Mitotic Spindle Apparatus Abnormalities in Chronic Obstructive Pulmonary Disease Cells

Author Manuscript Published OnlineFirst on July 12, 2020; DOI: 10.1158/1940-6207.CAPR-19-0557 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Mitotic Spindle Apparatus Abnormalities in Chronic Obstructive Pulmonary Disease cells:

A Potential Pathway to Lung Cancer

Jose Thaiparambil1*, Lingyun Dong2*. Diana Jasso1, Jian-An Huang3, Randa A. El-Zein1§

1Houston Methodist Cancer Center and Department of Radiology, Houston Methodist Research

Institute; 2 Department of Respiratory Medicine, Affiliated Wujiang Hospital of Nantong

University, Suzhou, 215200, China. 3 Department of Respiratory Medicine. The First Affiliated

Hospital of Soochow University, Suzhou, 215123, China.

* Contributed equally to this article

Running Title: Mitotic spindle apparatus abnormalities in COPD

§Corresponding Author:

Randa A. El-Zein, MD, PhD

Scientist, Houston Methodist Research Institute Professor, Department of Radiology, Cancer Center Adjunct Professor, Department of Epidemiology The University of Texas MD Anderson Cancer Center R11-113 670 Bertner Ave Houston, TX, 77030 Office: 713-441-6062

Downloaded from cancerpreventionresearch.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 12, 2020; DOI: 10.1158/1940-6207.CAPR-19-0557 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract Chronic obstructive lung disease (COPD) is a long-term lung disease characterized by

irreversible lung damage resulting in airflow limitation, abnormal permanent air-space

enlargement and emphysema. Cigarette smoking is the major cause of COPD with 15% to 30%

of smokers developing either disease. About 50% - 80% of lung cancer patients have pre-

existing COPD and smokers who have COPD are at an increased risk for developing lung

cancer. Therefore, COPD is considered an independent risk for lung cancer, even after adjusting

for smoking. A crucial early event in carcinogenesis is the induction of the genomic instability

through alterations in the mitotic spindle apparatus. To date the underlying mechanism by which

COPD contributes to lung cancer risk is unclear. We hypothesized that tobacco smoke

carcinogens induce mitotic spindle apparatus abnormalities and alter expression of crucial genes

leading to increased genomic instability and ultimately tumorigenesis. To test our hypothesis, we

assessed the genotoxic effects of a potent tobacco-smoke carcinogen (4-(methylnitrosamino)-1-

(3-pyridyl)-1-butanone, [NNK]) on bronchial epithelial cells from patients with COPD and

normal bronchial epithelial cells and identified genes associated with mitotic spindle defects and

chromosome missegregation that also overlap with lung cancer. Our results indicate that

exposure to NNK leads to a significantly altered spindle orientation, centrosome amplification

and chromosome misalignment in COPD cells as compared to normal epithelial cells. In

addition, we identified several genes (such as AURKA, AURKB and MAD2L2) that were upregulated and overlap with lung cancer suggesting a potential common pathway in the

transition from COPD to lung cancer.

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity worldwide affecting about 8% to 10% of the general adult population. In the United States, approximately

11 million people are diagnosed with COPD and a similar number of people remain undiagnosed

(1). The World Health Organization predicts that by 2030, COPD will become the third leading cause of death and the fifth cause of disability worldwide.

COPD is characterized by irreversible lung damage resulting in airflow limitation, abnormal permanent air-space enlargement and emphysema. Increased oxidative burden and release of reactive oxygen species, triggers inflammatory processes leading to airway obstruction. Once begun, the inflammatory response is maintained and progresses indefinitely. The inflammation is accompanied by a continual cycle of DNA damage and repair, and a higher rate of cell turnover increases likelihood for genetic errors. Such chronic inflammatory reaction, has been implicated in lung cancer pathogenesis (2). Similar to lung cancer, cigarette smoking is the major cause of

COPD with 15% to 30% of smokers developing either disease. It has been reported that the prevalence of COPD is about 20% in the general smoking population, and between 50%-80% of lung cancer patients have preexisting COPD (3,4,5). In addition, studies consistently have shown that smokers who have COPD are at an increased risk for developing lung cancer (6). Therefore, in addition to being a major cause of death and disability, COPD is also an independent risk for lung cancer, even after adjusting for smoking. Moreover, Wang et al., using mediation analysis methods, reported that COPD is a mediating phenotype that explains part of the effect of smoking exposure on lung cancer (7).

