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Chloroquine induces transitory attenuation of proliferation of human lung cancer cells through regulation of mutant P53 and YAP

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Abstract

Background

Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer-associated deaths worldwide. Though recent development in targeted therapy has improved NSCLC prognosis, yet there is an unmet need to identify novel causative factors and appropriate therapeutic regimen against NSCLCs.

Methods and results

In this study, we identify key molecular factors de-regulated in NSCLCs. Analyze their expression by real-time PCR and immunoblot; map their localization by immuno-fluorescence microscopy. We further propose an FDA approved drug, chloroquine (CQ) that affects the function of the molecular factors and hence can be repurposed as a therapeutic strategy against NSCLCs. Available NSCLC mutation data reflects a high probabilistic chance of patients harboring a p53 mutation, especially a gain of function (GOF)-R273H mutation. The GOF-P53 mutation enables the P53 protein to potentially interact with non-canonical protein partners facilitating oncogenesis. In this context, analysis of existing transcriptomic data from R273H-P53 expressing cells shows a concomitant up-regulation of Yes-associated protein (YAP) transcriptional targets and its protein partner TEAD1 in NSCLCs, suggesting a possible link between R273H-P53 and YAP. We therefore explored the inter-dependence of R273H-P53 and YAP in NSCLC cells. They were found to co-operatively regulate NSCLC proliferation. Genetic or pharmacological inhibition of YAP and GOF-P53 resulted in sensitization of NSCLC cells. Further analysis of pathways controlled by GOF-P53 and YAP showed that they positively regulate the cellular homeostatic process- autophagy to mediate survival. We hence postulated that a modulation of autophagy might be a potent strategy to curb proliferation. In accordance to above, autophagy inhibition, especially with the FDA-approved drug- chloroquine (CQ) resulted in cytoplasmic accumulation and reduced transcriptional activity of GOF-P53 and YAP, leading to growth arrest of NSCLC cells.

Conclusion

Our study highlights the importance of GOF-P53 and YAP in NSCLC proliferation and proposes autophagy inhibition as an efficient strategy to attenuate NSCLC tumorigenesis.

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Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

BAF:

Bafilomycin

CMA:

Chaperone mediated autophagy

CQ:

Chloroquine

EGFR:

Epidermal growth factor receptor

EV:

Empty vector

GDC:

Genomic data commons

GOF:

Gain of function

LTR:

Lysotracker red

MDC:

Monodansylcadaverine

NSCLC:

Non-small cell lung carcinoma

PFT-α:

Pifithrin-alpha

SCLC:

Small-cell lung carcinoma

VP:

Verteporfin

WT:

Wild type

YAP:

Yes-associated protein

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Acknowledgements

The authors acknowledge BITS Pilani, Pilani campus, for providing them with infrastructural support. Heena Saini thanks CSIR for providing a student fellowship.

Funding

We thank DST-SERB Fast Track Project of RC (Sanction order No. SB/FT/LS-233/2012), DBT-Pilot Project of RC (BT/PR/8799/MED/30/1067/2013), ICMR of RC (2020-1404/ADHOC-BMS) and SERB of RC (EMR/2016/006788) for providing funding support for conducting the experiments.

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Author notes

  1. Heena Saini and Mahima Choudhary have contributed equally to the work.

Authors and Affiliations

  1. Department of Biological Sciences, Birla Institute of Technology and Science, Pilani Campus, Pilani, Rajasthan, 333031, India

    Heena Saini, Mahima Choudhary, Harshita Sharma, Shibasish Chowdhury, Sudeshna Mukherjee & Rajdeep Chowdhury

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  1. Heena Saini
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Contributions

HS-I performed and analyzed the experiments, MC helped with wet-lab experiments and revision, HS-I, HS-II and SC performed the in silico and statistical analysis; SM helped in experimental analysis and manuscript correction; RC received funding for research; HS-I and RC designed the study plan and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Rajdeep Chowdhury.

