Griseofulvin Radiosensitizes Non-Small Cell Lung Cancer Cells and Activates cGAS

Abstract

Extra copies of centrosomes are frequently observed in cancer cells. To survive and proliferate, cancer cells have developed strategies to cluster extra-centrosomes to form bipolar mitotic spindles. The aim of this study was to investigate whether centrosome clustering (CC) inhibition (CCi) would preferentially radiosensitize non-small cell lung cancer (NSCLC). Griseofulvin (GF; FDA approved treatment) inhibits CC, and combined with radiation therapy (RT) resulted in a significant increase in the number of NSCLC cells with multipolar spindles, and decreased cell viability and colony formation ability in vitro. In vivo, GF treatment was well tolerated by mice, and the combined therapy of GF and RT resulted in a significant tumor growth delay. Both GF and RT treatment also induced the generation of micronuclei (MN) in vitro and in vivo and activated cyclic GMP-AMP synthase (cGAS) in NSCLC cells. A significant increase in downstream cGAS-STING pathway activation was seen after combination treatment in A549 radioresistant cells that was dependent on cGAS. In conclusion, GF increased RT efficacy in lung cancer preclinical models in vitro and in vivo. This effect may be associated with the generation of MN and the activation of cGAS. These data suggest that the combination therapy of CCi, RT and immunotherapy could be a promising strategy to treat NSCLC.

Introduction

Lung cancer is the most common cause of cancer mortality in the United States and across the world (1,2). NSCLC accounts for 80% of lung cancers and patients with unresectable stage III NSCLC comprise approximately 40% of all lung cancers. The standard treatment, with concurrent platinum-based chemoradiation and adjuvant immune checkpoint blockade, is not curative in the vast majority of patients in part due to radioresistance of the primary tumor (3,4). In particular, patients presenting with an oncogenic mutation in the KRAS gene – about one quarter of NSCLC patients – remain recalcitrant to current therapeutic options. This reveals an urgent need to identify novel molecular targets to increase lung cancer radiosensitivity and increase anti-tumor immune systemic effects.

Centrosomes are cytoplasmic organelles composed of a pair of centrioles, which nucleate and anchor microtubules (MTs). During mitosis, normal cells possess two centrosomes each migrating to opposite poles of the cell in order to form a bipolar mitotic spindle. Interestingly, in contrast to normal cells, cancer cells frequently contain extra-centrosomes that can be generated during the course of tumorigenesis or be induced by treatments like radiotherapy (5,6). The presence of supernumerary centrosomes often leads to multipolar mitotic spindle formation and subsequent stress and inviable mitotic division. Cancer cells have developed strategies to cluster supernumerary centrosomes into two functional spindle poles (pseudo-bipolar) enabling tumor cells to divide and survive. Therefore, preventing centrosome clustering (CC) provides a means to selectively kill cancer cells with extra-centrosomes by forcing them into detrimental multipolar divisions without affecting the divisions of normal cells with normal numbers of centrosomes (7). Griseofulvin (GF), an orally active antifungal drug in human, has attracted considerable interest as a potential anti-cancer agent owing to its low toxicity and efficiency in inhibiting the proliferation of different types of cancer cells (8–12). Recently, it has been shown that GF induced multipolar spindle formation in tumor cells with supernumerary centrosomes and resulted in cell death (9,11,13,14). Furthermore, GF treatment arrested tumor cells at the G2/M phase of cell cycle and inhibited progression at metaphase and anaphase of mitosis (9,11,13,14). Cancer cells are most sensitive to radiation in the G2/M phase of the cell cycle (15–17). Thus, CCi could result in the selective radiosensitization of cancer cells with supernumerary centrosomes by reducing clonogenic potential while sparing healthy tissues with normal centrosome numbers.