Cigarette smoke is estimated to contain 7,357 different chemical compounds, with at least 70

known carcinogens including polycyclic aromatic hydrocarbons, aromatic amines and N-

nitrosamines among other lung carcinogens, tumor promoters, and co-carcinogens (8). Tobacco- specific nitrosamines are found in high concentrations in mainstream smoke (8). The most potent

carcinogenic member of this group, is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

(9). NNK forms DNA adducts by inducing deleterious mutations in oncogenes and tumor suppression genes, which could be considered as tumor initiation (10). NNK is known to activate

phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB/AKT) (11) protein kinase C (PKC)

(12), nuclear factor kappa B (NF-κB) (13), and other signaling pathways (14), all of which promote cell proliferation, survival, and angiogenesis, contributing to the development of smoking-associated lung cancers. Our group has previously shown a differential sensitivity to the genotoxic effects of NNK, in a lung cancer case control study, using the cytokinesis blocked

micronucleus assay for assessing genomic instability (15,16,17). A crucial early event in

carcinogenesis is the induction of the genomic instability phenotype, which enables an initiated

cell to evolve into a cancer cell by achieving a greater proliferative capacity. Alterations in

mitotic spindle apparatus play a major role in the generation of genomic instability through

promoting chromosome mis-segregation and aneuploidy (18). In addition, it has recently been

reported that NNK increases chromosomal instability by disrupting spindle microtubule

attachment to the kinetochore (KT) and spindle dynamics (19). We have previously assessed the

extent of genomic instability among smokers with COPD as compared to smokers with lung

cancer and cancer-free COPD-free smokers and reported that COPD smokers harbored

intermediate levels of genomic instabilities as compared to the other two groups (20). To date it

is unclear how COPD contributes to lung cancer risk or whether both COPD and lung cancer are

the result of common underlying exposures. To address this gap in knowledge gap, we assessed the genotoxic effects of NNK on bronchial epithelial cells from patients with COPD as compared to normal bronchial epithelial cells and identified genes associated with mitotic spindle defects and chromosome missegregation that also overlap with lung cancer and could therefore play a significant role in the transition from COPD to lung cancer. We hypothesize that NNK induces mitotic spindle abnormalities and alters expression of crucial spindle apparatus associated genes in COPD cells leading to increased genomic instability that may ultimately lead to tumorigenesis.

Materials and Methods

Cells and cell culture: Normal human bronchial epithelial cells (Beas-2B) were purchased from

ATCC® (Cat# ATCC-CRL-9609). Chronic obstructive pulmonary disease cells (COPD) were purchased from Lonza® (Cat.# 195275). Both cell lines were authenticated from ATCC and

Lonza respectively, and obtained in 2019 and used first few passages (3-5). Both cell lines were grown in a special media, BEGM bullet kit (CC-3170-Lonza) containing 0.5 ng/ml human recombinant epidermal growth factor, 50 μg/ml bovine pituitary extract, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10 μg/ml transferrin, 0.5 μg/ml epinephrine, 0.1 ng/ml retinoic acid, and 6.5 ng/ml tri-iodothyronine. Before plating Beas-2B cells, the flasks/dishes/plates were coated with a mixture of 0.01 mg/ml fibronectin (Sigma®), 0.03 mg/ml PureCol (Sigma®), and

0.01 mg/ml bovine serum albumin (Sigma®) dissolved in BEBM at 37°C for at least 2 h. After vacuum aspirating the mixture to dry, Beas-2B cells were cultured in BEGM completed growth medium containing the same components as mentioned above. COPD cells were plated directly

without any additional steps. Cells were then incubated at 37°C in 95% air and 5% CO2 atmosphere until reaching 80% confluence.

XTT assay: Beas-2B and COPD cells were seeded into 96-well plates at a density of 1×104 cells/well. Cells were treated with NNK at different concentrations for 48 and 72h time points.

After incubation 50 μl of XTT labeling mixture (ATCC # 30-1011K) was added to each well.

Finally, the optical density was measured at 490 nm with a reference wavelength at 650 nm in a microplate reader (Biotech®, plate reader). Triplicate wells were used for each treatment.

Cell synchronization and NNK treatment: To synchronize Beas-2B and COPD cells in mitosis, a double thymidine block was used. Cells were incubated in thymidine (2.5 mM) for 24 h at 37°C and released from thymidine for 14 h, and treated with 2.5 mM thymidine for another

24 h. Beas-2B and COPD cells were treated with 100nM NNK [Sigma® cat # B-1760] for 2 h.

