Citation: Zanini, G.; Selleri, V.; De Gaetano, A.; Gibellini, L.; Malerba, M.; Mattioli, A.V.; Nasi, M.; Apostolova, N.; Pinti, M. Differential Expression of Lonp1 Isoforms in Cancer Cells. Cells 2022, 11, 3940. https://doi.org/10.3390/ cells11233940 Academic Editor: Natascia Tiso Received: 2 September 2022 Accepted: 5 December 2022 Published: 6 December 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Differential Expression of Lonp1 Isoforms in Cancer Cells Giada Zanini 1,† , Valentina Selleri 1,2,† , Anna De Gaetano 1 , Lara Gibellini 3 , Mara Malerba 4 , Anna Vittoria Mattioli 2,4 , Milena Nasi 4 , Nadezda Apostolova 5,6 and Marcello Pinti 1, * 1 Department of Life Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy 2 National Institute for Cardiovascular Research (INRC), 40126 Bologna, Italy 3 Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, 41124 Modena, Italy 4 Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, 41124 Modena, Italy 5 Department of Pharmacology, University of Valencia, 46010 Valencia, Spain 6 FISABIO—Hospital Universitario Dr. Peset, 46017 Valencia, Spain * Correspondence: marcello.pinti@unimore.it; Tel.: +39-059-205-5386 † These authors contributed equally to this work. Abstract: Lonp1 is a mitochondrial protease that degrades oxidized and damaged proteins, assists protein folding, and contributes to the maintenance of mitochondrial DNA. A higher expression of LonP1 has been associated with higher tumour aggressiveness. Besides the full-length isoform (ISO1), we identified two other isoforms of Lonp1 in humans, resulting from alternative splicing: Isoform-2 (ISO2) lacking aa 42-105 and isoform-3 (ISO3) lacking aa 1-196. An inspection of the public database TSVdb showed that ISO1 was upregulated in lung, bladder, prostate, and breast cancer, ISO2 in all the cancers analysed (including rectum, colon, cervical, bladder, prostate, breast, head, and neck), ISO3 did not show significant changes between cancer and normal tissue. We overexpressed ISO1, ISO2, and ISO3 in SW620 cells and found that the ISO1 isoform was exclusively mitochondrial, ISO2 was present in the organelle and in the cytoplasm, and ISO3 was exclusively cytoplasmatic. The overexpression of ISO1 and, at a letter extent, of ISO2 enhanced basal, ATP-linked, and maximal respiration without altering the mitochondria number or network, mtDNA amount. or mitochondrial dynamics. A higher extracellular acidification rate was observed in ISO1 and ISO2, overexpressing cells, suggesting an increase in glycolysis. Cells overexpressing the different isoforms did not show a difference in the proliferation rate but showed a great increase in anchorage-independent growth. ISO1 and ISO2, but not ISO3, determined an upregulation of EMT-related proteins, which appeared unrelated to higher mitochondrial ROS production, nor due to the activation of the MEK ERK pathway, but rather to global metabolic reprogramming of cells. Keywords: Lon protease; mitochondria; SW620; mitochondrial DNA 1. Introduction Alternative splicing is a tightly regulated process allowing for the expression of multiple RNA and protein isoforms from one gene, and its dysregulation has recently been described in several diseases, most of them due to either the alteration in spliceoso- mal/splicing regulatory factors expression or genetic mutation in canonical RNA splicing sites [1,2]. Recurrent somatic mutations in components of the human splicing machinery have occurred in neurological diseases, such as Parkinson’s disease [3,4] and Alzheimer’s disease [5], infectious and immunological diseases, such as cardiovascular disease [6,7], systemic lupus erythematosus [8], diabetes mellitus [9,10], viral infections [11], and inflam- matory response [12], solid tumours, and hematological malignancies [1]. A dysregulated splicing program in cancer can represent a driving force in the development and main- tenance of cancer [13]. For instance, tumour suppressor genes that undergo alternative splicing in cancer, such as p53, can lose partially or totally their function and generate Cells 2022, 11, 3940. https://doi.org/10.3390/cells11233940 https://www.mdpi.com/journal/cells