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. 2023 Jul 25;18(7):e0289098. doi: 10.1371/journal.pone.0289098

α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling

Murat Oz 1,#, Justin R King 2,#, Keun-Hang Susan Yang 3, Sarah Khushaish 1, Yulia Tchugunova 1, Maitham A Khajah 1, Yunus A Luqmani 1, Nadine Kabbani 2,4,*
Editor: Israel Silman5
PMCID: PMC10368273  PMID: 37490473

Abstract

Chronic smoking is a primary risk factor for breast cancer due to the presence of various toxins and carcinogens within tobacco products. Nicotine is the primary addictive component of tobacco products and has been shown to promote breast cancer cell proliferation and metastases. Nicotine activates nicotinic acetylcholine receptors (nAChRs) that are expressed in cancer cell lines. Here, we examine the role of the α7 nAChR in coupling to heterotrimeric G proteins within breast cancer MCF-7 cells. Pharmacological activation of the α7 nAChR using choline or nicotine was found to increase proliferation, motility, and calcium signaling in MCF-7 cells. This effect of α7 nAChR on cell proliferation was abolished by application of Gαi/o and Gαq protein blockers. Specifically, application of the Gαi/o inhibitor pertussis toxin was found to abolish choline-mediated cell proliferation and intracellular calcium transient response. These findings were corroborated by expression of a G protein binding dominant negative nAChR subunit (α7345-348A), which resulted in significantly attenuating calcium signaling and cellular proliferation in response to choline. Our study shows a new role for G protein signaling in the mechanism of α7 nAChR-associated breast cancer growth.

Introduction

Epidemiological studies show that smoking of tobacco products can significantly increase a woman’s risk of developing breast cancer [1–4]. Thus, although cigarette smoke consists of a complex mixture of > 3000 chemicals, nicotine is a primary bioactive component of tobacco contributing to cancer risk [5]. A large body of published work relates nicotine exposure and the proliferation as well as metastatic potential of cancer cells within tissue such as lung and breast [6]. Molecular studies show that nicotine increases the proliferation of cancer cells through the activation of nicotinic acetylcholine receptors (nAChRs) expressed at the cell surface [7]. In some cases, nAChR expression can be altered during cancer cell growth and through hormones or toxin exposure [8].

The nAChR channel is a pentameric protein consisting of combinations of 16 isoforms of subunits including a subunits α1–α10, β1–β4, γ, δ, and/or ε [9, 10]. Homopentameric nAChRs including α7 and α9 exhibit high calcium permeability and thus can directly signal long-term cellular growth [11, 12]. Indeed, nicotine has been shown to activate α7 [13–17] and α9 [18–21] nAChRs in breast cancer cells. In cooperation with calcium influx, ligand activation of α7 nAChRs drives metabotropic signaling cascades important for growth and cytoskeletal motility [22–25]. In this study, we explored the role α7 nAChR interaction with G proteins in MCF-7 breast cancer cell proliferation, motility, and calcium signaling.

Materials and methods

Cell culture and transfection

Michigan Cancer Foundation-7 (MCF-7) cell line (ATCC, Manassas, VA, USA) cells was maintained as monolayers in advanced Dulbecco’s minimum essential medium (DMEM) containing phenol red and supplemented with 5% fetal bovine serum (FBS), 600 μg/ml L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 6 ml/500 ml 100 x non-essential amino acids (all from Invitrogen, CA, USA), and grown at 37°C in an incubator of 5% CO2 and 95% humidity. MCF-7 is a human breast cancer cell line with estrogen, progesterone and glucocorticoid receptors and is derived from the pleural effusion of a 69-year-old metastatic adenocarcinoma. MCF-7 cells were transfected with constructs encoding human α7345-348A or the control human α7 nAChR in pEYFP-C1 (Addgene) [24], GCaMP5G [26] using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA). DNA was purified using a maxi prep kit (Xymo Research, Irvine, CA, USA).