Cells were then washed with PBS and allowed to grow for 48, and 72 h. NNK concentration was selected on the basis of our cytotoxicity study using XTT (ATCC) (Fig S1) and reported literature (21). DMSO (0.1% of final volume) was used as a control in all experiments. The treatment was conducted under low light and the experiment was repeated three times. The mitotic abnormalities were examined at each time point and the results compared to those of untreated cells (control).

Immunofluorescence and antibodies for spindle apparatus detection: Immunofluorescence was performed following standard protocols (22). Briefly, cells were allowed to adhere overnight on no.1.5 coverslips placed in tissue culture plates. Cells were fixed in PHEMO buffer,

consisting of 3.7% formaldehyde, 0.05% glutaraldehyde, 60 mM PIPES [piperazine-N,N'-bis(2- ethanesulfonic acid)], 25 mM HEPES, 10 mM EGTA, and 4 mM MgSO4, for 10 min. Cells were then washed in PBS and blocked with 10% bovine serum albumin for 15 min. Coverslips were then incubated with the appropriate primary antibody overnight at 4°C. Cells were washed again with PBS, and Alexa Fluor-conjugated secondary antibody was added at 1:500 for 1 h at room temperature. Antibody incubations were then sequentially repeated for each additional primary- secondary-antibody pair.

Primary antibodies were used as follows: Monoclonal mouse anti-α-Tubulin 1:1,000 (Sigma #T-

9026) and rabbit polyclonal anti-pericentrin (Abcam, # ab4448). Secondary antibodies were

Alexa Fluor 488 or 555 (Invitrogen®) at a dilution of 1:500 and incubated for 1 h at room temperature. Nuclear staining was performed by incubating cells with DAPI (4′, 6-diamidino-2- phenylindole) containing mounting media (Vectashield®).

Centrosome amplifications: We used pericentrin antibody to stain centrosomes or centrioles

(mentioned above). The number of centrioles in 100 mitotic cells were recorded using confocal microscopy and the number of cells having two or more centrosomes were noted. Mitotic cells with engaged centrioles have 2 centrosomes with 2 centrioles each, whereas cells with evidence of amplification either have 3 centrosomes or 4 centrosomes each with a single centriole (23).

Confocal imaging

Image acquisition and analysis: Fluorescence image acquisition was performed using a Nikon

A1R confocal imaging system with an oil immersion Plan-Apo _60 numerical aperture 1.40 lens

(Nikon) controlled by the Nikon NIS Elements software (Nikon®). The Images were acquired as

Z-stacks at 0.2-mm intervals and maximum-intensity projections were generated using the NIS

Elements software (Nikon®). Confocal z stacks were acquired with sections ranging from 0.5 μm

to 1.1 μm. Image acquisition settings were constant throughout the experiments.

Spindle orientation, centroid measurements, and image analysis: Spindle angle

measurements were adapted from previously published method (24). Briefly, the three-

dimensional (3-D) distance (across the x, y, and z planes) between the two spindle poles and the

2-D distance (across the x and y planes) of the spindle were measured. The spindle angle was

then calculated using the cos−1 (arccosine). Image analysis was done using confocal z stacks with

Nikon NIS Elements software (Nikon®), using the 3-D length tool.

Real-Time PCR: In order to identify the genes associated with mitotic spindle defects and

chromosome missegregation, we used a custom designed 96 well plate for selected mitotic

spindle associated genes in duplicates (Fig S2) on a CFX96 Touch Real-Time (RT) PCR

Detection System (Bio-Rad®). Total RNA was extracted from both BEAS-2B and COPD cells

(Qiagen-RNAeasy plus mini kit-(Qaigen Kit® Cat#74104) according to the manufacturer’s

instructions. RT-PCR was performed using the iScript cDNA synthesis kit (BioRad

Cat#1708841) using 500ng RNA and Sso advancedTM Universal SYBR® Green Supermix (Cat#

172-5271). The following cycle was used: 95 °C for 10min (1 cycle), 95 °C for 15s, 60 °C for 1 min, 95 °C for 15s for 40 cycles followed by 95 °C for 15s and 60 °C for 1 min. Specificity of the PCR products were confirmed by melt curve analysis. Data were normalized to Ct values of

GAPDH and Actin from the same sample and the fold-changes in the expression of each gene were calculated using the ΔΔCt method. Analysis of data was performed using CFX Manager

Software (Bio-Rad®).

