FAM46C controls antibody production by the polyadenylation of immunoglobulin mRNAs and inhibits cell migration in multiple myeloma

Abstract FAM46C, frequently mutated in multiple myeloma (MM), has recently been shown to encode a non‐canonical poly(A) polymerase (ncPAP). However, its target mRNAs and its role in MM pathogenesis remain mostly unknown. Using CRISPR‐Cas9 technology and gene expression analysis, we found that the inactivation of FAM46C in MM down‐regulates immunoglobulins (Igs) and several mRNAs encoding ER‐resident proteins, including some involved in unfolded protein response and others that affect glycosylation. Interestingly, we show that FAM46C expression is induced during plasma cell (PC) differentiation and that Ig mRNAs encoding heavy and light chains are substrates of the ncPAP, as revealed by poly(A) tail‐length determination assays. The absence of the ncPAP results in Ig mRNA poly(A) tail‐shortening, leading to a reduction in mRNA and protein abundance. On the other hand, loss of FAM46C up‐regulates metastasis‐associated lncRNA MALAT1 and results in a sharp increase in the migration ability. This phenotype depends mainly on the activation of PI3K/Rac1 signalling, which might have significant therapeutic implications. In conclusion, our results identify Ig mRNAs as targets of FAM46C, reveal an important function of this protein during PC maturation to increase antibody production and suggest that its role as a tumour suppressor might be related to the inhibition of myeloma cell migration.

using next-generation sequencing has revealed novel mutations in genes whose function in MM pathogenesis is largely unknown, such as FAM46C and DIS3. 8 FAM46C is one of the most commonly mutated genes in MM, with somatic point mutations having been identified in about 10% of newly diagnosed MM cases. 9,10 The vast majority of these mutations are of an inactivating nature, frameshift or non-sense mutations, which indicates that FAM46C may function as a tumour suppressor. 9 In addition, FAM46C is located in cytoband 1p12, which is known to be deleted in approximately 20% of MM patients. Loss of heterozygosity or mutations in FAM46C has been associated with shorter survival. 11 Moreover, the acquisition of FAM46C mutations over time, as described in some longitudinal studies, suggests that loss of function of FAM46C might be a progression event in MM. 12,13 A recent study has demonstrated that FAM46C encodes an active non-canonical poly(A) polymerase 14 and therefore may increase gene expression by extending the poly(A) tails of some mRNAs in the cytoplasm. 15 Its authors showed that overexpression of FAM46C in human myeloma cell lines (HMCLs) carrying the mutated gene led to polyadenylation and stabilization of several mRNAs, and induced cell death. 14 Another recent study found that overexpression of FAM46C induced substantial cytotoxicity in MM cells, up-regulated genes involved in unfolded protein response (UPR) and increased Ig light chain production. 16  This finding demonstrates that the loss of polyadenylation activity is the mechanism by which antibody production is decreased in FAM46C KO clones.

| Cell lines
The human myeloma cell lines (HMCLs) JJN3 and RPMI-8226 were acquired from DMSZ, and U266 from ATCC. The cell lines were cultured as previously described. 17 Cell line identity was con-  FACSCalibur flow cytometer. To favour the growth of single cells, 50% filtered conditioned medium and 20% FBS were added to the culture medium. Isolated clones were expanded in culture over a period of 1 month, in the case of JJN3, or 2 months for U266 and RPMI-8226, and then genomic DNA was extracted. Clones were analysed by PCR using the primers FAM46C-FOR and FAM46C-REV (Table   S1). Sanger sequencing was used to evaluate the alterations resulting from non-homologous end-joining (NHEJ) at the cut site.

| Cell migration and invasion assays
MM cells were washed in serum-free culture medium and resuspended at a final concentration of 10 6 cells/mL. In the migration assays, 1.5 mL of cell suspension was seeded in the upper chamber of a 6-well, 8-µm pore Transwell plates (Corning-Costar) and 2.6 mL of RMPI-1640 with 20% serum was placed in the lower compartment.
Invasion assays were performed using BioCoat Matrigel Invasion 24well, 8.0-μm pore Transwell chambers (Corning-Costar). For these experiments, 500 µL of cells resuspended at 10 6 cells/mL in serumfree medium was seeded in the upper chamber, and 750 µL of medium containing 20% serum was used as the chemoattractant in the lower chamber. After 20 hours, cells migrating into the lower chambers were collected, resuspended in 400 μL PBS and counted using a BD Accuri C6 flow cytometer.

