and CF clones using the GenElute Mammalian Genomic DNA Miniprep Kit (Merck Sigma-Aldrich, #G1N350). For clinical cancer specimens, uterine cervical cancer (UCC) and normal tissue adjacent to the tumor (NAT) parts of FFPE sections were scratched using 23G needles, and genomic DNA was extracted using Nucleospin DNA FFPE XS (Takara, #740980.50). The genomic DNA fragments encompassing codon 100 of OR1N2 were amplified by PCR using the following primer set: 5′-CAGCTCTGACCCACACCTCC-3′ and 5′-GTGCACAGAGCTGGGGACTCATAG-3′. The PCR products were then directly sequenced using following primers: 5′-CACTTCTGCCTCCATCCCC-3′ for coding strand sequencing and 5′- GGTTGGCAGATGGCCACATAGCGGTC-3′ for template strand sequencing.

Glutathione S-transferase (GST)-tagged fusion protein purification

cDNA for the furin wild-type (WT) and S421D mutant encompassing the catalytic and P domains (amino acids 108–578) was subcloned into the pGEX-2T vector (GE Healthcare, #28-9546-53). GST-tagged furin fusion proteins were expressed in Escherichia coli strain BL21(DE3)pLysS (Promega, #L119B) by adding 200 μM isopropyl-β-D-thiogalactopyranoside (IPTG) overnight at 18 °C. The cell pellets were resuspended in PBS containing 5 mM EDTA, 1 mM dithiothreitol (DTT), and a Protease Inhibitor Cocktail (Merck Sigma-Aldrich, #P8340) and then sonicated using an ultrasonic homogenizer (VP-050N; TAITEC). The lysates were centrifuged at 15,000 rpm for 20 min at 4 °C and GST-tagged furin proteins in the supernatant were captured with glutathione-agarose beads (Thermo Fisher Scientific Pierce, #16100). GST fusion proteins were eluted from the beads with 20 mM reduced glutathione (Fujifilm Wako, #073-02013) and dialyzed with 3500 MWCO Slide-A-Lyzer G3 Dialysis Cassettes (Thermo Fisher Scientific, #A52966) against 20 mM Tris–HCl (pH 8.0) containing 10 % glycerol. The furin proteins were then concentrated with 3000 MWCO Amicon Ultra centrifugal filters (Merck Millipore, #UFC200324), and aliquots were stored at −80 °C.

Furin in vitro assay

A furin protease assay was performed using a furin protease assay kit (BPS Bioscience, #78040) according to the manufacturer's protocol with the following modifications. Prepared purified GST-tagged furin proteins were mixed with 2 µM of the fluorogenic furin substrate and incubated in the assay buffer at room temperature for 30 min. The fluorescence intensity resulting from substrate cleavage was measured at an excitation wavelength of 380 nm and an emission wavelength of 460 nm using a TriStar2 LB 942 multimode microplate reader (Berthold Technologies).

Statistical analysis

For statistical analysis, data were evaluated using a paired Student's t test. One-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was used to analyze differences between multiple experimental groups. A p-value of <0.05 was considered statistically significant, and p-values are represented in the figures as *p < 0.05, **p < 0.01.