Drug preparation

Choline, nicotine, cotinine, nornicotine, methyllycaconitine citrate (MLA) and α-bungarotoxin (BTX) were from Sigma Aldrich (St. Louis, MO, USA). Choline, MLA, and BTX were dissolved in distilled water. Nicotine, cotinine, and nornicotine were dissolved in ethanol. YM 254890 and pertussis toxin (PTX) were purchased from Tocris-Bio-Techne Corporation (Minneapolis, MN, USA) and dissolved in DMSO and distilled water, respectively. α-Conotoxin RgIA (CTX) was obtained from Alomone Labs (Jerusalem, Israel) and dissolved in distilled water.

Cellular fluorescence

Cell were permeabilized with 0.05% Triton X-100 then blocked with 10% goat serum (Life Technologies, Carlsbad, CA, USA) [27]. Surface α7 nAChRs were visualized in non-permeabilized cells using 100 nM Alexa Fluor (488 or 647) conjugated BTX as described [28, 29]. Imaging was performed on an inverted Zeiss LSM800 confocal microscope (Carl Zeiss Oberkochen, Germany). Analysis was carried out in ImageJ (NIH, Bethesda, MD, USA).

Cell proliferation (MTT assay)

Approximately 104 cells were seeded into triplicate wells of 12-well plates and allowed to attach overnight. Growth was assessed by an MTT assay after 3 days of drug incubation. Briefly, 1 ml of MTT [3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide] reagent (Promega) (0.5 mg/ml) was added to each well and incubated at 37°C for 30 min before the addition of 1 ml acidic isopropanol then vigorous re-suspension of the converted blue crystals. Cells were optically counted with a hemocytometer (Thermo Fisher) or the absorbance of the suspension was measured at 595 nm with background subtraction at 650 nm. Results are expressed as mean ± standard error of the mean (S.E.M.).

Calcium imaging

Cells were transfected with GCaMP5G [26] and the signal was detected using a Zeiss LSM 800 at an acquisition rate of 1 frame per 256 ms for 75 sec at 2 x 2 binning as described previously [23]. Drugs were applied to the recording chamber via a gravity fed perfusion at a flow rate of 1 ml/sec. Regions of interest (ROIs) were normalized as ΔF/Fθ and analyzed using ImageJ (NIH) as described [23]. A total of 20–30 cells were imaged per experimental condition and experiments were performed in triplicate.

Cell motility assay

Cells were plated on 6-well plates at 80–90% confluence with complete DMEM containing vehicle or drug. The following day, a scratch was created in the cell monolayer using a sterile p1000 pipette tip. A photograph of the scratched area was taken immediately (0 h) and after a 24 h incubation in a 37°C, 5% CO2. Cell motility was determined by calculating the width of the scratch at 24 h as a percentage of 0 h.

Statistical analysis

Student’s two-tailed unpaired t-test, or one-way ANOVA test followed by Bonferroni post hoc test were used to compare means of individual groups with p < 0.05 as statistically significant.

Results

Nicotinic receptor ligands promote proliferation and motility in MCF-7 cells

Nicotine has been shown promote cancer cell proliferation within various cell lines and in animal systems [6, 7, 20]. We examined the effect of a 3-day treatment with nicotine (50 nM-1 μM) on MCF-7 cell proliferation (Fig 1A). Treatment with nicotine was found to produce a concentration dependent increase in cell proliferation (p<0.05; ANOVA). Similarly, treatment with the selective α7-nACh receptor agonist choline increased proliferation of MCF-7 cells (Fig 1B). Cotinine, is the primary metabolite of nicotine and can persist longer in the body that nicotine [30]. Nornicotine, like nicotine, is found in tobacco products and has carcinogenic properties [31]. Both cotinine and nornicotine activate mammalian nAChRs and contribute to addiction [32]. We tested the effect of a 3-day treatment with cotinine (0.1–10 μM) or nornicotine (0.1–10 μM) on cell proliferation. We found that both cotinine and nornicotine increased cell proliferation to levels comparable with choline and nicotine at the concentration range of 1–10 μM (Fig 1C and 1D).

