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Integrated analysis of ALK higher expression in human cancer and downregulation in LUAD using RNA molecular scissors

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Abstract

Purpose

Anaplastic lymphoma kinase (ALK) is an endorsed molecular target in ALK-rearranged carcinomas, including lung adenocarcinoma. However, the clinical advantage of targeting ALK using druggable inhibitors is almost universally restricted by the development of drug resistance. Therefore, a strategy for combating ALK overexpression remains paramount for ALK-driven cancer.

Methods

We systemically analyzed the overexpression pattern of ALK and its clinical consequences, genetic alterations, and their significance in cancer hallmark genes, and correlation using integrated multidimensional approaches. The LwCas13a RNA molecular scissors was used to downregulate ALK-rearrangement by leveraging two target guide RNAs in lung adenocarcinoma (LUAD) cells. Immunocytochemistry, immunoblotting, and MTT assays were conducted to validate the downregulation.

Results

We found elevated levels of ALK in several malignancies, including LUAD, than in normal tissues. Higher expression of ALK was significantly associated with worse or shorter survival than patients with lower expression. We identified numerous genetic alterations in ALK, which potentially alter the cancer hallmark genes, including STAT1 and CTSL, in patients with LUAD. Next, we observed that the LwCas13a molecular scissors robustly downregulated both phosphorylated and total ALK chimera protein expression in LUAD cells compared to the control. Furthermore, we found that downregulation of ALK chimera protein substantially inhibited cell viability and induced cell death, including apoptosis.

Conclusion

Our findings suggest a basis for ALK as a prognostic biomarker and the LwCas13a molecular scissors successfully downregulated the onco-driver ALK-rearrangement protein, which will potentially pave the way toward the development of novel therapeutic strategies for ALK-driven cancer.

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Data availability

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary file.

Abbreviations

LwCas13a:

CRISPR-associated protein 13a originated from Leptotrichia wadei

RAS-MAPK:

Rat sarcoma–mitogen-activated protein kinase

PI3K-AKT:

Phosphatidylinositol 3 kinase–protein kinase B

JAK-STAT:

Janus kinase–signal transducer and activator of transcription

MEK-ERK:

Mitogen-activated protein kinase–extracellular signal = regulated kinase

CRKL-C3G:

CRK-like proto-oncogene–guanine nucleotide-releasing factor 2

PTPN11:

Tyrosine-protein phosphatase non-receptor type 11

Src:

Proto-oncogene tyrosine-protein kinase Src

FAK:

Focal adhesion kinase

Shc-GRB2:

Shc-transforming protein–growth factor receptor bound protein 2

IRS2:

Insulin receptor substrate 2

GSK-3a:

Glycogen synthase kinase-3 alpha

FRS2:

Fibroblast growth factor receptor substrate 2

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Acknowledgements

Saifullah gratefully acknowledges the scholarship from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan for economic assistance. We acknowledge professor Fujimoto lab, School of Materials Science, JAIST, Japan for their support in image acquisition of immunoblotting. We also acknowledge professor Takeshi Suzuki and assistant professor Akihiko Ishimura, Cancer Research Institute, Kanazawa University, Kanazawa, Japan for their help in apoptosis assay. We are thankful to Mr. Jiarui Li, School of Materials Science, JAIST for technical help in confocal microscopy.

Funding

This research was funded by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (Grant number: 21H02067).

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Author notes

  1. Saifullah

    Present address: Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Kodaira City, Tokyo, 187–8502, Japan

Authors and Affiliations

  1. Area of Bioscience and Biotechnology, School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1–1 Asahidai, Nomi City, Ishikawa, 923–1292, Japan

    Saifullah & Toshifumi Tsukahara

  2. Division of Transdisciplinary Science, Japan Advanced Institute of Science and Technology (JAIST), 1–1 Asahidai, Nomi City, Ishikawa, 923–1292, Japan

    Toshifumi Tsukahara

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Contributions

S performed all the experiments, integrated analysis, data interpretation, statistical analyses, and wrote the first draft of the manuscript. TT supervised, acquired funding, reviewed and edited the writing. All authors read and approved the final manuscript.

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Correspondence to Toshifumi Tsukahara.

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Conflict of interest

The authors have declared no competing interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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No human and animal samples were used in this study. This study falls under the category of "Genetic engineering experiments". This research has been approved by the Life Science Committee, Japan Advanced Institute of Science and Technology (JAIST).

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Saifullah, Tsukahara, T. Integrated analysis of ALK higher expression in human cancer and downregulation in LUAD using RNA molecular scissors. Clin Transl Oncol 24, 1785–1799 (2022). https://doi.org/10.1007/s12094-022-02835-6

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