ASPM induces radiotherapy resistance by disrupting microtubule stability leading to chromosome malsegregation in non-small cell lung cancer

doi:10.21203/rs.3.rs-2091604/v1

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ASPM induces radiotherapy resistance by disrupting microtubule stability leading to chromosome malsegregation in non-small cell lung cancer

https://doi.org/10.21203/rs.3.rs-2091604/v1

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Radiotherapy (RT) resistance remains a substantial challenge in cancer therapy. Although physical factors are optimizing, the biological mechanisms for RT resistance are still elusive. Herein, we explored potential reasons for this difficult problem by generating RT-resistant models for in vitro and in vivo experiments. We found that abnormal spindle-like microcephaly-associated protein (ASPM) was highly expressed in RT-resistant samples and significantly correlated with disease advance in lung adenocarcinoma. Mechanistically, ASPM helps RT-resistant cells to evade spindle checkpoint surveillance and complete cell division after irradiation through destruction of microtubule stability, with subsequent increases in chromosome mis-segregation and deteriorating chromosomal stability during mitosis. Depletion of ASPM stabilized microtubules and significantly decreased chromosome mis-segregation, rendering RT-resistant cells renew sensitive to radiation. We further found, with bioinformatics analysis, amino acid sequence 963–1263 of ASPM as a potential new drug target for overcoming RT resistance and identified 9 drug pockets within this domain for clinical translation. Our findings suggest that ASPM is a key regulator with an important role in promoting RT resistance in non-small cell lung cancer, and that suppressing or blocking its expression could be worth exploring as therapy for a variety of RT-resistant cancers.

ASPM

radiotherapy

radiotherapy resistance

chromosomal malsegregation

Radiotherapy (RT) is one of the main modes of treatment for non-small cell lung cancer (NSCLC) [1], with about 60% of all newly diagnosed cases requiring RT as first-line treatment [2]. Nevertheless, NSCLC in some patients is resistant to RT and is unlikely to respond to this treatment. Previous studies of RT resistance primarily focused on tumor-cell killing mediated by immune cells [3, 4], but changes in the tumor cells themselves that render resistance to RT have not been fully revealed. Moreover, validated biomarkers for response to RT in NSCLC are lacking [5, 6], and as such overtreatment with RT in RT-resistant disease contributes an unnecessary burden and contributes to treatment-induced morbidity [7–9]. Thus, additional information is needed on the molecular regulators of sensitivity to radiation, with detailed mechanistic studies to dissect the complexities of these interactions.

Many characteristics of cancer cells are lost or perturbed in RT-resistant cancer cells [10, 11], including tumor-cell survival after RT that may be related to improper segregation of chromosomes [12]. Defects in chromosome segregation lead to aneuploidy, which in turn leads to genomic instability via defects in mitosis, which is associated with disease progression [13]. Genomic instability is thought to increase the likelihood of generating combinations of chromosomes that confer a selective growth advantage [14]. Although mitotic cells have ways of ensuring correct segregation of chromosomes, those pathways may not be fully operational in RT-resistant tumor cells [15]. Intrigued by this, we wanted to know whether and how these RT-resistant cancer cells achieve incorrect chromosome segregation.

Although a growing body of evidence underscores the importance of cytokinesis in chromosome segregation [16–19], whether cytokinesis has a significant role in the biology of RT resistance is still an open question. Cytokinesis causes the physical separation of the cytoplasm of a mother cell into two daughter cells, with each having the correct complement of chromosomes. This process depends on having a stable microtubule cytoskeleton and correspondingly on the integrity and function of the spindle complex [20–23]. Stable microtubules form a structurally symmetric spindle about metaphase-plate–positioned chromosomes during mitosis [24]. Essential to the chromosomal segregation process is that all chromosomes bi-orient on the mitotic spindle so that the two daughter cells receive equal sets of chromosomes as they divide. A cell model that can recapitulate RT resistance would be quite useful for functional investigations of cytokinesis in RT and resistance to clarify the contribution of RT resistance to disease progression.