Fig 1. Dose-response effect of nicotine, choline, and nicotine metabolites on breast cancer cell proliferation.

Fig 1

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Approximately 104 MCF-7 cells were seeded into microwell plates and grown for 3 days in the presence of vehicle (control) or increasing concentrations of nicotine (A), choline (B), cotinine (C), and nornicotine (D). Cells were harvested and growth was determined by an MTT assay. Bars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from control with p < 0.05.

Nicotine can activate calcium signaling and lead to cell proliferation and the regulation of apoptosis within cancer cells [20]. We investigated the involvement of α7 and α9-nAChRs in breast cancer proliferation in response to 10 mM choline application, which activates both homopentameric receptor types [33]. Co-application of α7-nAChR antagonists: methyllycaconitine (10 μM, MLA) or α-bungarotoxin (100 nM, BTX), was found to significantly reduce cell proliferation in this experiment. Conotoxin RgIA (100 nM, CTX), an antagonist with higher potency for α9 and α10-nAChRs [34], also inhibited choline mediated proliferation (Fig 2).

Fig 2. Choline effect on proliferation in MCF-7 cells.

Fig 2

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Approximately 104 MCF-7 cells were seeded into microwell plates and grown for 3 days in the presence of vehicle (control), choline (10 mM), choline + methyllycaconitine (10 μM, MLA), choline + α-bungarotoxin (100 nM, BTX), and choline + α-Conotoxin RgIA (100 nM, CTX). Cells were harvested and growth was determined by an MTT assay. Bars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from choline treated group with p < 0.05.

The wound healing (or scratch) assay can measure cancer cell migration in vitro [35]. Based on earlier studies that show that α7 nAChR activation can modify the cytoskeleton and cell motility as well as structural change [23, 36], we tested the effect of choline on the motility of MCF-7 cells. A 24-hour treatment with 10mM choline was found increase cell motility by 2-fold when compared to the control. This effect of choline on motility was virtually eliminated by co-application of 100 nM BTX (Fig 3).

Fig 3. Choline increases cell motility.

Fig 3

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(A) Images of MCF-7 cells at the start (0 hr) and end (24 hr) of the motility assay. Experimental groups: vehicle (control), choline (10 mM), and choline with α-bungarotoxin (100 nM, BTX). (B) Average motility measures at 24 hr from 3 independent experiments. Asterisk denotes significance p < 0.05. Scale = 100μm.

G protein coupling to α7 nAChR drives intracellular calcium signaling and cell proliferation

Ligand stimulation of α7 nAChRs impacts receptor synthesis and trafficking to the cell surface in cancer cells [37]. We examined α7 nAChR expression at the cell surface after a 3-day treatment with 10 mM choline. In this experiment, we used a BTX-Alexa Fluor 488 conjugate to label non-permeabilized MCF-7 cells (S1 Fig), comparing choline treated to control cells. As shown in Fig 4, treatment with choline was associated with an increase in the BTX-Alexa Fluor 488 signal at the cell surface in comparison to the control condition.

Fig 4. An effect of choline on surface labeling for the nAChR.

Fig 4

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(A) MCF-7 cells were incubated with 100 nM α-bungarotoxin (BTX) Alexa Fluor 488. Images of representative cells showing labeling at 72 hr in control and choline (10 mM) treated cells. Scale = 5μm (B) Average BTX fluorescence values. Asterisk denotes significance p < 0.05 (n = 24–27).