Protein extraction and Western blot analysis: To further confirm the RT-PCR results, we performed western blot analysis on a selected panel of gene products identified from the PCR arrays. Cells were lysed and centrifuged. Samples containing equal amounts of protein (50 μg) were separated by SDS-PAGE and electroblotted onto PVDF membrane (Bio-Rad). All antibodies for Immunoblotting were purchased from Abcam®. After incubation with enhanced chemiluminescence, the membranes were exposed to X-ray film. Each Western blot analysis was repeated at least 3 times.

RNA interference with small interfering RNAs (siRNAs) For FOXM1 silencing, Beas-2B and

COPD cells were transiently transfected with siRNA SMART pool reagents purchased from

(Thermo Scientific Dharmacon) using the transfection reagent Lipofectamine (Thermo Fisher

Scientific) according to the manufacturer's instructions. SMART pool siRNAs used were: siRNA

FOXM1 (L-009762-00-0020) and the non-specific (NS) control siRNA.

Statistical analysis: Statistical analyses were conducted using Prism 7 (GraphPad® Software

Inc.). Two-way ANOVA analysis was used to compare the NNK treated and untreated experimental conditions. Bonferroni’s multiple comparison test was used to analyze the significance at each experimental time point. Two-tailed unpaired t test was used to compare spindle angle. Differences were considered significant with a P-value<0.05.

Results

NNK induces aberrant amplification of centrosomes during mitosis [at interphase &

metaphase]

Since accurate chromosome segregation requires two centrosomes to move towards opposite

poles, we assessed the effect of NNK on centrosomes using confocal immunofluorescence

analysis and by counting the number of pericentrin dots in interphase and metaphase cells. NNK

exposure induced more than two centrosomes in Beas-2B and COPD cells at 48 and 72 hr

indicating that abnormal amplification occurred in the presence of NNK (Fig 1 A, B, C and D).

The quantitative data (Fig 1 E) shows that in NNK exposed cells, 40% of COPD cells had

centrosome amplification at 72 h as compared to 25% of Beas- 2 B cells at interphase. Similarly,

NNK treated metaphase cells showed 60 % centrosome amplification in COPD cells as

compared to 40% in Beas-2B cells (Fig 1 F).

Recent studies (25) reported that aberrant centrosome amplification maybe due to up regulation of PKL4. Results from our western blot experiments show that NNK induces PLK4 at 48 and 72

hr in COPD cells. While Beas-2B cells exhibited PLK4 increase only at 48 hr followed by a

decrease at 72 hr (Fig 1 G and H). Our data suggest an increased expression of PLK4 that

persists in COPD cells.

NNK induces misaligned chromosomes and spindle misorientation at the metaphase

Chromosome alignment at metaphase is highly critical to maintain a proper spindle orientation

for faithful chromosome segregation. Using confocal microscopy we assessed the effect of NNK

on chromosome alignment and spindle orientation in COPD and Beas-2B cells. In untreated

cells, over 90% of the cells recorded for both the COPD and Beas-2B cells were properly aligned

(92% and 95 %, respectively). Figure 2A shows the confocal z sections with a higher proportion

of severe misaligned chromosomes in COPD cells compared to Beas-2B cells. Following NNK

exposure, the COPD cells showed a significantly higher percentage of misaligned cells (Fig 2B)

(38% and 58% at 48 and 72 h, respectively) as compared to the Beas-2B cells (21% and 38 % at

48 and 72 h, respectively).

Since chromosome misalignment cause spindle misorientation, we further examined confocal planar z sections of tubulin in vehicle and NNK treated COPD and Beas-2B cells. Figure 2C shows the bipolar spindle that is oriented parallel to the substratum, with spindle poles in similar z planes. In contrast, NNK treated mitotic cells had bipolar spindles that appeared to be misoriented relative to the substratum, whereby spindle poles were in drastically different confocal z planes.

To determine the extent of spindle misorientation, we quantitated the angle of spindle rotation in

NNK treated versus control cells using a standard method (24). Perfectly aligned spindle poles

would have 0° of rotation with a spindle oriented perpendicular to the substratum would have

90° of rotation. Figure 2 D&E, show that the NNK treatment lead to a significantly misoriented

spindle in both COPD and Beas-2B cells as compared to untreated cells (31.5° and 27.6 °

compared to 14.9° and 17.6°, respectively (P < 0.001, Fig 2 D&E)). In addition, the data shows

that the  change in angle misorientation over control is larger in the COPD treated cells (16 °) as compared to the Beas-2B (10°) treated cells (p < 0.0001).