In the present study, we report an RT-resistant non-small cell cancer (NSCLC) model with significant chromosomal segregation defects consistent with the results we found in human RT-resistant NSCLC. Specifically, we found that disordered levels of abnormal spindle-like microcephaly-associated protein (ASPM) can enhance RT resistance by affecting microtubule stability and increasing chromosome mis-segregation. Under physiological conditions, these RT-resistant cells can evade spindle-checkpoint surveillance and complete cell division after irradiation. Mechanistically, the dynamics of intracellular microtubule cytoskeleton formation and stability were disrupted, leading to spindle misorientation in mitotic cells that promoted cell survival by increasing chromosomal mis-segregation. We further identified, using bioinformatics analysis, that the amino acid sequence 963–1263 of ASPM could be an effective drug target to overcome RT resistance, and we found 9 drug pockets within this domain. Our findings revealed that chromosome mis-segregation can directly regulate the viability of RT-resistant cancer cells during RT treatment, which could have substantial implications for future treatments involving radiation.

ASPM expression in RT-resistant cells correlates with disease progression in lung adenocarcinoma

To investigate the clinical phenomenon of radiation resistance in NSCLC, we first sought to create a system suitable for interrogation of contributors to RT resistance. We exposed A549 cells to 30 rounds of 2 Gy, with stepwise selection of resistant cells (A549-R) (Fig. 1A). We then exposed the resistant cells to another 2 Gy and measured the ratio of apoptotic cells. As expected, the percentage of apoptotic cells decreased sharply in the A549-R cells but significantly increased in A549 cells (Fig. 1B). These results were confirmed by counting the numbers of A459 and A549-R clones after the irradiation (Fig. 1C). To extend our characterization of characteristics possibly related to RT resistance in A549-R cells, we measured the expression of γH2AX and cleaved caspase-3 in A549 and A549-R cells after a 2-Gy irradiation and found declines in both in the A549-R cells (Figs. 1D, 1E). We next injected A549 and A549-R cells subcutaneously into the flanks of nude mice and tested the RT resistance of the resulting tumors in vivo. Irradiation of the implanted tumors to 2 Gy led to significant reduction in A549 tumor volumes but not the A549-R tumors (Fig. 1F). These in vivo results are consistent with our in vitro results and confirm the RT resistance in our A549-R cell model.

As a next step in examining the molecular contributors to RT resistance in NSCLC, we compared mRNA expression in A549 cells and A549-R cells (Fig. 1G) and identified differences in genes associated with microtubule cytoskeleton organization and chromosome segregation (Fig. 1H). These findings led us to consider ASPM for further study. ASPM expression levels in A549-R cells in culture and in subcutaneous tumors in mice were higher than in A549 cells (Fig. 1I, 1J). This result was verified with core biopsy samples of lung tissue from patients with known RT-resistant NSCLC (with sensitivity vs resistance defined by CT imaging findings) (Fig. 1K). Next, we analyzed the Gene Expression Profiling Interactive Analysis (GEPIA) database for ASPM expression in relation to survival and found that patients with lung cancer and high ASPM expression had higher mortality rates than patients with low ASPM expression (Fig. 1L). These findings prompted us to investigate APSM further for its possible involvement in regulating RT resistance in NSCLC.