Results

Establishment of cervical cancer cell lines exhibiting multinucleation

To explore the genetic mutations related to cancer progression in cervical cancer, we introduced unpredictable genetic mutations into human cervical cancer HeLa cells and genetically analyzed the mutated clones exhibiting malignant characteristics. To this end, we focused on off-target mutations using genome editing technology. Cytosine base editors (CBEs) create substantial off-target mutations [27,28], and the fourth-generation CBE, CBE4, generates large-scale off-target single nucleotide variants (SNVs) [29]. We first transfected the CBE4 plasmid into HeLa cells and then isolated and propagated 30 single cells. Similar to the parental HeLa cell line, the vast majority of cells were mononuclear in 25 of 30 monoclonal cell lines. In cells of clone 1, one of the 25 clones, more than 98 % of the total cells were uninucleated, while cells with multiple nuclei were observed in the other five cell lines, including clone 2 (Fig. 1A and B). We then investigated whether this multinucleation was driven by cell fusion or acytokinetic mitosis using live-cell monitoring and found that it was induced by cellular membrane fusion (Fig. 1C; Video S1). Hereafter, we refer to 25 cell lines composed of mononuclear cells as non-fusion (NF) clones and the other five cell lines displaying cell fusion as constitutive fusion (CF) clones. Clones 1 and 2 were used in experiments as representatives of NF and CF clones, respectively. Additionally, mononuclear cells were generated from multinucleated cells via cellular division in the CF clone (Fig. 1D; Video S2). It appeared to occur without nuclear division because the total number of nuclei remained the same after the cellular division. In contrast, mononuclear cells displayed normal cellular division in which individual nuclei were divided into two daughter cells (Fig. 1E; Video S3). We next examined clinical specimens of advanced cervical cancerous tissues invaded into the submucosal layer (cancer diagnosis; stages ⅠB2 and ⅡA). Comparing them with normal tissues adjacent to the tumors (NATs), nuclear enlargement and increased nuclear-to-cytoplasmic ratio were recognized as striking differences between advanced cancer and normal cells (Fig. 1F and G). Notably, multinucleated cells were observed in these cancerous tissues, as detected in a wide variety of human cancers (e.g., lung, breast, and prostate cancers) [[20], [21], [22]].Fig. 1 Establishment of cervical cancer cell lines exhibiting multinucleation. (A–E) HeLa cells were transfected with CBE4 cytosine base editor plasmid, and then single cell-derived clones were expanded. (A) Cells from clones 1 and 2 were immunostained with an anti-pan cadherin antibody (green). DAPI for nuclear staining (blue). The bottom images show magnifications of the boxed areas in the top images. Scale bar, 200 μm. (B) Box plots present the percentage of cells with two (left) or more than two (right) nuclei versus the total number of cells (≥1000 cells) (n = 5 independent experiments). **p < 0.01 by Student's t test. (C) Serial images for cell fusion were captured from clone 2 cells. Scale bar, 20 μm. Arrowheads indicate the position of cell fusion. (D and E) Serial images of cell division were captured from multinucleated (D) and mononuclear (E) CF (clone 2) cells. The number of nuclei in the cells is shown. Scale bars, 20 μm. (F and G) UCC tissues (F, stage ⅠB2; G, stage ⅡA) and NATs were excised from patients with cancer. Hematoxylin and eosin stained images (left) and magnified images of the boxed areas in the left images (right) are shown. Arrowheads indicate the multinucleated cells. Scale bars, 200 μm.

Fig 1

In multinucleated cells, nuclear abnormalities can occasionally occur due to aberrant nuclear fusion and division [22,23]. When delineating the nuclear envelope by tracing A-type lamins (lamins A and C), we found nuclei of atypical sizes and shapes in CF cells (Fig. S1A and B). We then investigated the genomic integrity of CF cells by analyzing chromosome numbers in individual nuclei; karyotyping data revealed that nuclei with more than or less than 46 chromosomes were present in CF cells (Fig. S1C). These results suggest that fusion-induced multinucleation of CF cells participates in aneuploidy formation.

The olfactory receptor OR1N2 gene is mutated in multinucleated cervical cancer cells

To elucidate the molecular mechanism underlying cell fusion in CF cells, we aimed to identify CF cell-specific mutations using whole exome sequencing (WES), given that cell fusion in CF cells was conceivably triggered by de novo off-target mutations induced by CBE4 introduction. After aligning the sequencing reads to the human reference sequence, 9939 and 9857 non-synonymous SNVs (nsSNVs) were detected in NF and CF cells, respectively (Fig. 2A). Furthermore, 9787 of these overlapped, and 70 nsSNVs were selectively detected in CF cells (Fig. 2A; Table S1). To explore the causative mutations underlying cell fusion, these 70 nsSNVs were filtered by verification with other NF and CF clones. Heterozygous thymidine-to-cytosine substitution in the olfactory receptor OR1N2 gene, which causes cysteine-to-arginine conversion at amino acid position 100 (C100R mutation), was detected in all five CF clones but not in the other 25 NF clones (Fig. 2B). This thymidine-to-cytosine substitution in OR1N2 has not yet been deposited in the human OR mutation database (hORMdb), a repository of natural variations in human ORs [30], nor the Cancer Genome Atlas (TCGA), a catalog of genomic alterations associated with cancer. This heterozygous mutation was verified by direct sequencing in the reverse direction and confirmed in all CF clones (Fig. 2B). To address the clinical relevance of the C100R mutation in the OR1N2 gene, we validated this mutation in clinical specimens, confirming its presence in multinucleated cells regionally isolated from advanced uterine cervical cancer (UCC) tissues, although it was absent in genomic DNA isolated from NATs (Fig. 2C and D).Fig. 2 The olfactory receptor OR1N2 gene is mutated in multinucleated cervical cancer cells. (A) Whole exome sequencing of NF (clone 1) and CF (clone 2) cells was performed. Venn diagram illustrates the total counts and comparison of nsSNVs in the coding sequence (CDS) region. (B) Direct sequencing of coding and template strands of genomic DNA fragments, including codon 100 of OR1N2 (underline) from NF and CF clones. (C and D) Direct sequencing of genomic DNA fragments, including codon 100 of OR1N2 from UCC tissues and NATs (C, stage ⅠB2; D, stage ⅡA). (E) Parental HeLa cells, NF cells, and CF cells were lysed and separated into soluble and insoluble fractions. The insoluble fractions were then lysed directly in 1 × Laemmli sample buffer, sonicated, and analyzed by immunoblotting to evaluate the protein expression level of OR1N2. NS points to a nonspecific band that displays equal protein loading. (F) The amino acid sequence of human OR1N2 protein surrounding the third and fourth predicted transmembrane (TM) domains, as well as the first and second extracellular loop (ECL) and the second intracellular loop (ICL) regions. Asterisks indicate cysteine residues required for the disulfide bond formation. (G) Ribbon diagrams of OR1N2 WT (blue) and C100R mutant (yellow) viewed from the extracellular space were obtained using AlphaFold2. Red spheres represent disulfide bonds. (H) The superposition between ribbon diagrams of OR1N2 WT (blue) and C100R mutant (yellow) are shown as viewpoints parallel to the membrane and from the intracellular space. (I) The model represents structural and transitional differences between the OR1N2 WT and C100R mutant. (J) Cell lysates from parental HeLa cells, NF cells, and CF cells were subjected to immunoblotting with an antibody against phosphorylated PKA substrates (p-PKA sub.). β-actin was used as a loading control. (K and L) CF cells were transfected with control (Ctrl) siRNA or siRNA against human OR1N2 for 48 h. (K) Cells were analyzed as described in (E). (L) Cell lysates were subjected to immunoblotting with antibodies indicated.