Effect of NNK on mitosis and DNA damage response

Since NNK induces metaphase misalignment that could interfere with mitotic spindle assembly,

we tested the protein levels of several genes involved in spindle assembly and orientation.

Aurora A showed a marked increase in expression in both Beas-2B and COPD cells at 48 and 72

hrs (Fig 3A). In Beas-2B cells, Aurora B increased only at 48 hrs as compared to COPD cells which showed an increase expression at both 48 and 72 hrs (Fig 3A). Although modest, PLK1

[which plays a major role in spindle orientation] was down regulated in response to NNK

treatment in COPD cells at 72 h as compared to Beas-2B where PLK1 was not affected (Fig 3A

& B). Our real time PCR data showing significant increase of both Aurora A and Aurora B at 72

hr time point (>2 fold) in COPD cells correlated well with our western blot data. Assessing the

level of CHK1 which is crucial to S-to-G2/M phase transition (26), we observed a modest up regulation at 48 and 72 h in COPD cells compared to Beas-2B where it decreased at 72 h (Fig 3A

&B). The overexpression of CHK1 suggests a delay in mitosis in response to NNK in COPD cells. A similar increase of CHK1 was noticed in Beas-2B cells only at 48 h time point (Fig

3A&B).

Since it has been shown that alterations in mitosis causes DNA damage, we assessed the extent

of DNA damage in NNK treated cells using γ-H2AX expression at 48 and 72 h. We observed

persistent hyper-phosphorylation of γ-H2AX in COPD cells (Fig 3C) as compared to Beas-2B

cells, indicating a defective repair mechanism in COPD. Since the γ-H2AX phosphorylation depends on the nucleotide excision repair factor XPC (27), we assessed the levels of XPC1 and

observed a similar persistent increase at 48 and 72 h in COPD cells in as compared to Beas-2B

(Fig 3 C) cells that showed a decrease at 72 hrs.

Centrin-2 has been shown to bind to XPC (alpha helical region at the C-terminal) and enhances

the binding capacity of XPC to DNA damage recognition sites. Figure 3D shows persistent

increase in centrin-2 levels at 48 and 72 h time points in COPD cells in contrast to Beas-2B,

where centrin-2 expression was not evident at 48 h and later it increased at 72 h.

Effect of NNK on key genes involved in genomic stability

In order to define the molecular underpinnings of NNK induced mitotic abnormalities in COPD,

we used RT-PCR custom designed plates to determine the changes in expression of key genes

associated with mitotic spindle apparatus and chromosome segregation. Figure 4, shows the key

genes that were altered in response to NNK treatment in COPD and Beas-2B cells. Figure 4A

and B shows the genes that were unique to COPD and Beas-2B respectively. Also Fig S3 shows

the fold change of all the genes for Beas-2B and COPD.

Upregulation of three mitotic spindle associated genes, DCTN6, DYNC112 and MAD2L2 was

only seen in COPD cells. On the other hand, upregulation of CLASP2 and down regulation of

TUBB4A genes was only observed in Beas-2B cells (Fig: 4B). We further confirmed the

expression of MAD2L2 in COPD and a down regulation of TUBB4A in Beas-2B cells by our

western blot analysis (Fig 4C).

Overlap between COPD and Non-Small Cell Lung Cancer

Since a significant percentage of lung cancer patients have pre-existing COPD, we used our real

time PCR data to identify genes with altered expressions in both COPD and non-small cell lung

cancer both (adenocarcinoma-H2342) and squamous cell carcinoma (H1703). Figure 4D shows the gene expression profile depicting genes common between COPD and NSCLC cell lines

[Adeno and Squamous]. There were 8 and 12 common genes identified between COPD and

adeno and squamous cell carcinomas respectively. We identified the genes that were upregulated

in both COPD and NSCLC and were involved with mitotic spindle apparatus abnormalities in

both diseases. Aurora kinases A&B and CCNB1 (cyclin B1) were upregulated in COPD cells as well as in both adeno and squamous cell carcinoma cell lines suggesting that these genes may be

critical in transitioning from COPD to lung cancer.