ASPM regulates microtubule stability in RT-resistant cells

Previous studies have shown that ASPM localizes at the “minus ends” of microtubules, recruits katanin, and promotes katanin-mediated severing of growing microtubules [25]. Given our finding of high ASPM expression levels in A549-R cells, we reasoned that ASPM may participate in RT resistance by regulating microtubule dynamics. This notion was supported by our findings on immunofluorescence co-localization analysis with α-tubulin, γ-tubulin, and acetylated α-tubulin expression (Fig. S1 and 2A, 2B), which revealed that ASPM binds along microtubules during interphase and co-localizes with the spindle complex during mitosis. Acetylated α-tubulin levels were also significantly lower in A549-R cells than in A549 cells, indicating that stable microtubules had been destroyed in A549-R cells. To gain further insight into the role of ASPM in A549-R cells, we next examined its effects on microtubule formation during interphase. Immunofluorescence analysis revealed that ice-induced microtubule depolymerization was exacerbated in A549-R cells (Fig. 2C). After microtubule depolymerization was complete, we incubated cells at 37°C to induce regrowth of microtubules, and we found that regrowth was consistently attenuated in A549-R cells (Fig. 2D). We also found that irradiating HeLa cells stably expressing GFP-α-tubulin (HeLa-TUBA) with 2 Gy led to substantial changes in microtubule length (Fig. 2E-2G). To substantiate the role of ASPM in microtubule stability, we knocked down ASPM expression in A549-R cells and found that this knockdown led to increased microtubule acetylation. That is, microtubule stability was enhanced after ASPM knocked down in A549-R cells. (Fig. 2H, 2I). Collectively, these findings indicate that ASPM has a crucial role in regulating microtubule stability.

Spindle misorientation in high-ASPM-expressing RT-resistant cells

Stable microtubules are necessary for correct orientation of the mitotic spindles, which is important for ensuring the accuracy of subsequent chromosome segregation [26, 27]. To investigate the adverse effects triggered by abnormal microtubule stability, we measured spindle orientation in A549-R cells (Fig. 3A). Fluorescence microscopy revealed that the spindles were misoriented in A549-R cells (Fig. 3B-3D). To determine whether this misorientation could be reversed by downregulating ASPM, we introduced two different siRNAs to ASPM into A549-R cells (Fig. 3E) and found drastic changes in the spindle angles after siASPM treatment, with orientation returning to nearly that of non-resistant A549 cells (Fig. 3F-3H). These functional assays demonstrated that high ASPM expression promoted spindle misorientation in lung cancer cells, which would be expected to negatively affect the equal segregation of chromosomes.

Chromosomal mis-segregation can be repaired by suppressing ASPM expression in RT-resistant cells

Next, to understand the downstream effects of misoriented spindles and how they might contribute to RT resistance in cancer cells, we studied chromosome segregation conditions during cytokinesis. As noted previously, failures in cytokinesis lead to increased genomic instability, which increases the likelihood of chromosome combinations that may provide a selective growth advantage (Fig. 4A). Consistent with this notion, we saw substantial increases in chromosomal mis-segregation in A549-R cells relative to A549 cells (Fig. 4B). Importantly, chromosomal mis-segregation has also been observed in samples from patients with RT-resistant NSCLC (Fig. 4C), which were verified as taking place in individual cells through co-staining with γ-tubulin (Fig. 4D). In the meantime, we calculated ploidy scores for lung adenocarcinoma samples from The Cancer Genome Atlas dataset and found that high ASPM expression was more likely to be linked with high ploidy scores (Fig. 4E). To further examine this observation, we knocked down ASPM in A549-R cells and found drastic reductions in chromosomal mis-segregation and fewer micronuclei (Fig. 4F-4H). Given the common presence of high ASPM expression in A549-R cells, we next proposed that ASPM can disrupt microtubule dynamics and affect genomic instability by offering RT-resistant cells the possibility to establish an “escape route” to survive.