Fig 2

Next, we examined the implications of the C100R mutation in OR1N2 in cell fusion-induced multinucleation by exogenously expressing the OR1N2 C100R mutant. To promote the functional cell surface expression of ORs, we first retrovirally introduced the OR accessory protein receptor-transporting protein 1 (RTP1), which functions as a chaperone and facilitates OR trafficking to the cell surface membrane [31,32] and then stably expressed OR1N2 wild-type (WT) and C100R mutant into parental HeLa cells. ORs are widely distributed throughout the human body, including the uterus [16,17], and the expression of endogenous OR1N2 and exogenetically increased OR1N2 was detected in HeLa cells (Fig. S2A). Comparing the proportion of cells with two nuclei and cells with more than two nuclei, we found a significant increase in the number of OR1N2 WT-overexpressing cells (Fig. S2B and C). Moreover, this increase was accelerated in OR1N2 C100R mutant-overexpressing cells, accompanied by the presence of cellular membrane fusion (Fig. S2B–D), suggesting that the C100R mutation in OR1N2 likely contributes to the induction of cell fusion.

To examine the effect of the C100R mutation in OR1N2 in CF cells, we first compared the expression levels of OR1N2 between NF and CF cells, and found them to be nearly comparable (Fig. 2E). Mutations in cell surface receptors can lead to structural changes that enhance or reduce their responsiveness to signaling molecules [11,12]. These structural changes also induce the continuous activation or disruption of downstream pathways, resulting in anomalous cellular behavior and disease. We then analyzed the impact of the C100R mutation on the OR1N2 structure. ORs are the largest subfamily within the class A G protein-coupled receptors (GPCRs) and are integral membrane proteins composed of seven transmembrane-spanning domains (7TM) [33,34]. Disulfide bond formation between the cysteine residue at the top of the third transmembrane domain (TM3) and that in the second extracellular loop (ECL2) is widely conserved in class A GPCRs, and the presence of an intra-ECL2 disulfide bond has been predicted for ORs [33,34]. The C100R mutation was located in the cysteine residue at the top of TM3 (Fig. 2F), and 3D modeled structures of the OR1N2 WT and C100R mutant suggested the destruction of the disulfide bridge by this mutation (Fig. 2G). The TM3-ECL2 disulfide bridge in ORs drives ECL2 toward their odorant-binding pocket; accordingly, their overall structure is correctly folded [33,34]. It is conceivable that the destruction of the disulfide bridge provokes structural changes. The OR1N2 WT and C100R mutant 3D models displayed similarity or correspondence in a greater part of their 7TM structures, although superposition data showed that the tilt angles of the TM6 helix notably differed between them (Fig. 2H).