In this study, we investigated the effect of GF-induced CCi on radiation treatment of NSCLC in vitro and in vivo. Our results showed that CCi forced the formation of multipolar spindles in NSCLC cell lines but not in normal cells (immortalized human bronchial epithelial cells, HBEC). CCi decreased cell viability and inhibited the proliferation of NSCLC cells in vitro. The combination of CCi and RT significantly sensitized NSCLC cells to RT treatment. In vivo, GF treatment was well tolerated, and the combination therapy of GF and RT sensitized tumors, compared to RT alone, resulting in a significant tumor growth delay. Interestingly, GF treatment induced the generation of micronuclei (MN) and promoted the translocation of cyclic GMP-AMP synthase (cGAS) into these MN, with the most significant increase seen after combined treatment of GF and RT. MN formation has been previously shown to activate the cytoplasmic double-stranded DNA sensor pathway cGAS, which in turn activates an innate immune response (18). We show that GF treatment activated the cGAS-STING pathway and increased gene expression of the downstream cytokine CCL5 in multiple NSCLC cell lines. Interestingly, the level of CCL5 increased the most with combined RT and GF treatment in the radioresistant cell line A549, and this CCL5 upregulation (as well as ISG15 and TNFalpha) was dependent on cGAS.

Materials & Methods

Cell culture, antibodies, and reagents

Human KRAS mutant lung cancer cell lines (H460 (CVCL_0459), H358 (CVCL_1559), A549 (CVCL_0023)) were purchased from ATCC (Manassas, VA, USA). The immortalized normal human bronchial epithelial cell (HBEC; CVCL_X491) line was a gift from Dr. Stephen Baylin. All cancer cell lines were authenticated by short tandem repeat (STR) profiling at the Genetic Resources Core Facility at the Johns Hopkins University (JHU). Cells were tested (Genetic Resources Core Facility - JHU) and treated when found positive with plasmocin (InvivoGen) routinely to avoid Mycoplasma contamination. Cell culture methods and the antibodies used were listed in Supplementary Materials and Methods. Griseofulvin (Sigma-Aldrich, G4753), HTdsDNA (Sigma, D6898), Polyoxyl-35 castor oil (Sigma-Aldrich, C5135) and Trypan blue solution (T8154, Sigma) were purchased from Sigma-Aldrich. For Caspase-3 and cleaved Caspase-3 assays, cells were harvested after incubation with GF (30uM) after 48h. For cGAS assays, cells were harvested after treatment with GF and irradiation after 48h.

Cell Viability Assay

Cell viability was determined at indicated time points based on the Trypan blue exclusion method as described previously by Strober W. (19). 3×10⁴ cells were seeded into each well in the 6-well plates. The numbers of viable cells were manually counted daily.

Radiation Treatment (RT)

In vitro, cells were irradiated with 0 to 8Gy at room temperature using the CIXD Biological X-rays Irradiator (Xstrahl; SKCCC-Experimental Irradiators Core - JHU) or the X-RAD320 irradiator (Precision X-ray - University of Maryland Baltimore-DTRS). For in vivo experiments, mice were treated with X-rays using the Small Animal Radiation Research Platform (SARRP) (Xstrahl; SKCCC-Experimental Irradiators Core - JHU), (20,21). Hind-flank tumors received 2x 2Gy irradiation daily using a circular 1 cm diameter beam.

Clonogenic Assay

The procedure (22) was described in the Supplementary Methods.

Immunoblot analysis

The Western-Blot procedure was described in the Supplementary Methods.

Immunofluorescence (IF) Staining

IF was performed as described previously (23), (Supplementary Methods). Spindle polar detection: All nuclei were detected and segmented using DAPI staining (total cell number). Mitotic spindle formation was confirmed by α-tubulin staining. Pericentrin staining was used to identify centrosomes. Bipolar spindles present with one centrosome per pole (2 centrosomes per cell). Mitotic cells with greater than two division poles were classified as multipolar spindles. Centrosomes clustering was defined by the presence of a pseudo-bipolar spindle associated with more than one centrosome per pole.