Knocking down ASPM in RT-resistant cells promotes RT-induced apoptosis

Our findings thus far support the notion that ASPM affects genomic instability in A549-R cells. Although the importance of genomic stability in cancer development and progression is well established [28, 29], the question of whether downregulating ASPM expression can increase apoptosis in RT-resistant cells such as A549-R remains unanswered. To address this question, we analyzed apoptosis in A549-R cells treated with siRNA to ASPM. Notably, downregulating ASPM did not affect apoptosis in A549-R cells (Fig. S2). However, when siASPM cells were treated with 2 Gy irradiation, γH2AX expression was significantly increased relative to the non-irradiated group (Fig. 5A, 5B), leading us to analyze apoptosis again under these conditions. Intriguingly, cleaved caspase-3 expression was increased and numbers of clones were decreased compared with unirradiated RT-resistant cells (Fig. 5B, 5C), indicating a sharp increase in numbers of apoptotic cells. These results were confirmed by flow cytometry (Fig. 5D). We further collected subcutaneous tumors from mice implanted with ASPM-siRNA tumor cells and treated with 2 Gy and analyzed expression of acetylated α-tubulin and cleaved caspase-3 by western blotting. Consistent with the findings from the cultured siASPM-treated cells, expression of both acetylated α-tubulin and cleaved caspase-3 increased (Fig. 5E). Moreover, tumor volumes were significantly smaller in the mice with siASPM A549-R implants treated with 2 Gy (Fig. 5F). Collectively, these findings show that ASPM has similar effects in vivo and in vitro and suggest that ASPM in RT-resistant cells has a conserved role in the pathogenesis and progression of lung adenocarcinoma by affecting the stability of microtubules (Fig. 5G).

Targeting the 963–1263 amino acid region of ASPM as anticancer therapy

The anticancer drug paclitaxel and its analogues act by directly targeting and stabilizing microtubules; however, this strategy can still result in drug resistance [30–32]. To circumvent this problem, we explored potential drug targets within the ASPM protein as follows. First, we searched the SWISS-MODEL database for homologous sequences of ASPM and found its 963–1263 amino acid sequences to be highly matched (Fig. 6A). We next created model structures based on this region (Fig. 6B) and discovered 9 candidate drug-target sequences there (Fig. 6C). We then used this information to predict the location of “drug pockets” and to estimate their shapes and sizes. We found 9 such drug pockets for ASPM by using this strategy (Fig. 6D, 6E). At this time, we speculate that these regions participate in the inhibition of ASPM in RT-resistant cancer cells. We are currently constructing different truncated mutations of ASPM to identify the functional region(s) responsible for RT resistance in future studies. In the meantime, we are actively developing a new small-molecule inhibitor of ASPM for treating RT-resistant disease, an effort that is expected to have important implications for improving the diagnosis and treatment of patients with RT-resistant NSCLC.

RT resistance remains a major cause of failure of clinical cancer therapy [33–35]. Knowledge of the mechanisms underlying RT resistance is urgently needed so that patients who have RT-resistant disease (and hence are unlikely to respond to RT) can be spared the morbidity and expense of ineffective treatment, ideally before that treatment is begun. Given the growing interest in using ASPM as a target for cancer therapy [36–38], we examined its potential involvement in the therapeutic response to RT-resistant cancer. In the present study, we used in vitro cell culture models, in vivo mouse models, and analysis of human genome databases to clarify the importance of ASPM in radiation-resistance in NSCLC. We found that ASPM has a crucial role in RT resistance by affecting microtubule stability and chromosomal mis-segregation during mitosis. Indeed, high expression of ASPM correlated directly with chromosome mis-segregation and increased numbers of micronuclei, suggesting that high expression of ASPM in NSCLC is more likely to be intrinsic and integral to RT resistance rather than incidental. This notion is also supported by recent comprehensive functional analyses of ASPM in different types of tumors in patients [39–41]. We propose that mis-segregation of chromosomes could have much broader effects extending beyond radiation resistance in cancer, which underscores the broad clinical value of understanding the exact role of the genomic instability in cancer progression and therapy.

Regulation of microtubule dynamics is a crucial aspect of a wide variety of physiological and pathological processes [42, 43]. Abnormal microtubule dynamics in cells represent an obstacle to normal physiological activity. Many microtubule-associated proteins can affect microtubule dynamics. Any promotion of anomalies in microtubule dynamics via changes in genes or microtubule-associated proteins can have uncertain results that may have catastrophic consequences to organisms [44, 45]. Although some drugs currently in clinical use such as paclitaxel and docetaxel can directly target microtubules, such drugs can also lead to or worsen drug resistance. Our findings provide a new target strategy for RT-resistant NSCLC cells—that is, to use ASPM inhibition to decrease micronucleus formation by stabilizing microtubule dynamics during mitosis. Moreover, this mechanism is not restricted to NSCLC; high ASPM levels have been linked with tumor development and progression in colorectal cancer as well [46]. Therefore, ASPM could be a target molecule for preventing RT resistance in a broad range of cancer types, prevention being a much better alternative than having to treat resistant cancer after it develops.

