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LIVE CELL ANALYSIS Live Cell Imaging. IncuCyte ZOOM live cell imaging systems (Room N136 and Room N232, Anderson Stuart Building, Room 616, Molecular Bioscience Building G08) (funded by Sydney Cancer Research Fund, Cancer Institute of NSW and USYD Collaborative Research Equipment grant) (User Manual) (IncuCyte ZOOM software version 2016A download) - online booking.

Introduction Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear.

We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated.

Results MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability. The most important DNA mismatch repair (MMR) protein commonly dysregulated in colon cancer is MLH1. MLH1 is the main component of the heterodimer MutLα, formed by MLH1 and PMS2.

Germline mutations in MLH1 are responsible for 50% of a hereditary form of colorectal cancer (CRC) called Lynch syndrome [ ]. In addition, 13-15% of sporadic CRCs are caused by MLH1 deficiency based on somatic promotor hypermethylation [, ]. Looking at functionality, MutLα is mainly involved in the correction of base-base mismatches and insertion-deletion loops resulting from defective DNA replication [ ].

Besides, recent studies suggest that MLH1 also participates in other important fundamental cellular functions beyond its primary role in MMR, e.g., the regulation of cell cycle checkpoints and apoptosis [ ], but also in meiotic reciprocal recombination and meiotic mismatch repair [ ]. Several MLH1 interacting proteins have been published, which might be essential for signaling DNA damages to different cellular processes [,,,, ]. Amongst them we identified non-erythroid spectrin αII (SPTAN1) as a novel interaction partner of MLH1 and found evidence for the involvement of both proteins in cytoskeletal and filamental organization [ ]. SPTAN1 belongs to a superfamily of F-actin cross-linking proteins (scaffolding proteins) which, first identified as membrane-skeleton components in erythrocytes, are ubiquitously expressed in metazoan cells [ ].

Spectrins contribute to cell adhesion and migration [ ], interact with structural and regulatory proteins [,, ] and are involved in the regulation of DNA repair [, ]. Deregulation of spectrins, especially of SPTAN1 seriously affects cellular behavior and promotes tumor progression. Upregulated SPTAN1, e.g., was demonstrated in various types of tumors [,,, ] and shown to be associated with local aggressiveness and metastic behavior of soft tissue carcinomas [ ]. Moreover, enhanced SPTAN1 was linked to tumor progression and malignancy in ovarian cancer [ ] and described to be involved in the carcinogenesis of sporadic CRC [ ]. After i) identification of MLH1-SPTAN1 protein-protein interaction [ ], ii) knowledge of MLH1 capacity to stabilize its partner proteins [, ] and iii) indications that MLH1 deficient tumors are less aggressive and distant metastasis are less common than in MMR proficient forms [, ], we propose a MLH1 dependent role of SPTAN1 for cellular motility, metastasis and aggressiveness of CRC.