FOXM1 as a regulator that controls spindle orientation metaphase chromosome alignment

To determine the potential pathway that controls spindle orientation and chromosome alignment,

we used Ingenuity Pathway analysis software (IPA-Qiagen) to define the upstream regulator of

aurora kinases. We used two scores an ‘enrichment’ score [Fisher’s exact test (FET) P-value]

that measures overlap of observed and predicted regulated gene sets, and a Z-score assessing the

match of observed and predicted up/down regulation patterns to determine FOXM1 as an

upstream regulator (Fig 5A). Our western blot showed an upregulation of FOXM1 in COPD

cells exposed to NNK at 48 and 72 h, in contrast to Beas-2B where, FOXM1 increased only at 48

h and then decreased (Fig 5B). To further confirm the role of FOXM1, we performed a siRNA

knockdown of FOXM1 in the presence and absence of NNK and found that aurora kinases were

downregulated in either conditions at 72 h (Fig 5C).

Discussion Proper functioning of mitotic spindle apparatus is essential for development, cell fate, and tissue

organization by ensuring an accurate distribution of genetic material and a normal division plane.

The centrosome duplicates during the S phase to yield two centrosomes that instruct formation of

the bipolar spindle. Formation and correct positioning of a bipolar mitotic spindle is essential for

correct chromosome alignment and subsequent segregation (28). Alterations in the regulatory

mechanisms that govern centrosome duplication result in centrosome amplification, which could

lead to aberrant mitoses and chromosome segregation errors (29). Centrosome amplification has

been reported in solid and hematological cancers (29,30). In addition, amplified centrosomes

have been reported in both early and late stages of tumorigenesis and are associated with poor

prognosis (31, 32).

Our data shows that centrosome amplification was significantly higher in NNK treated COPD

cells [in both interphase and metaphase] as compared to Beas-2B cells. Polo like Kinase 4

(PLK4) plays a major role in controlling centriole duplication and it has been shown that

elevated PLK4 expression results in the centrosome amplification (33, 34), abnormal mitosis and chromosomal instability all of which are hallmarks of carcinogenesis. In our study, NNK exposure induced PLK4 upregulation that correlated with significantly higher levels of

centrosome amplification observed at 72 hours in COPD cells in contrast to Beas-2B. These

results are consistent with a report (35) showing similar overexpression of PLK4 triggers

aberrant centrosome amplification at the mitosis and suggest a role for PLK4 with genetic

instability and carcinogenesis in COPD cells.

Aurora kinases play a major role in spindle assembly and chromosome segregation (36). It has

been recently reported that aurora A localizes to the duplicate centrosomes from the beginning of

S phase and shifts to the bipolar spindle microtubules during mitosis while aurora B, is required

for correct chromosome segregation and cytokinesis (37). In our experiments we observed a

significantly higher number of misaligned chromosomes at metaphase in NNK treated COPD

cells as compared to Beas-2B cells. Real-time PCR analysis showed an overexpression of both

aurora A and aurora B in COPD cells which was further confirmed using western blots. Beas-2B

cells failed to show an alteration of aurora A and aurora B based on the real time PCR results

although western blot showed an overexpression of aurora A at 48 and 72h and aurora B only at

48 and then decreased at 72 h. This may imply that misaligned chromosomes at metaphase in

COPD could be due to a sustained overexpression of aurora B at 48 and 72 h. This observation

was further supported with our immunofluorescence data showing a significant increase of

misalignment at 72h in COPD cells. We observed an increase of misaligned chromosomes at 48

hr in Beas-2B cells followed by a decrease at 72 hr consistent with our western blot data. The

initial increase of misaligned chromosomes in Beas-2B cells may be due to elevated level of

aurora B at 48 h.

Since alterations in mitotic apparatus may cause DNA damage, we assessed the extent of DNA

damage using γ-H2AX as a marker (38). Histone H2AX, one of several variants of the

nucleosome core histone H2A becomes phosphorylated on serine 139 in response to DNA

damage that involves formation of DNA double strand breaks (γ-H2AX) and is widely used as a sensitive double strand break (DSB) marker. Incorrect repair of DNA may give rise to genomic instability, therefore, the recognition and repair of DSB is of critical importance to mitosis. In our study, we observed a sustained increase level of γ-H2AX at 72 h in COPD cells as compared to Beas-2B cells. It has been reported that γ-H2AX phosphorylation after UV exposure in the G1 phase of the cell cycle largely depends on the nucleotide excision repair factors XPA and XPC

(39). In our study, we observed a steady increase in XPC levels in the COPD cells which correlated well with the increase in γ-H2AX. Both γ-H2AX and XPC increased at 72 h in response to NNK in COPD cells, suggesting a role for XPC in the formation of γ-H2AX. XPC levels were sustained in the Beas-2B cells and did not correlate with the decreasing levels of γ-

H2AX suggesting a more efficient damage repair mechanism in the Beas-2B cells as compared

to the COPD cells. In addition, we observed the same pattern of response with the centrin -2

(that has been shown to augment the DNA damage-recognition activity of XPC) in COPD in

contrast to Beas-2B cells where it increased only at 72 h.