| RNA extraction and microarray data analysis
Total RNA was extracted from three independent FAM46C WT or KO clones using an RNeasy mini kit (Qiagen). RNAs were then processed and used to hybridize Affymetrix PrimeView Human Gene Expression Arrays following the manufacturer's instructions. Raw data were background-adjusted, normalized and log 2 -transformed using the RMA algorithm 18 available in the Affymetrix expression console (v. 1.4.1). Microarray data were deposited in Gene Expression Omnibus (GEO) under accession number GSE114984. We compared the gene expression of the three FAM46C KO clones with each of the three control samples, resulting in nine comparisons. Genes with an absolute value of the fold change (FC) greater than 1.5 were selected for further analysis. A second approach using an absolute FC > 1.2 was carried out to assess the overrepresentation of gene ontology categories.

| Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
Total RNA was reverse-transcribed to cDNA using a cDNA Reverse Transcription Kit from Applied Biosystems. qRT-PCR was performed using an iQ™ SYBR ® Green Supermix kit (Bio-Rad), the iQ5 PCR detection system and the following gene-specific primers: FAM46C-FOR and FAM46C-REV2, GAPDH-FOR and GAPDH-REV (Table S1).
Alternatively, and for other genes, expression was assessed using TaqMan qRT-PCR assays (Applied Biosystems). Relative gene expression was calculated by the 2 −ΔCt method using GAPDH as the reference gene for normalization or 18S rRNA when indicated.

| siRNA
HMCLs were transfected with 25 nmol/L of on-TARGET plus™ control pool or on-TARGET plus SMART pool Human FAM46C (Dharmacon). Transfections were carried out using the Amaxa Cell Line Nucleofector Kit V, the Amaxa Nucleofector device (Lonza) and program G-016.

| Cell proliferation and cell viability assays
Cell proliferation and cell viability in the absence or presence of drugs was assessed by the MTT assay. Apoptosis was measured using annexin V-fluorescein isothiocyanate/propidium iodide (PI) doublestaining (Immunostep) according to the manufacturer's procedure.

| Knockout of FAM46C slightly affects the gene expression profile
To investigate the consequences of FAM46C inactivation for the pathogenesis of MM, we employed CRISPR-Cas9 technology to delete endogenous FAM46C in MM cells. The experiments were performed in the JJN3 cell line, which bears a wild-type (WT) Recent studies have shown that FAM46C encodes a non-canonical mRNA poly(A) polymerase that may affect the stability of its target mRNAs. 14,19 In order to identify putative FAM46C substrates, total RNAs were extracted from FAM46C WT and KO clones and subsequently processed for microarray hybridization. Surprisingly, we found few genes whose levels of expression in all FAM46C KO clones changed relative to WT clones. Probe sets corresponding to eight genes (MAGED1, LRPAP1, RHOBTB1, GPX7, CKAP4, PCOLCE2, CD55 and HACD1) were underexpressed in the nine comparisons (three FAM46C KO cells compared with three controls) using a FC < −1.5 (Table S2), and 80 probe sets corresponding to 54 genes decreased their expression 1.2-fold (Table   S3). On the other hand, no gene was up-regulated using a FC > 1.5, although 12 genes were overexpressed 1.

2-fold in all FAM46C
KO cells compared with controls (Table S4). When we considered at least six comparisons, nine genes were up-regulated 1.5-fold: and TRA2 (Table S5).