The conformational transition between the active and inactive conformations of class A GPCRs is associated with the patterns of global movement of transmembrane helices, particularly the inter-helical movements between TM3 and TM6 [35,36]. Given that the OR1N2 C100R mutation causes notable positional changes in the cytoplasmic end of TM6 (Fig. 2H and I), the activity of the non-canonical olfactory pathway, provoked by the spontaneous conformational transition between active and inactive conformations, may differ between the OR1N2 WT and C100R mutant. Moreover, because cAMP-dependent protein kinase A (PKA) is the major target of intracellular cAMP in the non-canonical olfactory pathway [13,14,37], we evaluated PKA activity in NF and CF cells and found that several PKA substrate proteins were more phosphorylated in CF cells (Fig. 2J). This increased phosphorylation of PKA substrates was also detected in OR1N2 WT-overexpressing cells and was more prominent in OR1N2 C100R mutant-overexpressing cells (Fig. S2E). Furthermore, depletion of OR1N2 expression diminished PKA activation in CF cells (Fig. 2K and L). Taken together, these observations suggest that the C100R mutation in OR1N2 in CF cells triggers PKA activation via transduction of the non-canonical olfactory pathway.

Furin plays a crucial role in cell fusion of CF cells

Next, we investigated the functional role of PKA activated by the C100R mutation in the OR1N2 gene in the fusion of CF cells. In the non-canonical olfactory pathway, cAMP response element-binding protein (CREB), a transcription factor that regulates diverse cellular responses [38], is the principal PKA substrate [13,14,37]. The results of immunoblot analysis demonstrated that phosphorylation of CREB at Ser133, a major phosphorylation target site of PKA [38], was intensified in CF cells (Fig. S3A); treatment with the PKA inhibitor H89 suppressed this augmented phosphorylation (Fig. S3B). ERVW-1 and ERVFRD-1 genes are envelope protein (env) genes of the human endogenous retroviral origin and encode syncytin-1 and -2, which possess fusogenic properties and participate in trophoblast fusion during placental morphogenesis [39,40]. Syncytin functions in cancer cell fusion and contributes to the progression of various cancer types [20,23,41,42]. Given that ERVW-1 and ERVFRD-1 are transcriptionally induced in response to activation of PKA/CREB signaling and promote cell fusion in human placental BeWo cells [[43], [44], [45]], we compared the expression levels of ERVW-1 and ERVFRD-1 between NF and CF cells; however, no significant changes were detected (Fig. S3C). To determine whether the expression of CREB target genes was increased in CF cells, we compared 155 genes exhibiting increased expression in CF cells with putative CREB target genes (4084 genes), defined by the presence of a conserved gene promoter element, CRE [46], revealing that only 10 genes met these criteria (Fig. S3D; Table S2). We further investigated whether the transcriptional activity of CREB was elevated in CF cells using a CREB-dependent luciferase reporter plasmid and found no significant difference in luciferase activity between NF and CF cells (Fig. S3E). Collectively, these findings suggest that CREB phosphorylation alone is not reliable for predicting its transcriptional activation in CF cells and that the recruitment of additional transcriptional regulatory partners to the promoters may be required for its target gene expression [38,46].

Considering the fusogenic function of syncytin in cancer cells [20,23,41,42], although no significant transcriptional differences were detected between NF and CF cells (Fig. S3C), we further confirmed their expression levels using immunoblot analysis. Although full-length syncytin-2 levels remained almost unaltered, levels of the cleaved form of syncytin-1 increased concomitantly with a decrease in full-length syncytin-1 in CF cells (Fig. 3A). Syncytins are produced as precursor proteins and are cleaved into two mature proteins, the surface (SU) and transmembrane (TM) subunits (Fig. 3B), by a furin endoprotease. These two proteins are allocated to apposing cellular membranes, and their interactions via disulfide bonds bring the membranes into close proximity, eventually leading to cellular membrane fusion [39,41]. Cleavage of syncytin by furin initiates cell fusion, and furin introduction induces syncytin cleavage and subsequent cell fusion in cancer cells [47]. We then examined whether furin activity was required for syncytin-1 cleavage and cell fusion in CF cells. Treatment with a furin inhibitor decreased the cleaved form of syncytin-1 in CF cells and increased the uncleaved form (Fig. 3C). Consistent with this observation, treatment with the furin inhibitor reduced the proportion of multinucleated CF cells (Fig. 3D and E). Thus, these findings suggest that furin activity is sufficiently enhanced to induce syncytin-1 cleavage and that its activation is essential for cell fusion in CF cells.Fig. 3 Furin plays a crucial role in cell fusion of CF cells. (A) Cell lysates from parental HeLa cells, NF (clone 1) cells, and CF (clone 2) cells were subjected to immunoblotting with indicated antibodies. The solid and open arrowheads indicate the position of full-length and cleaved form syncytin-1, respectively. (B) Schematic represents the domain structure of syncytins. The positions of the disulfide bond formation motifs, the furin cleavage sites, signal peptides, fusion peptides, transmembrane domains (TMD), surface (SU) subunits, and transmembrane (TM) subunits are indicated. The immunogen ranges for syncytin antibodies are also indicated. (C–E) CF cells were treated with furin inhibitor (furin INH; 10 μM) for 96 h. The media were changed, and furin inhibitor was repeatedly added every 24 h. DMSO was used as a vehicle. (C) Cell lysates were subjected to immunoblotting with indicated antibodies. (D) Cells were immunostained with an anti-pan cadherin antibody (green). DAPI for nuclear staining (blue). The bottom images show magnifications of the boxed areas in the top images. Scale bar, 200 μm. (E) Box plots present the percentage of cells with two (left) or more than two (right) nuclei versus the total number of cells (≥1000 cells) (n = 5 independent experiments). **p < 0.01 by Student's t test.