Immunohistochemistry (IHC)

IHC was described in the Supplementary Methods (23).

Assessment of micronucleus and cGAS-positive micronuclei frequency

For in vitro, the percentage of cells with micronuclei was determined by microscopy under blinded conditions. Micronucleated cells were classified manually by distinct staining by DAPI of structures outside of the main nucleus. Cells with an apoptotic appearance were excluded. For in vivo micronucleus assay, H&E staining procedures were performed by the JHU Tissue Core Facility. Slides were observed under a Nikon Eclipse Ni microscope equipped with a Nikon digital site DS-U3 camera under 40X magnification. All micronuclei grading was performed in a double-blinded fashion. At least 500 cells per core were counted. The criteria for the micronuclei included: a) the same staining as the main nucleus; b) smaller than the diameter of the main nucleus, and; c) not attached to the main nucleus. To enumerate cGAS positive micronuclei, these structures were counted manually for each field and expressed as a percentage of total cells within the field. Total cells were counted manually based on the DAPI and cGAS. More than 500 cells were quantified for each sample.

Xenograft mouse model

Mice were purchased from Harlan Laboratories, and housed and maintained in accordance with the guidelines from our Institutional Animal Care and Use Committee at the JHU School of Medicine (Approved protocol number MO18M195/MO21M205). Female and male nude (NCRNU, sp/sp) mice 4-5 weeks old (eight subjects per condition) were injected subcutaneously in the flanks with 1x10⁶ H358 cells in 100μL of Hank’s solution and Matrigel (BD Biosciences) mixed 1:1. Once tumors reached 100mm³, mice were treated or not with GF (50mg/kg) and/or RT (2Gy x2). GF was dissolved in 100% DMSO firstly and then diluted with 10% castor oil (final 9% DMSO). The mice received Vehicle or GF daily (five days a week) via intraperitoneal injections (IP), (11). Three days after starting GF treatment, mice were irradiated. After 3 days of irradiation, two of the mice in each group were sacrificed and the tumors were assessed by HE, Ki67 and cleaved-Caspase 3 staining. After 21 days of treatment, all mice were sacrificed. The tumor volume ((length x width²)/2) and animal weight were measured every 2-4 days. Tumor growth curve was developed based on the tumor size. Tumor weight was measured after mice were sacrificed.

Quantitative RT-PCR and siRNA

The primers, siRNAs and reagents were detailed in Supplementary Materials & Methods.

Statistics

All in vitro experiments were repeated at least three times. All in vivo experiments were using groups of eight mice per arm. Data were analyzed by Student’s t-test or one-way ANOVA. A p value <0.05 was considered statistically significant. All statistical analyses and graphs were carried out by using Graphpad Prism v8.4.3 (GraphPad Software, San Diego, CA, USA; SCR_002798).

Study Approval

All animal experiments were performed in accordance with the Johns Hopkins Institutional Animal Care and Use Committee (IACUC) protocols (Approved protocol number MO18M195/MO21M205). Animals were housed in a pathogen-free environment in the animal facility of the JHU School of Medicine, SKCCC, Cancer Research Building.

Data Availability Statement

The data generated in this study are available within the article and its supplementary data files.

Results

Centrosome clustering inhibition (CCi) induced multipolar spindle formation in NSCLC cells