Ligand stimulation of the α7 nAChR rapidly increases intracellular calcium levels by extracellular calcium influx through open nAChRs, the activation of voltage-gated calcium channels, and the activation of calcium induced calcium release (CICR) as well as inositol induced calcium release (IICR) from the ER [22]. We examined the effect of 10 mM choline on intracellular calcium within MCF-7 cells. As shown in Fig 5A, application of choline resulted in a calcium transient that appeared within 0.5 sec of drug application and lasted for ~1 second. This calcium transient was not seen when BTX was present in the application solution. Statistical analysis of calcium transient peaks between drug treatment groups confirms that BTX abolishes choline-associated calcium responses within MCF-7 cells (Fig 5B).

Fig 5. Choline mediated change in intracellular calcium in MCF-7 cells.

Fig 5

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(A) Calcium transients (ΔF/Fθ) measured using GCaMP5G in response to drug application. Vehicle (control), choline (10 mM), and choline with α-bungarotoxin (100 nM, BTX). (B) Average calcium peaks across experimental groups. Choline with pertussis toxin (5 μg/ml, PTX) (n = 21–25 cells). Statistical significance: *p<0.05; **p<0.01, ***p<0.001.

In previous studies we have shown that G protein interaction is involved in α7 nAChR-mediated IICR [22]. We tested the effect of the Gαi/o inhibitor PTX on choline-mediated calcium transients. Co-application of PTX was found to reduce the choline calcium transient by over 30% an effect that was found to be statistically significant (Fig 5B). We further explored G protein activity in nAChR-mediated cell growth. The 3-day proliferation assay was repeated in MCF-7 cells treated with 10 mM choline alone, choline with PTX (5 μg/ml), or choline with the Gαq inhibitor YM 254890 (1 μM). Analysis indicates that application of PTX or YM 254890 indicates a significant reduction in choline-mediated proliferation when G protein blockers are present (Fig 6).

Fig 6. G-protein inhibitors block choline-mediated MCF-7 cell proliferation.

Fig 6

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Approximately 104 MCF-7 cells were seeded into microwell plates for 3 days then assessed via an MTT assay: (A) vehicle (control), pertussis toxin (5 μg/ml, PTX), choline (10 mM), and choline with PTX, (B) vehicle (control), YM 254890 (1 μM), choline (10 mM), and choline with YM 254890. Bars represent means ± SEM of 3 independent experiments. Asterisk denotes significant difference from choline treated group (p < 0.05).

We have shown that α7 nAChRs directly bind G proteins through a binding sequence that is located within the intracellular loop region of the receptor [24]. Site directed mutagenesis of 4 amino acids at this site creates a dominant negative α7 subunit (α7345–348A) that is unable to bind G proteins [24]. In previous work, the expression of α7345–348A is sufficient to impair wild-type α7 nAChR calcium signaling and cell growth [36]. We transfected MCF-7 cells with α7345–348A and as shown in Fig 7, the expression of this mutant was seen sufficient in abolishing calcium transient responses to choline application. We also examined the effect of α7345–348A on proliferation in MCF-7 cells. An analysis of α7345–348A expression indicates that this mutant subunit does not significantly alter MCF-7 growth in the absence of a cholinergic ligand (Fig 7B). However, in cells transfected with α7345–348A, a 3-day treatment with choline did not increase proliferation as evidenced by a comparison of choline effect on cell number between wild-type α7 and α7345–348A expressing cells (Fig 7B and 7C).

Fig 7. G protein regulation of α7 nAChR calcium signaling.

Fig 7

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(A) Representative calcium transients following application of vehicle (control), choline (10 mM), and choline with α-bungarotoxin (100 nM, BTX) in cells transfected with wild-type α7 or the mutant α7345-348A subunit. Inset: average of calcium transient peak values (ΔF/Fθ) across experimental groups (n = 21–25). (B) Approximately 104 MCF-7 cells were seeded into microwell plates for 3 days then assessed via the MTT assay: vehicle (control) or choline (10 mM) in cells transfected with wild-type α7 or the mutant α7345-348A subunit. (C) A comparison of choline mediated proliferation in cells transfected with wild-type α7 or the mutant α7345-348A subunit relative to the vehicle control. Statistical significance **p<0.01, ***p<0.001.