Fig 3

Phosphomimetic furin mutant at Ser421 induces cell fusion

To elucidate how furin protease activity is enhanced in CF cells, we hypothesized that activated PKA phosphorylates furin, regulating its protease activity. A search for the R-R/K-X-S/T consensus PKA phosphorylation motif was then performed against furin [48], and the applicable sequence, RKVS, was exclusively identified within the C-terminal portion of its catalytic domain (Fig. 4A). To examine whether PKA targets the serine residue at position 421 in this predicted phosphorylation motif of furin, we prepared parental HeLa cells stably expressing PKA WT and the constitutively active form, PKA Cα mutant (PKA CA), displaying catalytic activity even in the absence of cAMP [49]. Phosphorylated furin at Ser421 was detected in PKA WT-overexpressing cells and was more evident in PKA CA-overexpressing cells, similar to the phosphorylation of CREB at Ser133 (Fig. 4B). Phosphorylation of furin at Ser421 was intensified in CF cells when compared with that in NF cells (Fig. 4C), and H89 treatment reduced this band intensity (Fig. 4D), suggesting that Ser421 of furin is a potential PKA phosphorylation target in CF cells.Fig. 4 Phosphomimetic furin mutant at Ser421 induces cell fusion. (A) The domain structure of furin and sequence alignment of the consensus motif for PKA-mediated phosphorylation in furin and known PKA substrates, CREB and BAD, are shown. X, any amino acid. (B) HeLa cells were infected with retroviruses expressing PKA WT, PKA CA mutant, or control retrovirus. Cell lysates were analyzed by immunoblotting with indicated antibodies. (C) Cell lysates from parental HeLa cells, NF (clone 1) cells, and CF (clone 2) cells were subjected to immunoblotting with indicated antibodies. (D) CF cells treated with H89 (4 μM) for indicated time periods were subjected to immunoblotting with indicated antibodies. H89 was repeatedly added every 24 h. (E–H) HeLa cells were infected with retroviruses expressing furin WT, furin S421D mutant, or control retrovirus. (E) Cells were immunostained with an anti-pan cadherin antibody (green). DAPI for nuclear staining (blue). The bottom images show magnifications of the boxed areas in the top images. Scale bar, 200 μm. (F) Box plots present the percentage of cells with two (left) or more than two (right) nuclei versus the total number of cells (≥1000 cells) (n = 5 independent experiments). Data were analyzed by one-way ANOVA, followed by Tukey's multiple comparison test. *p < 0.05 and **p < 0.01. (G) Serial images for cell fusion were captured from furin S421D mutant-expressing cells. Arrowheads indicate the position of cell fusion. (H) Cell lysates were analyzed by immunoblotting. The solid and open arrowheads indicate the position of full-length and cleaved form syncytin-1, respectively. The relative intensity (RI) of full-length syncytin-1 and -2 proteins normalized to β-actin protein were evaluated. (I) The cleavage of fluorogenic furin substrates by recombinant furin proteins was evaluated using furin in vitro assay. The fluorescence intensity values of GST control protein were subtracted from those of GST-tagged furin proteins. Data are shown as the mean of three independent experiments ± SD and were analyzed by Student's t test. **p < 0.01. Recombinant proteins used in the reactions were analyzed by immunoblotting.