Mammalian somatic cells typically possess one centrosome which is duplicated in coordination with DNA replication. Each centrosome then migrates to a cell pole during cell division, forming a bipolar spindle to ensure symmetric chromosome segregation. The number of centrosomes in cancer cells can be quite heterogeneous, even within the same cell population. Hence, a cancer cell population can contain a mix of bipolar spindles, multipolar spindles with asymmetric division, and pseudo-bipolar spindles with centrosomes clustering (CC) as a pro-survival mechanism. NSCLC tumor tissues and cancer cell lines contain various levels of centrosome amplification (CA) across the entire population, and levels can range from ±5% to ±40% depending on the cell line (24–26). To examine the effect of CC-inhibiting drug GF on KRAS mutated-NSCLC cells and normal cells, three KRAS mutated-NSCLC cell lines, A549, H358, and H460, and one non-cancer cell line, HBEC, were used. The basal levels of CA were heterogeneous within each population, and the average frequency of CA (percentage of cells with >2 centrosomes over the total number of cells) was <1% in HBEC cells and higher, ±10%, in NSCLC cells (Fig. s1A). Then, mitotic cells were characterized as monopolar spindle, bipolar spindle or multipolar spindle by immunostaining with anti-α-tubulin and anti-pericentrin (Fig. 1A). GF significantly reduced the mitotic cells with bipolar spindles and increased the ratio of multipolar spindles in all cell lines at the highest dose tested of 30 uM. However, GF caused significantly more multipolar spindles in NSCLC cells than in HBEC, (40-80% in NSCLC cells vs. 17% in HBEC with 30 uM GF treatment), (Fig. 1B). These results demonstrate that GF preferentially induced multipolar spindles in NSCLC cells over normal cells.

Figure 1. Centrosome clustering inhibition (CCi) induced multipolar spindle formation in NSCLC cells.

(A) Representative images showing mitosis with amplified centrosomes. Cells were immunostained with α-tubulin (microtubules, green), pericentrin (centrosomes, red) and DAPI (DNA, blue). (B) The proportions of monopolar spindles, bipolar spindles, and multipolar spindles in the control and GF treated cells were statistically analyzed. Data were shown as average value ± S.D. calculated from three independent experiments (one-way ANOVA; ***P < 0.001, ****P < 0.0001).

CCi reduced cell viability and could induce apoptotic cell death

GF treatments resulted in a time- and dose-dependent inhibition in cell growth of NSCLC and HBEC cells (Fig. s2A). GF 10 uM treatment significantly inhibited cell proliferation in NSCLC cells, but only slightly slowed the proliferation of HBECs. Higher concentrations of GF (30 uM) severely inhibited growth of all cell lines (Fig. s2A). Trypan blue exclusion assay showed that the percentage of dead cells was significantly higher in H358 and H460 cells after GF treatment compared to that in A549 and HBEC (Fig. s2B). After a genotoxic or microtubule-toxic stress, plural cell responses concomitantly exist within the cell population including mitotic catastrophe but also apoptosis, the induction of a senescent or an arrested phenotype. We also observed an induction of cleaved-caspase 3 in H358 and H460 with GF treatment consistent with the induction of cell death (Fig. s2C). The fact that we do not observe a cleaved-Caspase 3 induction for A549 might suggest an induction of a senescent or arrested phenotype, or possibly also mitotic catastrophe which could lead to a loss of these cells from the overall population at the time-point of analysis.

GF reduced centrosome clustering in NSCLC cells after radiation treatment

Irradiation can induce CA in cancer cells, and clustering these extra-centrosomes is a strategy for cancer cells to avoid multipolar mitoses. We examined CC events in NSCLC cells after RT. CC was identified via the presence of pseudo-bipolar spindles involving more than one centrosome per pole (Fig. 2A). As expected, the number of mitotic cells with multipolar spindles was significantly increased after RT (0-5% in control vs. 36-53% after RT; Fig. 2A,B). The number of mitotic cells with CC was also increased after RT (2-6% in control to 16-21% in RT treated NSCLC cells). GF treatment significantly reduced the number of mitotic cells with clustered centrosomes from 16-21% after RT alone to 4-5% in RT+GF treated NSCLC cells, and further increased the ratio of cells with multipolar spindles (36-53% in RT alone to 63-69% in RT+GF treated NSCLC cells). CC was observed less in HBEC cells (10%) after RT and there was no difference following GF treatment (Fig. 2A,B).

Figure 2. Griseofulvin (GF) reduced centrosome clustering in NSCLC cells after radiation treatment.