Given the extent of spindle apparatus alterations and DNA damage observed in the COPD cells

and given that between 50%-80% of lung cancer patients have pre-existing COPD, we decided to

determine the overlap in mitotic spindle regulation genes in COPD as compared to non-small cell

lung cancer cell lines. Our real time PCR analysis showed overexpression of AURKA, AURKB

and CCNB1 in COPD as well as in adenocarcinoma and squamous cell carcinoma cells (Fig 4

D). In addition, we observed an overlap between COPD and squamous cell carcinoma cells in terms of MAD2L2 overexpression. We further used the Ingenuity Pathway Analysis (IPA)

software (Qiagen®) to generate a network connecting the genes of interest and to determine the

overlap between COPD and non-small cell lung cancer within these networks. Our results

identified the top canonical pathways as: 1) Mitotic roles of polo-like kinase (14.3% overlap, p

value-6.56E-7); 2) G2/M DNA damage checkpoint regulation (10.2% overlap, p value-5.14E-

09); 3) DNA damage induced 14-3-3α signaling (15.8 % overlap, p value-2.00E-06); 4) ATM signaling (4.2 % overlap, p value-7.56E-06), and 5) Cyclins and cell cycle regulation (3.8% overlap, p value-1.61E-06) We then used the causal network analysis from IPA to connect to the upstream regulators to generate a more complete picture of possible contributors to the observed

expression changes. Our data identified FOXM1 as an upstream regulator of aurora A and B which has been recently reported to recruit aurora kinase A (40). Our siRNA knockdown of

FOXM1 in the presence and absence of NNK led to downregulation of the aurora kinases suggesting that FOXM1 may interact directly with aurora B in the mitotic spindle leading to

chromosomes misalignment at metaphase and inducing genomic instability.

It has been reported that FOXM1 is a key downstream effector of the PI3K-AKT, ATM/p53-E2F and p38-MAPK-MK2 signaling cascades (41,42,43), which also induces overexpression of both

AURKA and AURKB, affecting chromosome alignment, mitotic spindle assembly and bipolar spindle orientation further supporting the proposed pathway. FOXM1 may also act through

PLK4 that was upregulated in our COPD cells and plays role in aberrant centrosome amplification inducing genomic instability.

In summary, our study identified a novel pathway in COPD where aurora kinases are regulated by FOXM1 and overexpression could result in genomic instability and potential tumorigenesis

(Fig 5). To our knowledge, this is the first report describing the aurora kinases overexpression in

COPD. These findings are promising since they provide a potential avenue for cancer prevention among COPD patients. Aurora kinases inhibitors have been shown to be effective in suppressing cell proliferation and progress of many cancers (44,45), and could serve as a target to reduction of genetic instabilities among COPD patients.

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Legend to Figures Fig: 1 Effect of NNK on centrosome. Immunofluorescence images of centrosome staining in two different cell lines in interphase and metaphase stages of cell cycle. Beas-2B cells (panel A) and COPD cells (panel B) with single centrosome staining (upper panel) and abnormal number of centrosomes (>1) in response to NNK exposure (lower panel). Merged images show centrosome and DAPI staining (scale bar = 10 μm). 1 C and 1 D show two stained centrosomes at the mitotic poles (upper panel) and multiple stained