Fig 4

To evaluate the functional effect of Ser421 phosphorylation on furin activity, we employed a mutant that mimics Ser421 phosphorylation by amino acid substitution with aspartic acid (furin S421D mutant). The proportion of multinucleated cells was elevated in furin WT-overexpressing cells and was further increased in furin S421D mutant-overexpressing cells, demonstrating the presence of cellular membrane fusion (Fig. 4E–G). Upon analyzing syncytin cleavage, we found that the cleaved form of syncytin-1 was increased in furin WT-overexpressing cells, and this increase, coupled with a decrease in uncleaved syncytin-1, was pronounced in furin S421D mutant-overexpressing cells (Fig. 4H). Moreover, the amount of uncleaved full-length syncytin-2 remained the same in furin WT-overexpressing cells, whereas it declined in furin S421D mutant-overexpressing cells (Fig. 4H), under which the cleaved form of syncytin-2 was undetected because the antigenic amino acid sequence for syncytin-2 crosses the furin cleavage site (Fig. 3B). These findings indicate that the phosphomimetic S421D mutation in furin enhances the protease activity against syncytin. Although furin can cleave syncytin-2 and syncytin-1, syncytin-1 may be preferentially cleaved over syncytin-2 in HeLa cells (Fig. 4H, comparison of full-length syncytin-1 and syncytin-2 levels in each lane). Given the increased expression of syncytin-1 in various types of cancers [20,41,42,50], this priority may be ascribed to the greater abundance of syncytin-1 than syncytin-2. To directly evaluate the protease activity of furin WT and S421D mutant proteins, we generated recombinant furin proteins containing the catalytic and P domains, which are essential for enzymatic activity [51]. Based on the in vitro furin protease assay results, a larger amount of fluorogenic furin substrates was cleaved by the furin S421D mutant than by the furin WT (Fig. 4I). Thus, these results suggest that PKA-mediated furin phosphorylation at Ser421 promotes protease activity against syncytin.

Prevention of multinucleation improves the efficacy of anticancer drugs

Cell fusion is associated with malignant characteristics of advanced cancers, including anticancer drug resistance [[21], [22], [23]]. Accordingly, we then tested the anticancer drug sensitivity of NF and CF cells using cisplatin, the most common chemotherapeutic agent for patients with locally advanced cervical cancer [52]. We monitored the initial reaction to cisplatin treatment and detected a marked increase in dead NF cells. Conversely, the number of CF cells, irrespective of mononuclear and multinucleated cells, persisted after 24 h of treatment (Fig. 5A). According to the results of the cell viability assay, the percentage of cumulative dead NF cells reached 90 % and ranged between 42 and 63 % in CF cells (Fig. 5B). These results indicate that CF cells are intrinsically resistant to anticancer drugs, even when non-multinucleated. To evaluate activation of the cisplatin-induced cell death pathway, known as the apoptotic pathway, we examined the activity of caspases that are closely related to apoptosis induction [53]. In NF and parental HeLa cells, cisplatin induced an increase in the cleaved form of PARP, a substrate of caspases, although this was attenuated in CF cells (Fig. 5C). These results suggest that CF cells are less sensitive to cisplatin than NF cells.Fig. 5 Prevention of multinucleation improves the efficacy of anticancer drugs. (A–C) NF (clone 1) and CF (clone 2) cells were treated with cisplatin (40 µM) for 24 h (A and B) or 16 h (C). (A) Representative bright-field images are shown. Scale bar, 200 μm. (B) LDH release was measured using an LDH cytotoxicity assay. The percentage of cell death was calculated based on LDH release from cisplatin-treated and control cells. Data are shown as the mean of three independent experiments ± SD and were analyzed by Student's t test. **p < 0.01. (C) Cell lysates were subjected to immunoblotting with indicated antibodies. The solid and open arrowheads indicate the position of full-length and cleaved form PARP, respectively. (D) Serial images were captured from CF cells treated with cisplatin (40 µM) for indicated time periods. Yellow arrowheads indicate the multinucleated cells. Scale bar, 100 μm. (E and F) CF cells were treated with furin inhibitor (furin INH; 10 μM) for 96 h and then further treated with cisplatin (40 µM) for 40 h. The media were changed, and furin inhibitor was repeatedly added every 24 h. DMSO was used as a vehicle. (E) Representative bright-field images are shown. Scale bar, 100 μm. (F) The number of surviving cells after cisplatin treatment was counted using the trypan blue exclusion technique. Data are shown as the mean of three independent experiments ± SD and were analyzed by Student's t test. **p < 0.01.

Fig 5

PKA participates in the anti-apoptotic characterization of various types of cancers [54], and PKA/CREB signaling exerts an anti-apoptotic function by inducing Bcl-2, a key protein in inhibiting apoptosis [55,56]. Comparing the expression of BCL2 between NF and CF cells, we found that, as is the case with ERVW-1 and ERVFRD-1 (Fig. S3C), there was no increase in the expression of BCL2 in CF cells (Fig. S4A). Alternatively, given that phosphorylation of BAD at Ser118 by PKA supports the function of the pro-survival Bcl-xL protein and prevents apoptosis [57], we examined BAD phosphorylation at Ser118 and found that it was augmented in CF cells (Fig. S4B). In addition, BAD phosphorylation was suppressed by H89 treatment (Fig. S4B), suggesting that PKA, but not its downstream effector CREB, is functional in CF cells and may be involved in the intrinsic anti-apoptotic characteristics of CF cells.