(A) Representative images showing mitosis with normal bipolar, clustered supernumerary centrosomes and multipolar spindles. Cells were immunostained with α-tubulin (microtubules, green), pericentrin (centrosomes, red) and DAPI (DNA, blue). (B). Quantification of the percentage of normal bipolar spindles, centrosome clustering bipolar spindles and multipolar spindles. Data were shown as average value ± S.D. calculated from three independent experiments. (one-way ANOVA; *P < 0.05, **P < 0.01. NS: not significant)

Targeting centrosome clustering reduced NSCLC cell viability following irradiation

Next, we studied whether CCi could potentially radiosensitize tumor cells. The HSET protein, a key regulator of CC in cancer cells (7), was knocked-down with siRNAs in H460 and HBEC (Fig. 3A,B). Interestingly, HSET levels were significantly higher in cancer cells than in HBEC (Fig. s3). Consistent with other reports, reduced levels of HSET resulted in a significant increase in the number of cells with multipolar spindles in H460, but not in HBECs (Fig. 3C). When we treated these cells with RT, knockdown of HSET significantly reduced H460 cell viability after RT treatment, but not HBEC (Fig. 3D). The knockdown of HSET also significantly reduced the clonogenic potential and radiation response of A549 cells but not of HBEC (Fig. 3E). Therefore, genetically targeting CC demonstrated preferential effects on radiation-induced cell viability and clonogenic potential in NSCLC cells over non-cancer cells. Similarly, pharmacologic CCi with GF significantly enhanced the radiation-sensitivity in H358 and H460 cells, but not in HBEC cells (Fig. s4).

Figure 3. Genetically targeting centrosome clustering by knocking-down HSET reduced NSCLC cell viability following radiation treatment.

(A) qPCR of HSET gene expression and (B) Western blot (WB) of HSET protein levels after knocking-down HSET with siRNAs in H460 and HBEC cells. (C) Quantification of monopolar, bipolar and multipolar spindles after HSET knockdown. (D) Cell proliferation of H460 and HBEC after combined HSET knockdown and RT. (E) Clonogenic survival curves for A549 and HBEC treated with or without HSET siRNA 48h prior to irradiation. Data were shown as average value ± S.D. calculated from three independent experiments (t-test/one-way ANOVA nsP > 0.05; *P < 0.05; **P < 0.01; ***P<0.001).

GF treatment radiosensitized NSCLC in a xenograft mouse model

We also examined the radiosensitization effect of GF in a H358 xenograft mouse model. Mice bearing H358 flank tumors were treated with either GF, RT or both. GF or RT alone slowed tumor growth, but the combination of GF and RT showed further inhibition of tumor growth (Fig. 4A,B,C). GF treatment appeared well tolerated on the mice as determined by weight loss (Fig. 4D). We also collected tumor tissues after 3 days of irradiation and stained with Ki67 for proliferating tumor cells and cleaved-Caspase 3 for apoptotic cells. Consistent with the tumor growth delay results, less Ki67 positive cells were observed in GF alone or RT treatment groups compared to the control group. The lowest number of Ki67 positive cells was observed in the combination treatment group (Fig. 4E). Similarly, the combination treatment resulted in the highest ratio of apoptotic cells in the tumors (Fig. 4E). Overall, these results demonstrated that the combination of GF and RT significantly inhibited tumor growth in vivo compared to single treatment arms.

Figure 4. Griseofulvin (GF) treatment radiosensitized H358 NSCLC in vivo.

(A) Representative images showing the tumor sizes from each treatment group. (B) Plots of tumor growth curves. (C) Weight of the tumors from each treatment group. (D) Plots of body weights of tumor-bearing mice from day 0 to day 21. (E) Representative images of immunostaining of Ki67 and cleaved-Caspase 3, and quantification of positive stained cells in Ctrl, GF, RT and GF+RT tumors. Data were shown as average value ± S.D. calculated from three independent experiments (one-way ANOVA *P < 0.05; **P < 0.01; ***P < 0.001).