Discussion

Evidence indicates a link between tobacco product use and increased risk to oral, lung, and breast cancer [38]. While there maybe diverse mechanisms by which nicotine can promote cancer cell progression, they all involve the activation of nAChRs on target cells [7, 20, 39]. Indeed, various nAChR couple to cancer processes that regulate cell division, morphology, and can increase angiogenesis and modify inflammatory responses in microenvironment of the cancer cells [7, 25, 40]. Recently, nAChRs have been shown to promote MCF-7 breast cancer cell proliferation via the activation of ERK1/2 phosphorylation [14] and drive epithelial to mesenchymal transition (EMT) [17].

In various neuronal cells high calcium signaling through the homopentameric α7 nAChR directs actin associated cytoskeletal dynamics leading to observable changes synaptic growth and plasticity [25, 41]. An analogous mechanism for α7 nAChR-calcium signaling to the cytoskeleton exists in non-neuronal cells including immune and epithelial cells [42, 43]. In this case, nAChR activation may drive changes in cell proliferation and/or metastatic transition involving the regulation of the cytoskeleton in cancer cells [44]. Our observations suggest that cholinergic ligands (e.g., nicotine) can alter α7 nAChR expression at the cell membrane of breast cancer cells. This may explain aspects of chronic nicotine exposure on cancer risk in smokers and suggests that trafficking of nAChRs may contribute to cancer progression.

The metabotropic activity of the α7 nAChR is driven by protein interactions of the receptor’s intracellular (M3-M4) loop [45]. We have shown the existence of a G protein binding region within the α7 nAChR M3-M4 loop [24]. Coupling between the nAChR and various G proteins, including Gαi and Gαq, was found to participate in important aspects of nAChR signaling [25]. At present it is not clear if this G protein binding site within the α7 nAChR favors association with specific Gα subunits. Based on in vivo analysis of G protein interactions with the α7 nAChR in the adult rodent brain, various G protein subunits appear able to bind the nAChR [24].

In this study, functional interaction between the α7 nAChR and Gαi appears to contribute to MCF-7 cell proliferation and motility based on experiments that show that blocking Gαi/o activity with PTX can abolish choline-associated cell proliferation. In calcium imaging experiments however PTX was found to significantly reduce the choline calcium transient response but not completely abolish it thus suggesting additional (non-Gαi) contributions to the α7 nAChR calcium signal. This is consistent with experiments using the Gαq inhibitor YM 254890 showing an effect of the G protein subunit on calcium mediated MCF-7 growth. Our results are supportive of earlier finding that show that Gαq can bind the α7 nAChR and promote calcium store release [23] and Gαi can activate pathways important for breast cancer growth [46]

Mammalian α7, α9, and heteromeric α9α10 combinations are the nAChR types with the greatest Ca2+ permeability [11, 47, 48]. It is important to note that many cancer cells express homopentameric nAChRs that are found to stimulate cancer cell proliferation, metastasis, and inhibit cancer cell apoptosis [7, 20]. The α9 nAChR contributes to breast cancer growth and EMT through activation of PI3K or MAPK signaling pathways [49]. In this study we also find a significant effect of α-Conotoxin RgIA, that is known to have a higher potency for α9 than α7 nAChRs, on MCF-7 cell growth. Thus, in future studies it will be important to explore the involvement of G proteins in α9 nAChR signaling in breast cancer cells.

Supporting information

S1 Fig. Cell surface labeling of nAChRs using BTX.

MCF-7 cells were labeled with 100 nM BTX. Cell membranes were not permeabilized in these labeling experiments. Bottom panel shows a representative image of a labeled cell. Scale bar = 5μm.