To determine the reactivity of CF cells to anticancer drugs, the cells were treated with cisplatin for an extended period. Although no apparent differences were detected in cisplatin-induced cell death between mononuclear and multinucleated CF cells after treatment for 24 h (Fig. 5A), mononuclear cells, but not multinucleated cells, exhibited adequate features of cell death when continuously treated for up to 40 h (Fig. 5D; Video S4). These observations suggest that multinucleation due to cell fusion limits cisplatin-based chemotherapy and complicates complete cancer remission. Given that our data showed that furin inhibitor treatment prevented the multinucleation of CF cells (Fig. 3D and E), we treated CF cells with a combination of cisplatin and furin inhibitors. Some surviving cells were observed after cisplatin treatment alone, and most were multinucleated (Fig. 5E). Notably, combination treatment reduced the total number of surviving cells by ∼50 % (Fig. 5F). Taken together, these results suggest that the prevention of cell fusion by furin inhibitors promotes the eradication of cancer cells and provides promising therapeutic efficacy against advanced cervical cancers.

Discussion

Cervical cancer ranks among the most common gynecological cancers worldwide and adversely affects female reproductive organs along with uterine body cancer [58]. Because understanding and optimal management of these patients can improve their treatment outcomes and result in favorable prognoses, cervical cancer therapies must be optimized according to the malignant characteristics and causative genetic abnormalities. In the current study, we developed cervical cancer cells that underwent multinucleation by cell fusion: CF cells. Given that multinucleation plays a role in the malignant characteristics of cancer cells, such as anticancer drug resistance, the results of our study may provide new anticancer therapeutic strategies.

We demonstrated that cisplatin treatment was less effective in CF cells regardless of the number of nuclei (Fig. 5A). As the OR1N2 gene was mutated and the non-canonical olfactory pathway was subsequently activated in these cells, we postulate that PKA-mediated phosphorylation of BAD contributes to cell survival (Fig. S4B). We also propose the possible aneuploidy formation (Fig. S1C), in which aneuploid cells exhibit anticancer drug resistance. Aneuploidy can upregulate anti-apoptotic pathways and overexpress efflux pumps such as P-glycoprotein, which actively remove drugs from cells and prevent cell death triggered by chemotherapy [6]. However, given that the prolonged treatment of mononuclear CF cells with cisplatin could induce cell death (Fig. 5D), the most notable concern was the increased anticancer drug resistance observed in multinucleated cells. The principal mechanism of cisplatin-induced apoptosis involves the formation of covalent bonds between purine bases in the DNA double helix. These intra- or inter-strand cross-links disrupt DNA replication and stall replication forks, causing DNA damage and activating cellular stress responses, including apoptosis [53]. Although the precise molecular mechanism is yet to be elucidated, multinucleated cells may deviate from the normal cell cycle and evade cisplatin-induced apoptosis in a dormant state. For example, to adapt to morphological alterations induced by cell fusion, multinucleated cells must coordinate their metabolic pathways, including energy production and nutrient utilization [21]. Because enzymes involved in DNA synthesis and replication, such as DNA polymerases, helicases, and ligases, rely on ATP to drive their activities, these biological processes are energy-intensive. Therefore, if cellular energy levels drop considerably due to metabolic reprogramming, DNA synthesis and replication may slow down or stop simultaneously in multinucleated cells. This may be related to the absence of nuclear division during cellular division in multinucleated cells (Fig. 1D). Moreover, cell fusion in cell cycle control has been demonstrated using a vesicular stomatitis virus G glycoprotein (VSV-G)-mediated cell fusion system [59]. Cellular membrane fusion induces remodeling of the plasma membrane surface, including glucose transporter internalization, and decreases cytoplasmic glucose and ATP levels. This reduced energy state activates AMP-activated protein kinase (AMPK) signaling, leading to the persistent cytoplasmic retention of the transcription factor Yes-associated protein 1 (YAP1). As YAP1 promotes cell proliferation by directly activating the transcription of multiple genes required for cell cycle progression [60], cell fusion-induced YAP1 inhibition causes cell cycle arrest. One of the major undesirable cellular characteristics of cancer is uncontrolled cellular proliferation, and the survival of multinucleated cells in a growth-arrested state after anticancer drug treatment may not be of particular concern. However, considering that multinucleated cells can re-divide into mononuclear cells (Fig. 1D), these results indicate the risk of cancer recurrence. After withstanding anticancer drug treatment in the multinucleated state, mononuclear cells re-divided from multinucleated cells may restart uncontrolled proliferation, leading to a worse prognosis. Thus, from an alternate perspective, inhibiting multinucleated to mononuclear cell division could offer a new target for preventing cancer recurrence.

