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Nuclear IQGAP1 Promotes Gastric Cancer Cell Growth by Altering the Splicing Of bioRxiv preprint doi: https://doi.org/10.1101/2020.05.11.089656; this version posted May 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Nuclear IQGAP1 promotes gastric cancer cell growth by altering the splicing of 2 a cell-cycle regulon in co-operation with hnRNPM 3 Andrada Birladeanu1,5, Malgorzata Rogalska2,5, Myrto Potiri1,5, Vassiliki 4 Papadaki1, Margarita Andreadou1, Dimitris Kontoyiannis1,3, Zoi Erpapazoglou1, 5 Joe D. Lewis4, Panagiota Kafasla*,1 6 7 1Institute for Fundamental Biomedical Research, B.S.R.C. “Alexander Fleming”, 34 8 Fleming st., 16672 Vari, Athens, Greece 9 2Centre de Regulació Genòmica, The Barcelona Institute of Science and Technology 10 and Universitat Pompeu Fabra, Dr. Aiguader 88, 08003, Barcelona, Spain 11 3 Department of Biology, Aristotle University of Thessaloniki, Greece 12 4European Molecular Biology Laboratory, 69117 Heidelberg, Germany 13 5These authors contributed equally 14 *Correspondence: [email protected] 15 16 Summary 17 Heterogeneous nuclear ribonucleoproteins (hnRNPs) play crucial roles in alternative 18 splicing, regulating the cell proteome. When they are miss-expressed, they contribute 19 to human diseases. hnRNPs are nuclear proteins, but where they localize and what 20 determines their tethering to nuclear assemblies in response to signals remain open 21 questions. Here we report that IQGAP1, a cytosolic scaffold protein, localizes in the 22 nucleus of gastric cancer cells acting as a tethering module for hnRNPM and other 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.11.089656; this version posted May 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 spliceosome components. One of the consequences of the IQGAP1-hnRNPM 24 interaction is to change the alternative splicing of the anaphase promoting complex 25 (ANAPC), subunit 10 and is required for the hnRNPM response to heat-induced stress 26 signals. Together, IQGAP1 and hnRNPM promote gastric cancer cell growth, and 27 their genetic depletion leads to cell cycle arrest and halts tumour progression. We 28 propose that in response to stress-signals IQGAP1 translocates in the nucleus to 29 coordinate gastric tumour growth by recruiting spliceosome components that drive 30 cell cycle progression. 31 32 Introduction 33 In humans, more than 95% of multi-exonic genes are potentially alternatively spliced 34 (Pan et al., 2008; Wang and Burge, 2008). Precise modulation of Alternative Splicing 35 (AS) is essential for shaping the proteome of any given cell and altered physiological 36 conditions can change cellular function via AS reprogramming (Heyd and Lynch, 37 2011). The importance of accurate AS in health and disease, including cancer (El 38 Marabti and Younis, 2018; Kahles et al., 2018; Oltean and Bates, 2013; Sveen et al., 39 2015), has been well documented. However, evidence connecting AS regulation to 40 signalling comes mostly from few cases where localization, expression, or post- 41 translational modifications of specific splicing factors such as Serine-Arginine-rich 42 (SR) proteins or heterogeneous nuclear ribonucleoproteins (hnRNPs) (Heyd and 43 Lynch, 2011; Shkreta and Chabot, 2015) are altered. 44 An abundant, mainly nuclear protein of the hnRNP family is hnRNPM, with four 45 isoforms that arise from alternative splicing and/or post-translational modifications 46 (Datar et al., 1993). The only nuclear role so far reported for hnRNPM is in 47 spliceosome assembly and splicing itself (Kafasla et al., 2002; Llères et al., 2010), 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.11.089656; this version posted May 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 48 including regulation of AS of other as well as its own pre-mRNAs (Hovhannisyan and 49 Carstens, 2007; Huelga et al., 2012; Pervouchine et al., 2019). The association of 50 hnRNPM with the spliceosome is abolished under heat-induced stress (Gattoni et al., 51 1996; Llères et al., 2010), suggesting its involvement in stress-related signalling 52 pathways. 53 HnRNPM-regulated AS events have been linked to disease development and 54 progression. Specifically, hnRNPM-mediated AS is deterministic for the metastatic 55 potential of breast cancer cells, due to cell-type specific interactions with other 56 splicing factors (Harvey et al., 2018; Hu et al., 2020, p. 8; Xu et al., 2014). 57 Furthermore, in Ewing sarcoma cells, hnRNPM abundance and subnuclear 58 localization change in response to a chemotherapeutic inhibitor of the PI3K/mTOR 59 pathway, resulting in measurable AS changes (Passacantilli et al., 2017). Very 60 recently, hnRNPM was shown to respond to pattern recognition receptor signalling, 61 by modulating the AS outcome of an RNA-regulon of innate immune transcripts in 62 macrophages (West et al., 2019). 