In the future, targeting ASPM may have biological consequences distinct from targeting microtubules upon the appearance of RT-resistant tumor cells. Importantly, it may circumvent the problem of developing resistance to commonly used chemotherapeutic agents. ASPM has also been implicated in promoting homologous recombination-mediated DNA repair [47, 48], adding another layer of evidence in resistance to radiation. This may explain, at least in part, why cancer cells can evade spindle checkpoint monitoring. Abnormal microtubule dynamics in RT-resistant cells can have a variety of consequences, leading to complex outcomes. Given our positive results regarding the involvement of ASPM in RT-resistant cancer cells, we forecast its effectiveness as suggested by homology modeling. Future in-depth analyses of why ASPM expression differs, how to specifically inhibit it, and the development of ASPM-specific drugs will provide insights on how best to implement strategies for accurate diagnosis and selection patients for treatment of RT-resistant cancer.

Materials

Antibodies targeting α-tubulin (ab18251, ab7291), γ-tubulin (ab11316, ab11317), and Alexa Fluor-conjugated secondary antibodies were purchased from Abcam (Cambridge, UK). Antibodies targeting cleaved caspase-3 (9664S), γH2AX (9718S), and pan-keratin (4545) were purchased from CST (Danvers, MA, USA). Antibodies targeting ASPM (26223-1-AP), β-actin (20536-1-AP), and acetylated α-tubulin (66200-1-Ig) were purchased from ProteinTech (Rosemont, IL, USA). Horseradish peroxidase–conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 4´,6-diamidino-2-phenylindole (DAPI) was obtained from Sigma-Aldrich. Small interfering RNAs targeting ASPM (#1: 5´- GCAGCATGCCGTTTGTTTA − 3´; #2: 5´- CAGAGATGGTTTCGAGCAA − 3´) for in vitro and in vivo experiments were synthesized by RiboBio (Guangzhou, China).

Cell culture and treatment

A549 cells were obtained from the American Type Culture Collection. HeLa cells stably expressing GFP-α-tubulin (HeLa-TUBA) were obtained as we described previously[27]. Cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum. siRNAs were transfected with Lipofectamine RNAimax (Thermo Fisher Scientific). Cell cultures were irradiated with an RS2000 Pro-225 Biological X-ray irradiator (RadSource, Buford, GA, USA).

Animal experiments and ethical statement

Nude mice were obtained from Beijing Huafukang Biotechnology Development Co., Ltd. (Beijing, China). A549 and A549-R cells were injected subcutaneously into the flanks of the mice and allowed to form tumors. When subcutaneous tumor volumes reached about 300 mm³, the mouse tumors were irradiated with 2 Gy by using a Small Animal Radiation Research Platform (Xstrahl, UK). All applicable institutional and/or national guidelines for the care and use of animals were followed. The use of mice was approved by the Animal Care and Use Committee of Shandong University.

Fluorescence microscopy

Cells grown on coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 2% bovine serum albumin. Then, cells were sequentially probed with the primary antibodies, fluorophore-conjugated secondary antibodies, and DAPI. The coverslips were mounted with mounting medium and images obtained with an LSM 800 confocal microscope (Zeiss, Oberkochen, Germany). Spindle angles were measured as described previously [49]. Numbers of clones and γH2AX foci were counted with ImageJ software. For time-lapse microscopy, mitotic progression was recorded with a Cell Observer 7 Live cell optical fault microscopic monitoring system with a live-cell chamber as described method [50].