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Supplementary material 1 (TIF 5891.1 kb)

Supplementary Fig. 1. A Immunoblot post siRNA mediated ablation of P53 in R273H-P53-H1299 cells (n = 3, p < 0.0001). ‘Cntrl’ stands for scrambled siRNA. GAPDH served as loading control. B Transcript levels of p53 (n = 3, p = 0.0361), upon over-expression of YAP (YAP-OE) in SW480 cells. ‘∗’ indicates a significant difference compared to empty vector (EV) transfected control. C Immunoblot representing level of P53, upon overexpression of YAP (YAP-OE) in GOF-R156P-P53 harboring HOS cells. Actin served as loading control. D Cell viability, as measured through MTT assay after exposure to pifithrin-alpha (PFT-α) [n = 3; p > 0.05 (24 hours) and p < 0.001 (48, 72 and 96 hours) at 10 µM dose. ‘∗’ indicates a significant difference compared to untreated cells (Cntrl); one way ANOVA (vs Time). E Graph showing relation between expression of autophagy associated genes and 5-year survival (%) of NSCLC patients. Gene expression data analyzed from GDC portal. F Difference in LC3B-II protein expression between P53-Null and R273H-P53 expressing H1299 cells (n = 3, p = 0.0012) and immunoblot of P53 protein in H1299 null and H1299-R273H cells. GAPDH served as loading control. In the figures, wherever applicable, bar graphs indicate mean ± standard error of mean (SEM). Unless otherwise mentioned, T-test was used for analysis of statistical significance. The statistically significant differences in comparison to control are represented with * and their corresponding p values are given below: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Supplementary material 2 (TIF 30756.5 kb)

Supplementary Fig. 2. A The fluorescence intensity profile across the white line as analyzed from microscopic image for both green and red channels is shown in the graph representing YAP and R273H-P53 co-localization. B Immuno-fluorescence image showing YAP (Red) and R273H-P53 (Green) localization 48 h after si-ATG-5 transfection or C Baf (10 nM) treatment in R273H-P53 harboring H1299 cells. D Immuno-fluorescence image showing YAP localization post CQ treatment (50 µM) for 48 h in P53-Null H1299 cells. Scale bar: 10 μm. Objective: Plan Apochromat 63x/1.40 oil M27. E Immuno-fluorescence image showing YAP and mutant-P53 localization post CQ treatment (50 µM) for 48 h in HOS cells. DAPI (Blue) was used to stain the nucleus. T-test was used for analysis of statistical significance. The statistically significant differences in comparison to control are represented with * and their corresponding p values are given below: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Supplementary material 3 (TIF 4964.3 kb)

Supplementary Fig. 3. The fluorescence intensity profile across the white line as analyzed from microscopic images for both green and red channels is shown in the graphs representing protein co-localization. A The fluorescence intensity profiles for YAP and LTR co-localization (one way ANOVA, vs CQ); B YAP and LAMP-2a co-localization; C R273H-P53 and LAMP-2a co-localization; D HSC-70 and YAP co-localization post CQ exposure (50 µM) for 48 hours. In the figures, ‘Cntrl’ stands for untreated cells. The statistically significant differences in comparison to control/CQ are represented with */# and their corresponding p values are given below: */#p ≤ 0.05, **/## p ≤ 0.01, ***/###p ≤ 0.001

Supplementary material 4 (TIF 24634.8 kb)

Supplementary Fig. 4. A SEM images showing comparative cytoplasmic changes in cells cultured in CQ-free media (withdrawal; W). B Immuno-fluorescence image of YAP and R273H-P53 post CQ withdrawal. Scale bar: 10 µm. Objective: Plan Apochromat 63x/1.40 oil M27. White arrows indicate accumulation and co-localization. ‘∗’ or ‘#’ indicates a significant difference compared to untreated or CQ treated cells respectively. C Transcript levels of YAP (n = 4, p = 0.0426) and CYR61 (n = 3, p = 0.0106) after CQ withdrawal. CQ treatment (50 µM) was for 48 hours; however, for withdrawal experiments cells were exposed to CQ for 24 hours followed by culture in CQ free media for 24 hours. ‘#’ indicates a significant difference compared to CQ treated cells. In the figures, unless otherwise mentioned, T-test was used for analysis of statistical significance. The statistically significant differences in comparison to control/CQ are represented with */# and their corresponding p values are given below: */#p ≤ 0.05, **/##p ≤ 0.01, ***/###p ≤ 0.001.

Supplementary material 5 (TIF 7573.4 kb)

Supplementary Fig. 5. A schematic diagram representing the overall effect of CQ exposure as observed in our study

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Saini, H., Choudhary, M., Sharma, H. et al. Chloroquine induces transitory attenuation of proliferation of human lung cancer cells through regulation of mutant P53 and YAP. Mol Biol Rep 50, 1045–1058 (2023). https://doi.org/10.1007/s11033-022-08072-y

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