centrosomes (lower panel) in both Beas-2B and COPD cells, respectively. 1 E and 1 F represent the quantitative data for abnormal centrosomes in both interphase and metaphase, respectively in both cells. The percentage of abnormal centrosomes were higher in COPD compared to Beas-2B in both interphase and metaphase (*p<0.01, ***p<0.0004, respectively). 1 G, Western blots showing an increase in PLK4 in COPD at 72 h as compared to Beas-2B which decreased after 48 hrs. 1 H, mean intensity of PLK4 increases from 48 to 72 hr. in COPD in contrast to Beas-2B, which decreased after 48 hrs. Fig: 2 Effect of NNK on chromosome alignment. 2 A, normal metaphase with all chromosomes aligned at the metaphase plate for the vehicle treated control (upper panel) and misaligned chromosomes in response to NNK exposure in both Beas-2B and COPD cells (lower panel). 2B, Quantitative data for the misaligned chromosomes in metaphase in response to NNK exposure. Chromosome misalignment gradually increased from 48 to 72 h (****P<0.0001). 2C, confocal z sections showing vehicle and NNK treated spindles beginning at the top of the spindle and sectioning to the bottom. Numbers in the top right indicate the z distance from the top. Arrows indicate spindle poles (scale bar = 5 μm). Untreated Beas-2B and COPD cells showed two poles at z sections 1.76 and 7.76 respectively, compared to NNK treated, where two poles did not appear in the same sections rather in different z sections with first pole at 1.17 and second pole at 5.87 for Beas-2B and 6.57 and 9.55 for COPD. 2 D and 2 E, Comparison of the mean angle of rotation in vehicle treated and NNK exposed cells. The angle of rotation was significantly higher in COPD compared to Beas-2B cells (***, P < 0.001). The  change in angle misorientation over control is larger in the COPD treated with NNK compared to Beas- 2B (p<0.0001) Fig; 3 Effect of NNK on mitotic genes. 3A & B shows the changes in the level of key proteins that control the mitotic apparatus. Aurora A is upregulated in response to NNK in both Beas-2B and COPD cells at 48 and 72 h in contrast to aurora B, which showed an initial increase at 48 h followed by a decline at 72 h in Beas-2B cells. Whereas aurora B showed a gradual increase from 48 h and reached a plateau at 72 h in COPD cells. PLK1 increases at 48 h and later decreased in COPD cells compared to Beas-2B, where, it exhibited a gradual increase at 48 and 72 h time points. The COPD cells showed a gradual increase of CHK1 at 48 and 72 h in response to NNK exposure, whereas not much change in CHK1 expression in Beas-2B cells at 48 and 72 h. 3C shows the persistent increase of Gamma H2AX at 48 and 72 h in COPD cells in contrast to Beas- 2B, where it decreased at both time points. The level of XPC-1 gradually increased from 48 to 72 h in COPD cells in contrast to Beas-2B, where both decreased at 72 h time point. 3D centrin-2 gradually increased from 48 to 72 h in COPD cells in contrast to Beas-2B, where centrin-2 increased only at 72 h time point. Fig: 4 Effect of NNK on gene expression. Beas-2B and COPD cells were treated with 100nM NNK for 2 h and then washed with PBS and allowed to grow for 48, and 72 h. The treatment was conducted under low light and the experiment was repeated three times.

4A Real-time PCR analysis of NNK exposed COPD and Beas-2B shows several unique genes specific for each cell line. 4A, AURKA and AURKB increased more than 10 folds in COPD in response to NNK. In addition, MAD2L2, DCTN6 and DYNC112 showed a two fold increase. 4B, CLASP2 and HSP90B1 were upregulated and unique to Beas-2B while TUBB4A was downregulated in response to NNK exposure in Beas-2B cells. 4C, Western blot confirmed the RT-PCR data with an increase of MAD2L2 at 72 hr in COPD and a decrease of TUBB4A in Beas-2B and an increase TUBB4A in COPD. 4D, Overlap in upregulated genes between NNK exposed COPD cells and lung adenocarcinoma and squamous cell carcinoma cell lines (analysis between the different cell lines was conducted independently). AURKA, AURKB and Cyclin B1 were all upregulated in adeno carcinoma (3.5, 5.3 and 11.1, respectively) and (2.5, 11 and 6.7, respectively) for squamous in both lung cell lines and NNK treated COPD cells (12.7, 31 and 3.2, respectively). Fig: 5 A: Proposed pathway for regulation of chromosome misalignment and mitotic spindle orientation. FOXM1 as a master regulator that directly activates Aurora B and indirectly act on Aurora A through STMN1 and PLK1. In addition, it activates PLK4 and KIF20A was identified using casual network analysis. B: FOXM1 expression Beas-2B and COPD cells at 48 and 72 h; C: The western blot shows FOXM1 expression in siRNA knockdown at 48 and 72 h time points in the presence and absence of NNK in both Beas- 2B and COPD cells. Knock down of FOXM1 at 72 h showed a corresponding decrease of Aurora A and B in both COPD and Beas-2B cells indicating that FOXM1 acts through aurora kinases to control spindle orientation and chromosome misalignment.

Mitotic Spindle Apparatus Abnormalities in Chronic Obstructive Pulmonary Disease cells: A Potential Pathway to Lung Cancer

Jose Thaiparambil, Lingyun Dong, Diana Jasso, et al.

Cancer Prev Res Published OnlineFirst July 12, 2020.

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