63 Despite such growing evidence on the cross-talk between hnRNPM-dependent AS 64 and cellular signalling, how distinct signals are transduced to hnRNPM and the 65 splicing machinery remains unclear. Here, we present conclusive evidence of how this 66 could be achieved. We describe a novel interaction between hnRNPM and the 67 scaffold protein IQGAP1 (IQ Motif Containing GTPase Activating Protein 1) in the 68 nucleus of gastric cancer cells. Cytoplasmic IQGAP1 acts as a signal integrator in a 69 number of signalling pathways (Hedman et al., 2015; Smith et al., 2015), but there is 70 no defined role for the nuclear pool of IQGAP1. With IQGAP1 mRNA being 71 overexpressed in many malignant cell types, the protein seems to regulate cancer 72 growth and metastatic potential (Hu et al., 2019; Osman et al., 2013; White et al., 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.11.089656; this version posted May 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 73 2009). Moreover, aged mice lacking IQGAP1 develop gastric hyperplasia suggesting 74 an important in vivo role for IQGAP1 in maintaining the gastric epithelium (Li et al., 75 2000). In the present study we show that this novel, nuclear interaction between 76 hnRNPM and IQGAP1 links cellular signalling to hnRNPM-regulated AS in gastric 77 cancer, a cancer type that has been associated with a significantly high incidence of 78 AS changes (Kahles et al., 2018; Sveen et al., 2015). Additionally, we show that 79 depletion of both interacting proteins results in tumour growth reduction, which 80 makes them and their interaction interesting cancer drug targets. 81 82 Results 83 hnRNPM and IQGAP1 expression levels are significantly altered in gastric 84 cancer. 85 Analysis of the hnRNPM and IQGAP1 protein levels by immunofluorescence on 86 commercial tissue microarrays revealed increased hnRNPM and IQGAP1 staining in 87 tumour as compared to normal tissue, especially in adenocarcinoma samples (Figure 88 1A and Figure S1A-C). This agrees with TCGA data analysis indicating significantly 89 increased expression of both IQGAP1 and hnRNPM mRNAs in stomach 90 adenocarcinoma (STAD) vs normal tissue samples (Figure 1B). Furthermore, a 91 strong correlation between the expression of the two mRNAs (HNRNPM and 92 IQGAP1) in normal tissues (GTex) was revealed, which was reduced in normal 93 adjacent tissues (Normal) and eliminated in STAD tumours (Figure 1C). Detection of 94 hnRNPM and IQGAP1 protein levels in a number of gastric cancer cell lines by 95 immunoblotting (Figure 1D) identified cell lines with similar levels of hnRNPM and 96 low or high levels of IQGAP1 (MKN45, AGS and NUGC4, KATOIII), corroborating 97 the TCGA data. Two of those STAD cell lines were used for further studies (NUGC4 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.11.089656; this version posted May 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 98 and MKN45) since they have similar hnRNPM, but different IQGAP1 expression 99 levels. 100 An interaction between hnRNPM and IQGAP1 was detected in nuclear extracts from 101 both cell lines using immunoprecipitation with anti-IQGAP1 antibodies (Figure 2A). 102 Treatment with RNaseA showed that the two proteins interact independently of the 103 presence of RNA. This interaction also appeared DNA-independent (Figure S2A and 104 data not shown). Immunoprecipitation using anti-IQGAP1 antibodies from 105 cytoplasmic extracts did not reveal interaction with the minor amounts of cytoplasmic 106 hnRNPM (Figure S2B and data not shown), indicating that if the proteins do 107 interact in the cytoplasm, these complexes are less abundant compared to the nuclear 108 ones. 109 There are previous reports of a small fraction of IQGAP1 entering the nucleus in a 110 cell cycle-dependent manner (Johnson et al., 2011). Confocal imaging verified the 111 presence of IQGAP1 in the nucleus of both NUGC4 and MKN45 cells (Figure 2B) 112 and the interaction of nuclear IQGAP1 with hnRNPM in situ was confirmed using 113 proximity ligation assays (PLA) (Figure 2C-D). Quantification of the PLA signal per 114 cell confirmed that the interaction between the two proteins was clearly detectable in 115 the nucleus of both cell lines. Some cytoplasmic interaction sites were also detected, 116 but they were minor compared to the nuclear ones (Figure 2C-D) in agreement with 117 the immunoprecipitation results from cytoplasmic extracts (Figure S2B). 118 Together these results demonstrate the RNA-independent, nuclear interaction between 119 hnRNPM and IQGAP1 in gastric cancer cells where the levels of both proteins are 120 significantly deregulated. 121 122 Nuclear IQGAP1-containing RNPs are involved in splicing regulation.
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