GF treatment generated micronuclei and activated cGAS

Multipolar mitosis frequently results in micronuclei (MN) formation (27), as does RT treatment alone (28). We observed that GF or RT treatment alone induced MN generation in both NSCLC cell lines in vitro and xenograft tumor tissues in vivo (Figs. 5A, s5A). Interestingly, the combination treatment of GF + RT produced the most MN in vitro and in vivo (Figs. 5A, s5A). Generation of MN can activate the cytoplasmic double-stranded DNA sensor pathway cGAS-STING, which can trigger an innate immune response (29). We then observed that more micronuclei were stained positively with cGAS after combination treatment than with either single treatment in all three NSCLC cell lines in vitro (Fig. 5B,C, s5B). However, no cGAS-positive MN were observed in similarly treated HBECs. The activation of the cGAS-STING pathway includes the activation of downstream factors (e.g. TBK1, NFKB, IRF3) which cooperate together to induce the transcription of multiple target genes, including interferons and cytokines (e.g. CCL5, ISG15, IFNbeta, TNFalpha). We observed that the expression of STING and the phosphorylation of TBK1 were slightly upregulated after 6 Gy irradiation alone or after GF + 6 Gy combined treatment, and that the combined treatment also enhanced the phosphorylation of NF-KB compared with single treatment (Fig. 6A). We then analyzed the main downstream target genes of the cGAS signaling in NSCLC cells after the different treatments. We found that GF and RT single treatments significantly induced the expression of CCL5 cytokine in A549 and H460 (Fig. 6B). The combination GF+RT treatment further increased CCL5 levels in A549 cells (Fig. 6B). A similar profile in A549 was observed for additional cGAS pathway downstream transcriptional targets including IFNbeta1, ISG15 and TNFalpha (Fig. 6C–D and Fig. s6). Finally, the knockdown of cGAS in A549 and HBEC using siRNA against cGAS (smartpool siRNA cGAS) showed that the upregulation of these transcriptional targets (CCL5, ISG15, and TNFalpha) were cGAS-dependent in A549 (Fig. 6C–D). HBEC had low levels of cGAS and as expected demonstrated little change with or without treatment regardless of knockdown (Fig. 6C). Altogether, we show that Griseofulvin inhibits CC and results in micronuclei formation and activation of the cGAS-STING pathway that can be accentuated further with RT.

Figure 5. Centrosome clustering inhibition (CCi) induces micronuclei and activates cGAS.

(A) Representative H&E staining of the H358 tumor tissue showing regions of micronuclei (MN) positive cells (left panel), and the quantification were shown in the right panel; (B) Representative immunofluorescence images of DAPI (blue) and cGAS (red) after treatment (t+48h) in vitro (40X magnification) ; (C) Quantification of the percentage of cGAS-positive micronuclei (cGAS+) cells from Ctrl, GF (15 uM), RT (6 Gy) and GF+RT treatment groups in H460, H358, and A549. Data were shown as average value ± S.D. calculated from three independent experiments (one-way ANOVA *P < 0.05; **P < 0.01; ***P < 0.001).

Figure 6: The cGAS-STING pathway is activated after GF/RT treatment.

(A) Western-blot of cGAS, STING, TBK1, TBK1 phospho-Ser172, NF-KB, NF-KB phospho-Ser536 and Vinculin (loading control) protein expression in A549 after 15 uM GF and/or 6 Gy RT. (B) CCL5 mRNA expression levels were measured by qPCR after different treatments in vitro. (C) WB for cGAS and Vinculin (loading control) protein expression in A549 and HBEC without (Vehicle DMSO) or with 15 uM GF and/or 6 Gy RT in vitro, and with or without siRNA against cGAS. Herring Testis double-stranded DNA (HTdsDNA) single treatment (24h) was used as positive control of cGAS-STING downstream pathway activation and target gene induction. (D) CCL5, ISG15, TNFalpha mRNA expression levels (reported against control Vehicle (Veh) condition) were measured by qPCR after treatments in vitro with or without siRNA against cGAS in A549. Data were shown as average value ± S.D. calculated from at least three independent experiments (t-test/one-way ANOVA: nsP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 compared with Veh condition, unless bracket showing against siRNA control).