| Inactivation of FAM46C up-regulates oncogenic lncRNA MALAT1 and promotes cell migration and invasion in MM
Examining the functions of the genes deregulated in FAM46C KO clones, we found that some had previously been associated with cell migration and invasion: MAGED1 and RHOBTB1 inhibiting, [20][21][22] and lnc RNA MALAT1, PROK2 and TRA2 promoting these activities. [23][24][25] The deregulation of these five genes observed in the microarray analysis was validated by qRT-PCR ( Figure 2A). Moreover, reduced levels of MAGED1 and RHOBTB1 proteins were confirmed by Western blot ( Figure 2B). Then, we used the CRISPR-Cas9 technology to delete endogenous FAM46C in U266 and RPMI-8226, which also express wild-type FAM46C. In the same way as in JJN3, three WT and three FAM46C KO clones, confirmed by PCR, Sanger sequencing (data not shown) and Western blot ( Figure S1), were se-  Figure 3A,B). Moreover, FAM46C KO cells also exhibited a greater invasive ability relative to WT cells ( Figure 3C). To verify that the increase in cell mobility was independent of putative CRISPR-Cas9 off-target effects, we knocked down FAM46C expression by siRNA ( Figure 3D) and performed cell migration assays. Down-regulation of FAM46C in JJN3 significantly increased the migratory ability of the cells ( Figure 3E), confirming that loss of FAM46C promotes cell migration. Next, the in vitro cell migration assays were performed in the three RPMI-8226 FAM46C WT and KO clones. We found that the inactivation of the ncPAP induced a significant increase in migration, as previously demonstrated in JJN3 ( Figure 3F).

| Increased migration of FAM46C KO cells depends on PI3K activation
Next, we investigated molecular mechanisms that might underlie the increased migration and invasion of FAM46C KO cells. As matrix metalloproteinases (MMPs) are key mediators in cell invasion, we investigated whether the loss of FAM46C affected MMPs expression in MM cells. However, similar levels of MMP2 and MMP9 were found in FAM46C WT and KO cells ( Figure 4A). In MM, an activation of the epithelial-mesenchymal transition (EMT) similar to the phenomenon observed in solid tumours, which is considered a key process for metastasis, has been described. 26 EMT is characterized by the loss of E-cadherin, mediated by the up-regulation of its repressors such as Slug or Twist, and an increase in N-cadherin. 27 Although MM cells are not epithelial cells, some MM cell lines express N and/or E-cadherin. 28 We found that JJN3 cells, either FAM46C WT or KO, had no detectable level of N-cadherin, and a very low level of E-cadherin expression was observed in the different clones, with the exception of clone F2 ( Figure 4A).
Twist and Slug protein levels did not increase in FAM46C KO clones compared with WT cells. These results indicate that the increased rate of migration observed in FAM46C KO MM cells seems to be EMT-independent.
Previous studies have shown that MAPKs and PI3K can also regulate cell migration processes, 29,30 so we evaluated the influence of different kinase inhibitors on the migration ability of FAM46C WT and KO cells. We found that inhibition of MEK1/2 with U0126 did not affect migration of the cells and a small decrease in cell mobility was observed after inhibition of p-38 with SB203580 in JJN3 FAM46C KOs. However, a sharp reduction in cell migration occurred in FAM46C KO cells when PI3K was inhibited with Wortmannin or with the specific inhibitor LY294002 ( Figure 4B). PI3K acts via diverse downstream signalling components, including the GTPase Rac1 and the kinase Akt (PKB), to promote cell mobility. 31 We found that migration of FAM46C KO cells depended on Rac1 activation in these cells, as revealed by the fact that treatment with the specific inhibitor EHoP-016 also reduced cell mobility ( Figure 4B).

| Knockout of FAM46C does not increase proliferation rates or resistance to antimyeloma drugs
To determine whether loss of FAM46C affected cell growth rate, JJN3 and RPMI-8226 clones were cultured and proliferation was tested by the MTT assay. Growth rates did not increase in any of the FAM46C KO clones compared with controls. In fact, some of them, but not all, exhibited a slight decrease in growth ability ( Figure 5A), suggesting that differences in growth rates may be clone-dependent and not FAM46C-dependent. On the other hand, silencing FAM46C in JJN3 and RPMI-8226 did not affect cell growth under the conditions tested ( Figure 5B). We then evaluated the effect of common antimyeloma drugs in the different clones, and no significant differences were found in the sensitivity of FAM46C WT and KO clones to melphalan, bortezomib or dexamethasone ( Figure 5C).
It has recently been reported that depletion of FAM46C in the XG1 MM cell line results in increased expression of IRF4, Bcl2 and ERK signalling activation. 16   Zhu et al 16 Figure 6C). Therefore, we wondered whether Ig and BIP mRNAs might be direct targets of the non-canonical poly(A)-polymerase activity of FAM46C. As shown in Figure 6D