(TIFF)

Click here for additional data file. (453.8KB, tiff)

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

Funding provided by the Kuwait Foundation for the Advancement of Sciences (PN19-13PT-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Israel Silman

7 Jun 2023

PONE-D-23-15038α7 nicotinic acetylcholine receptor interaction with G proteins mediates breast cancer cell proliferation, motility, and calcium signalingPLOS ONE

Dear Dr. Kabbani,

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The experiment in figure 7B should be repeated with a control group for α7345-348a that was not exposed to choline to show that transfection with α7345-348a does not alter proliferation by itself.

The results in Figure 4 are not mentioned in the abstract or the discussion. It would be good if the authors discuss how the increase in α7 nAChR expression might affect the proliferation, motility, and/or calcium signaling of cancer cells. Additionally, the authors did not show if these effects could be blocked by MLA or another antagonist, or whether this upregulation is related to g-protein mediated signaling via α7 nAChRs. Further experiments to answer these questions would help to tie these results into the rest of the paper.

YM 254890 is mentioned in the abstract as a G protein blocker, but in the results section of the manuscript, the authors never define what specifically YM 254890 is or why it is being used.

The proliferation assay in Figure 7 plots Cell count x 105, whereas the other proliferation assays measure Net OD. Could the authors please explain why this assay was quantified differently?

There are two “B” panels in figure 5

The deltaF/F scales are not consistent across figures. For example, in Figure 5, A and C plot deltaF/F (x100), whereas panel B1 writes out the 100s, and the second panel B just has deltaF/F without the x100 clarification. Similarly, in Figure 7, panel A, the first graph is x100, while the second graph is not.

Scale bars should be added to Figure 3A and Figure 4A

Scale bar should be defined in Figure 7B.

In the figure legend for Figure 6 it is noted that the “Asterix denotes significant difference from control,” whereas the figure seems to indicate that the Asterix represents significant difference from the choline treated group. Could the authors clarify what comparisons are being measured in this figure, and what is significantly different from what?

The figure legend for Figure 2 states “Asterix denoted significant difference from control” but the figure seems to indicate that significance is measured as difference from the choline group.

Figure 7 has only 2 panels, A and B, but the figure legend has A, B, and C.

Figure 7 states “statistical significance relative to the control or α7 transfected group.” This is confusing. Could the authors clarify what comparisons are being made and what significance is being denoted. Does this mean that the α7345-348a group is significantly different from both the control and α7 group? If so, this should be denoted by two different symbols.

In the text, the mutant α7 is called “α7345-348a” whereas in the figure legend and figure it is “α7345-8a”, this nomenclature should be consistent throughout the paper

In the figure legend for Figure 5, the definition of a double Asterix is given, but there is no double Asterix in Figure 5

Reviewer #2: The paper by Oz et al, shows that in MCF-7 cells activation by choline of α7 receptor increases

proliferation, motility, and calcium signaling and this is partially blocked by the application of G protein blocker, Gαi/o inhibitor and by the presence of a G protein binding dominant negative α7 subunit (α7345-348A).

The work is interesting and well done, and it demonstrated that the α7 subtype is involved in mediating choline induced proliferation, motility, and calcium signalling. However the effect of choline was blocked using antagonists that act on both α7 and α9 subtypes. Why the authors did not used the α7 selective antagonist AR (Whitaker et al. 2007) and the α9 selective antagonist RGIA4 (Romero et al 2017) ? This certainly will help in clarifying the subtypes involved and their contribution to the effects reported

Minor points:

In the abstract it is written "Nicotine activates various nicotinic acetylcholine receptors (nAChRs)" and in the introduction it is written"Mammalian nAChRs assemble from α (α1–α6) and β (β2–β4) subunit combinations forming homo- or hetero-pentameric channels [9, 10].

Nicotine does not activates a1 containing receptors and nicotine activates various nAChRs

that assemble from α (α2–α7, α9, α10) and β (β2–β4) subunit combinations.