Most cases of cervical cancer are caused by persistent infection with high-risk human papillomaviruses (HPVs), such as HPV-16 and HPV-18. These viruses can induce cellular changes from normal to cancerous cervical cells by producing two main oncogenic proteins, E6 and E7, which contribute to cellular transformation [61]. In HPV infection of the basal cells of the cervical stratified squamous epithelium, the virus initially attaches to heparan sulfate proteoglycans (HSPGs). This induces a conformational change and exposes the furin cleavage site in minor capsid protein L2. The proteolytic processing of L2 by furin, which is present on the cell surface, induces an additional conformational change that allows the binding of HPV to a secondary cellular uptake receptor and subsequent receptor-mediated endocytosis [62,63]. Therefore, furin activity in cervical cells is required for HPV infection, and it is conceivable that females whose cervical cells have greater furin activity may be at a higher risk of cervical cancer. Herein, we demonstrated that the PKA-mediated phosphorylation of furin at Ser421 enhanced its protease activity and induced syncytin-1 cleavage, resulting in cell fusion. Although whether this mechanism can be applied to any type of cancer remains unexplored, it may be readily available for cervical cancers owing to their notable intrinsic furin activity. Furin protease activity in cervical cells contributes to cancer initiation via cleavage of HPV and also possibly to cancer progression via cleavage of synsytins, which could emerge as a potential therapeutic target for advanced cervical cancers.

Furin plays a role in infection with viruses other than HPV [62]. For example, furin is crucial for activating the spike glycoprotein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19). Furin cleaves the full-length S protein into S1 and S2 subunits, known to be responsible for receptor binding and membrane fusion, and this cleavage by furin facilitates its membrane fusion with host cells, allowing viral entry more efficiently [64]. Given that the inhibition of furin activity could reduce the viral infectivity of SARS-CoV-2 [65], it is likely to increase when furin activity is enhanced by PKA-mediated phosphorylation. Furin inhibitors have been explored as potential treatments for COVID-19, and the present study proposes their potential therapeutic benefits in advanced cervical cancers. Accordingly, furin inhibitors are promising candidates for widespread therapeutic applications.

Conclusions

On the basis of our data, we propose a breakthrough in anticancer drug treatment for malignant cervical cancers. In multinucleated cervical cancer cells, OR1N2 protein contains a cysteine to arginine substitution at position 100, leading to activation of the non-canonical olfactory pathway, which is OR-PKA signal transduction. Activated PKA phosphorylates furin protease at Ser421 and actuates its protease activity, provoking syncytin-1 cleavage. The cleaved syncytin-1 proteins, syncytin-1 SU and TM subunits, allocate to the apposing cell membrane surfaces and are linked by disulfide bonds, which leads to cell fusion-induced multinucleation. As multinucleated cells exhibit high resistance to anticancer drugs, inhibition of cell fusion by furin inhibitors may improve the therapeutic efficacy of anticancer drugs against multinucleated cervical cancer cells.

Funding

This work was supported by Grants-in-Aid for Scientific Research (Grant numbers 17K07185 and 22K09946 ) from the Japan Society for the Promotion of Science (JSPS).

CRediT authorship contribution statement

Keigo Araki: Writing – original draft, Visualization, Validation, Supervision, Project administration, Investigation, Funding acquisition, Data curation, Conceptualization. Takeru Torii: Visualization, Investigation, Data curation. Kohei Takeuchi: Investigation, Data curation. Natsuki Kinoshita: Investigation. Ryoto Urano: Investigation. Rinka Nakajima: Investigation. Yaxuan Zhou: Investigation. Tokuo Kobayashi: Resources. Tadayoshi Hanyu: Resources. Kiyoshi Ohtani: Supervision, Data curation. Kimiharu Ambe: Investigation. Keiko Kawauchi: Validation, Supervision, Project administration, Investigation, Data curation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

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Acknowledgments

We thank Nobuyasu Koga for his valuable insights into protein structure analysis; Motomi Osato and Hiroaki Hirata for discussions on this topic. R. N. is supported by scholarships from the TOBE MAKI Scholarship Foundation and the Yoshida Scholarship Foundation.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neo.2024.101044.
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