Discussion

Tumor radioresistance is a major cause of failure in locally advanced NSCLC treatment. The development of drugs that can enhance the sensitivity of tumor cells to radiation is of great importance to improve the outcomes of NSCLC therapy, particularly for KRAS mutant cases that have poor outcomes. Centrosome amplification in cancer cells can be caused by RT, and cancer cells have developed strategies to cluster extra-centrosomes to form pseudo-bipolar spindles for successful mitosis. In this study, we showed that GF radiosensitized NSCLC cells but not non-cancer HBEC cells. GF can promote CCi, and genetic CCi with knockdown of the CC protein HSET also decreased cell viability of NSCLC cells to radiation treatment. GF treatment also induced the generation of MNs, activating cGAS-STING pathway, and increased the gene expression of downstream targets in NSCLC cells. Interestingly, further increase in the level of certain cGAS-STING target genes after combined RT and GF treatment were seen in the most radioresistant of the cell lines, A549. Our data suggest that CCi can radiosensitize NSCLC and that combination of CCi, RT and immunotherapy is a potentially promising cancer treatment strategy that should be tested in NSCLC.

Griseofulvin (GF) is an oral drug, inhibiting fungal cell mitosis, largely used in human for the treatment of tinea capitis, as well as skin and nail dermatophytosis (12). GF has been shown to be able to alter cell cycle, to affect the stability of mitotic spindle, to inhibit cell proliferation, and induce apoptosis (9–11,13,14,30). Many previous studies have demonstrated that treatment with GF alone has anti-tumor activity (8,9,11,13,14,30). In this study, we found that when combined with RT, GF significantly radiosensitized NSCLC cells, but not normal cells. Similar effects of CCi in cancer selective radiosensitization has also been reported in breast cancer cell lines in vitro recently (31). Therefore, all these results support further testing of CCi in radiation therapy in the future.

The cGAS-STING pathway plays an essential role in antitumor immune response. Cyclic GMP-AMP (cGAMP) synthase (cGAS) can detect cytosolic DNA fragments and generate the second messenger cGAMP, which activates the adaptor STING and downstream innate immune responses (32). Micronuclei (MN) are indicators of DNA damage, and cGAS localizes to MN arising from genomic instability in mouse and human cancer cells (29,33). In our study, we presented the novel finding that GF and RT increased MN formation, over either treatment alone, resulting in more cGAS localization to MNs. Interestingly, the most intrinsically radioresistant NSCLC cell line in our study, A549, showed the largest increase in the gene expression of the cGAS downstream targets with the combination GF+RT treatment. Moreover, this was cGAS-dependent as shown by cGAS knockdown experiments. The mechanism and significance of this finding could potentially lead to future novel therapeutic option especially in cases of radiation-resistance, and deserves further study to allow selective enhancement of the cGAS-STING pathway with immunotherapy.

Conclusions

Cancer cells frequently have amplified centrosomes and have developed strategies to cluster those extra-centrosomes to divide successfully. Here, we evaluated whether centrosome clustering inhibition (CCi) can sensitize lung cancer cells (NSCLC) to radiation treatment. We used pharmacological and genetic methods to inhibit CC in lung cancer cells in vitro and in vivo. We demonstrated that CCi can selectively inhibit cancer cell proliferation, promote cell death and lead to radiosensitization. Finally, CCi and RT treatment resulted in increased MN and activation of the cGAS-STING pathway which can potentially promote innate immunity. These characteristics may serve as a novel strategy to concurrently increase radiation response and anti-tumor immunity in locally advanced NSCLC.