Fig 7: Please add C to the figure

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Jul 25;18(7):e0289098. doi: 10.1371/journal.pone.0289098.r002

Author response to Decision Letter 0

7 Jul 2023

Please find the revision of our manuscript entitled “α7 nicotinic acetylcholine receptor interaction with G proteins mediates breast cancer cell proliferation, motility, and calcium signaling” for consideration as a research article in PLOS One.

We have taken into consideration the comments of both reviewers and have addressed them throughout the revised manuscript. This is enumerated in a point-by-point response letter to the reviewers showing specific changes in the revised submission:

Reviewer #1:

• The experiment in figure 7B should be repeated with a control group for α7345-348a that was not exposed to choline to show that transfection with α7345-348a does not alter proliferation by itself.

RESPONSE: We thank the reviewer for this comment. In fact, the experiment was conducted as a control however it was not included in the original submission. Therefore, the revised manuscript shows data on mutant receptor subunit transfection within Figure 7 and corresponding text in Results page 8 (last sentences of the section) .

• The results in Figure 4 are not mentioned in the abstract or the discussion. It would be good if the authors discuss how the increase in α7 nAChR expression might affect the proliferation, motility, and/or calcium signaling of cancer cells. Additionally, the authors did not show if these effects could be blocked by MLA or another antagonist, or whether this upregulation is related to g-protein mediated signaling via α7 nAChRs. Further experiments to answer these questions would help to tie these results into the rest of the paper.

RESPONSE: We thank the reviewer for pointing this out and we have now incorporated a discussion of this on Page 9 (end of the second paragraph). As stated, alteration in nicotinic receptor expression at the cell surface is likely an important driver of calcium and other signaling responses that can support cancer cell growth, and the experiments suggested by the reviewer are clearly important. However, due to limitation in experimental resources, we are not able to perform these experiments currently. Furthermore, we would like to suggest that studies on the mechanisms that participate in the regulation of nAChR at the cell surface, fall outside of the scope of the current manuscript. We now mention this as an important direction in future study in the revised manuscript in the Discussion on Page 9 (end of the second paragraph).

• YM 254890 is mentioned in the abstract as a G protein blocker, but in the results section of the manuscript, the authors never define what specifically YM 254890 is or why it is being used.

RESPONSE: The correction is now included on Page 7, last sentences of the 3rd paragraph within the Results section. The clarification is made that YM254890 is a Gq inhibitor and used to confirm Gq involvement.

• The proliferation assay in Figure 7 plots Cell count x 105, whereas the other proliferation assays measure Net OD. Could the authors please explain why this assay was quantified differently?

RESPONSE: Proliferation assays in the manuscript were performed in the laboratory of Dr. Oz however those presented in Figure 7 were performed in the lab of Dr. Kabbani. The two assays are virtually identical (i.e., using the same cell line and plating and MTT counting protocol). The results are also highly robust and show a comparable effect of choline on MCF-7 cells proliferation when comparing % change in cell count relative to the control group. To clarify this in the revised manuscript a newly added Figure 7 C shows cell count results as “% control (vehicle)” as done in earlier figures.

• There are two “B” panels in figure 5

Response: This is now corrected.

• The deltaF/F scales are not consistent across figures. For example, in Figure 5, A and C plot deltaF/F (x100), whereas panel B1 writes out the 100s, and the second panel B just has deltaF/F without the x100 clarification. Similarly, in Figure 7, panel A, the first graph is x100, while the second graph is not. Scale bar should be defined in Figure 7B.

Response: These figures are significantly revised to correct for these errors.

• Scale bars should be added to Figure 3A and Figure 4A

Response: The scale bars are now added into Figs. 3 and 4.

• In the figure legend for Figure 6 it is noted that the “Asterix denotes significant difference from control,” whereas the figure seems to indicate that the Asterix represents significant difference from the choline treated group. Could the authors clarify what comparisons are being measured in this figure, and what is significantly different from what?