Immunoblotting analysis

Cells were lysed with cell lysis buffer (Beyotime Biotechnology, Shanghai, China), and the lysates were mixed with sodium dodecyl sulfate (SDS) sample buffer. Proteins were loaded onto 10% SDS-polyacrylamide gels, resolved by electrophoresis, and transferred onto polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% fat-free milk and sequentially incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Targeted proteins were visualized with an enhanced chemiluminescence substrate kit (Millipore, Burlington, MA, USA).

Apoptosis assays

To examine apoptosis, cells were stained with an FITC Annexin-V apoptosis detection kit (BD Biosciences) followed by flow cytometry (BD Biosciences) or stained with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis detection kit (Abcam) and imaged with an LSM 800 confocal microscope (Zeiss).

Statistical analysis

Prism 8 (Graph Pad) was used for data plotting and statistical analysis. All experiments were repeated independently at least three times, and data are expressed as means ± standard error of the mean (SEM). Student’s t tests were used for pairwise comparisons, and analysis of variance (ANOVA) for comparison of multiple groups, with the built-in analysis tools of Prism 8. Normal distribution, variations within each group, and sample independence were assumed to be met. All measurements were taken from distinct samples, and no data were excluded. P values are denoted by asterisks as: *P < 0.05; **P < 0.01; ***P < 0.001, with P < 0.05 considered to indicate statistical significance.

ACKNOWLEDGMENTS

This work was supported by the Academic Promotion Program of Shandong First Medical University (2019ZL002), Research Unit of Radiation Oncology, Chinese Academy of Medical Sciences (2019RU071), the foundation of National Natural Science Foundation of China (81627901, 81972863 and 82030082), the foundation of Natural Science Foundation of Shandong (ZR201911040452).

AUTHOR CONTRIBUTIONS

J.Y. and T.Z. conceived and designed the study. F.W. and M.W. performed the bioinformatic analysis. T.Z., J.W., S.X., S.L., C.X., W.Y., Y.C., X.G. and M.W. performed the experiments. T.Z., and D.W. wrote the paper. All authors reviewed the manuscript.

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Yes there is potential conflict of interest.

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Fig S1. ASPM co-localization with microtubules during the phases of mitosis. (A) Immunofluorescence staining for ASPM (green), α-tubulin (red), and DAPI (blue) in A549-R cells during mitosis. Scale bar, 12 μm. (B) Immunostaining for ASPM (green), γ-tubulin (red), and DAPI (blue) in A549-R cells during mitosis. Scale bar, 12 μm.
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Fig S2. ASPM deficiency does not affect apoptosis in A549-R cells. (A) Immunofluorescence staining and quantification of γH2AX foci inA549-R cells treated with control or ASPM siRNAs. Scale bar, 8 μm. (B) A549-R cells transfected with siControl or ASPMsiRNAs were stained with TUNEL to identify apoptotic cells. Scale bar, 20 μm. (C) A549-R cells were transfected with siControl or ASPM siRNAs, and protein levels were determined by immunoblotting with the indicated antibodies. (D) A549-R cells transfected with siControl or ASPM siRNAs were stained with Annexin V-FITC and propidium iodide, and apoptotic cells were detected by flow cytometry.

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ASPM induces radiotherapy resistance by disrupting microtubule stability leading to chromosome malsegregation in non-small cell lung cancer

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Abstract

Figures

Introduction

Results

ASPM expression in RT-resistant cells correlates with disease progression in lung adenocarcinoma

ASPM regulates microtubule stability in RT-resistant cells

Spindle misorientation in high-ASPM-expressing RT-resistant cells

Chromosomal mis-segregation can be repaired by suppressing ASPM expression in RT-resistant cells

Knocking down ASPM in RT-resistant cells promotes RT-induced apoptosis

Targeting the 963–1263 amino acid region of ASPM as anticancer therapy

Discussion

Materials And Methods

Materials

Cell culture and treatment

Animal experiments and ethical statement

Fluorescence microscopy

Immunoblotting analysis

Apoptosis assays

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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