| Inactivation of FAM46C decreases ER stress and down-regulates some genes involved in glycosylation
Functional enrichment analysis of the 54 genes down-regulated in JJN3 FAM46C KO cells (Table S3)  We observed the appearance of spliced XBP1 4 hours after treatment with tunicamycin in all the tested clones, and its subsequent disappearance over time ( Figure S4), as previously described. 39 However, the XBP1 unspliced form remained more intense at 4 and 8 hours after treatment with tunicamycin in all JJN3 FAM46C KO clones compared with WT cells ( Figure S4A). In RPMI-8226, the XBP1 unspliced forms reappeared after 30 hours of treatment and were also more intense in the FAM46C KO than in WT controls ( Figure S4B). These results suggest a reduced level of ER stress in FAM46C KO cells.
Finally, we took advantage of the MMRF CoMMpass study (research.themmrf.org) and obtained a list of genes down-regulated in  (Table S7). The enrichment analysis using WebGestalt identified 13 functional categories (Table S8), and one of them was the 'response to ER stress' category, which included 33 genes. On the other hand, cellular component analysis found that 124 genes were located in the ER. When we cross-checked this list with that including the genes down-regulated in JJN3 FAM46C KO cells (Table S3), 11 genes emerged as being commonly down-regulated in all FAM46C KO clones and in MM patients carrying FAM46C mutations (Table S9).
Interestingly, three of them participate in UPR (HSPA13, EDEM2 and PDIA6) and three are involved in glycosylation (DDOST, AGA and SSR4). These results suggest that inactivation of this non-canonical poly(A) polymerase may affect the glycosylation patterns of PCs.

| D ISCUSS I ON
The poly(A) tail plays an important role in the post-transcriptional control of gene expression as it regulates mRNA transport, stability and translation. In addition to canonical nuclear poly(A) polymerase (PAP), which adds poly(A) tails to most eukaryotic mRNAs, seven non-canonical PAPs (ncPAPs) have been identified. 15 FAM46C, which is frequently mutated in MM, has recently been described as a new ncPAP. 14,19 However, its function in myeloma cells remains to be elucidated. In this study, we showed that FAM46C is up-regulated during PC differentiation to increase antibody production by extending the poly(A) tail of Ig mRNAs. Moreover, we demonstrated that inactivation of FAM46C in MM sharply increased the migratory ability of PCs, which might explain the poor prognosis of MM patients with FAM46C abnormalities and the role of this gene as a tumour suppressor (Figure 7).
The terminal differentiation of B cells, which bear surface Ig, into antibody-secreting plasma cells is accompanied by a substantial increase in the abundance of the mRNAs of both Ig heavy and light chains. 40 It has been reported that this increase is not due to a higher transcription rate but to an extended half-life of Ig mRNAs. 40  Inactivation of FAM46C is expected to induce pleiotropic effects in the cells, not only by deregulation of mRNAs, but also because changes in poly(A) tail length may affect mRNAs translatability giving rise to modifications in the proteome. Here, we found that FAM46C inactivation substantially increased the migratory ability of MM cells. We found that some of the few genes deregulated in the FAM46C KO clones, such as MAGED1, RHOBTB1 and MALAT1, had previously been associated with cell migration 20,22,23,43 MALAT1 was found to be up-regulated by FAM46C inactivation in the three HMCLs used in this study. This lncRNA was overexpressed in a wide variety of solid tumours and also in haematological malignancies, including MM. [43][44][45] Moreover, high levels of expression of MALAT1 in MM have been associated with the onset of the disease, progression from normal PCs to MM and extramedullary dissemination. [44][45][46][47] MAGED1 was down-regulated in JJN3 FAM46C KO clones and RHOBTB1 in two (JJN3 and U266) out of the three HMCLs analysed.
The down-regulation of MAGED1 has also been reported in the XG1 HMCL knocked out for FAM46C. 16 Although the down-regulation of these factors by FAM46C inactivation might contribute to the great migration ability observed in JJN3 FAM46C KO cells, this effect seems to be cell type-specific and genetic background-dependent. Consistent with our findings, a recent study has demonstrated that overexpression of FAM46C in hepatocellular carcinoma reduced cell migration and invasion. 48 The authors showed that forced expression of the ncPAP suppressed EMT. However, we found here that the increased migratory ability of FAM46C KO MM cells seems to be EMT-independent. Conversely, we found that the increased rate of

CO N FLI C T O F I NTE R E S T
The authors declare no conflict interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. Additional methods are detailed in Appendix S1.