Response: Thank you for pointing out this error. We have revised the legends for figures 2, 6, and 7 with the new legends clearly stating which groups are used for statistical comparison.

• The figure legend for Figure 2 states “Asterix denoted significant difference from control” but the figure seems to indicate that significance is measured as difference from the choline group.

Response: This is now corrected.

• Figure 7 has only 2 panels, A and B, but the figure legend has A, B, and C.

Response: This is now corrected.

• Figure 7 states “statistical significance relative to the control or α7 transfected group.” This is confusing. Could the authors clarify what comparisons are being made and what significance is being denoted. Does this mean that the α7345-348a group is significantly different from both the control and α7 group? If so, this should be denoted by two different symbols.

In the text, the mutant α7 is called “α7345-348a” whereas in the figure legend and figure it is “α7345-8a”, this nomenclature should be consistent throughout the paper

Response: This is now corrected.

• In the figure legend for Figure 5, the definition of a double Asterix is given, but there is no double Asterix in Figure 5

Response: This is now corrected.

Reviewer #2: The paper by Oz et al, shows that in MCF-7 cells activation by choline of α7 receptor increases

proliferation, motility, and calcium signaling and this is partially blocked by the application of G protein blocker, Gαi/o inhibitor and by the presence of a G protein binding dominant negative α7 subunit (α7345-348A). The work is interesting and well done, and it demonstrated that the α7 subtype is involved in mediating choline induced proliferation, motility, and calcium signalling.

• However the effect of choline was blocked using antagonists that act on both α7 and α9 subtypes. Why the authors did not used the α7 selective antagonist AR (Whitaker et al. 2007) and the α9 selective antagonist RGIA4 (Romero et al 2017) ? This certainly will help in clarifying the subtypes involved and their contribution to the effects report.

Response: α-Conotoxin RgIA has higher potency for α9/α10 than α7 nAChRs (Ellison et al., 2008), and was thus chosen in complementary experiments to α bungarotoxin. The use of additional pharmacological ligands with greater selectivity, as suggested by the reviewer, is definitely important and to that end this point is now stated in the Discussion on Page 10, last two sentences.

Minor points: In the abstract it is written "Nicotine activates various nicotinic acetylcholine receptors (nAChRs)" and in the introduction it is written"Mammalian nAChRs assemble from α (α1–α6) and β (β2–β4) subunit combinations forming homo- or hetero-pentameric channels [9, 10].

• Nicotine does not activates a1 containing receptors and nicotine activates various nAChRs

that assemble from α (α2–α7, α9, α10) and β (β2–β4) subunit combinations.

• Fig 7: Please add C to the figure

Response: We thank the reviewer for pointing these mistakes and have corrected them within the revised manuscript.

Attachment

Submitted filename: Reviewer_Comments.docx

Click here for additional data file. (19.5KB, docx)

Decision Letter 1

Israel Silman

12 Jul 2023

α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling

PONE-D-23-15038R1

Dear Dr. Kabbani,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Israel Silman

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

**********

Acceptance letter

Israel Silman

17 Jul 2023

PONE-D-23-15038R1

α7 nicotinic acetylcholine receptor interaction with G proteins in breast cancer cell proliferation, motility, and calcium signaling

Dear Dr. Kabbani:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Israel Silman

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Cell surface labeling of nAChRs using BTX.

    MCF-7 cells were labeled with 100 nM BTX. Cell membranes were not permeabilized in these labeling experiments. Bottom panel shows a representative image of a labeled cell. Scale bar = 5μm.

    (TIFF)

    Click here for additional data file. (453.8KB, tiff)
    Attachment

    Submitted filename: Reviewer_Comments.docx

    Click here for additional data file. (19.5KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting information files.

    Articles from PLOS ONE are provided here courtesy of PLOS

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