Loss of KAP3 decreases intercellular adhesion and impairs intracellular transport of laminin in signet ring cell carcinoma of the stomach

Signet-ring cell carcinoma (SRCC) is a unique subtype of gastric cancer that is impaired for cell–cell adhesion. The pathogenesis of SRCC remains unclear. Here, we show that expression of kinesin-associated protein 3 (KAP3), a cargo adaptor subunit of the kinesin superfamily protein 3 (KIF3), a motor protein, is specifically decreased in SRCC of the stomach. CRISPR/Cas9-mediated gene knockout experiments indicated that loss of KAP3 impairs the formation of circumferential actomyosin cables by inactivating RhoA, leading to the weakening of cell–cell adhesion. Furthermore, in KAP3 knockout cells, post-Golgi transport of laminin, a key component of the basement membrane, was inhibited, resulting in impaired basement membrane formation. Together, these findings uncover a potential role for KAP3 in the pathogenesis of SRCC of the stomach.

www.nature.com/scientificreports/ Quantitative reverse transcription-PCR (qRT-PCR). Total RNA was extracted from each cell line using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Residual genomic DNA was removed by incubating the RNA samples with RNase-free DNase I (Takara Bio, Otsu, Japan) prior to performing qRT-PCR. The mRNA levels were quantified by qRT-PCR using the LightCycler 96 system (Roche Diagnostics, Mannheim, Germany) and the KAPA SYBR FAST Universal Kit (KAPA Biosystems, Cape Town, South Africa) according to the manufacturer's protocol. The endogenous control for mRNA was the ACTB transcript encoding the housekeeping protein β-actin. The primers used were as follows: KAP3 (KIFAP3) mRNA: forward, AGG AGC CAT AAG TCC CGA TT, and reverse, GTC CAA GAA TGC CAA CTG GT; RHOA mRNA: forward, AAG GAC CAG TTC CCA GAG GT, and reverse, GCT TTC CAT CCA CCT CGA TA; ACTB mRNA: forward, GTC CAC CTT CCA GCA GAT GT, and reverse, TGT TTT CTG CGC AAG TTA GG.
Immunohistochemistry. Immunohistochemistry was performed as described previously 22 . The anti-KAP3 antibody was used for immunohistochemistry at a dilution of 1:50. The GC tissue microarray (Cat. #NBP2-30308) was obtained from Novus Biologicals (Centennial, CO, USA). Expression levels were evaluated semi-quantitatively as follows: each sample was scored for two parameters. The first parameter was the percentage of positive cells, which was determined using a scale as follows: 0, absence of positive cells; 1, < 20% of cells are positive; 2, 20-50% positive; 3, > 50% positive. The second parameter was the intensity of the immunostaining, which was scored as follows: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. Scores for the two parameters were multiplied, yielding a product with a value ranging from 0 to 9. Representative examples are shown in Supplementary Fig. 1.
Immunofluorescence. Cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100. The following primary antibodies and dilutions were used for immunofluorescence staining: anti-ZO- Image analysis. The relative staining intensity was measured using line scans of fluorescence intensities with ImageJ software 23 . Lines were drawn across randomly selected cell-cell junctions, and peak fluorescence intensities were measured using plot profile function. Colocalization was quantified by calculating Mander's colocalization coefficient, using ImageJ Coloc 2 software 24 .
Cell aggregation assay. Cells were seeded in 60-mm non-adherent dishes (Nunclon Sphera Dishes, Thermo Fisher Scientific) at a density of 20,000 cells/mL and cultured to form cell aggregates. After 10 days of culturing, the medium containing the cells was transferred into a 15-mL conical tube, and the cells were allowed to settle (pellet) by gravity sedimentation for 5 min to separate cell aggregates from cells that remained in suspension. The supernatant was recovered by careful aspiration, and the number (N1) of cells in the supernatant was counted. The pellets were dissociated into single cells by incubation with trypsin/EDTA followed by pipetting, and the number (N2) of cells in the pellets was counted.

Results
KAP3 expression is decreased in SRCC of the stomach. We investigated the gene expression prolife of GC using a web tool called Reference Expression dataset (RefEx; http:// refex. dbcls. jp/) 25 and noticed that KAP3 mRNA expression, which is ubiquitous in mammalian cells, was markedly decreased in NUGC4 and KATOIII, cell lines that are derived from SRCC of the stomach. We confirmed that KAP3 expression, at both the protein and mRNA levels, was decreased in NUGC4 and KATOIII cells compared with cell lines derived from other histological subtypes of GC (i.e., tubular adenocarcinoma, MKN7 and MNK74 cells; poorly differentiated adenocarcinoma, MNK45 and NUGC3 cells), as determined by immunoblot and qRT-PCR analysis (Fig. 1A,B). Immunohistochemistry revealed that the expression levels of KAP3 were significantly lower in primary SRCC (sig) than in tubular adenocarcinoma (tub) and poorly differentiated adenocarcinoma (por) of the stomach (Fig. 1C,D). Phase contrast microscopy showed that KAP3 KO cells, unlike WT cells, had a clear halo artifact 26 around the cell boundaries (Fig. 2B), a finding suggestive of decreased ("looser") cell-cell adhesion 27 .
To further evaluate cell-cell adhesion, we performed a cell aggregation assay on cells grown in non-adherent dishes. Cell aggregates were smaller in KAP3 KO cells compared to WT cells (Fig. 2C), and the percentage of aggregated cells was significantly lower in the mutant than in the WT (Fig. 2D), consistent with the abovementioned loosened cell-cell adhesion of KAP3 KO cells.
To examine whether there was a difference in anoikis resistance between WT and KAP3 KO cells, cell viability assays were performed in non-adherent 96-well plates. No difference in cell viability was observed between WT and KAP3 KO cells (Fig. 2E), suggesting that the difference in cell aggregation was due to the difference in cell-cell adhesion, but not to anoikis resistance.
KAP3 depletion causes RhoA inactivation and consequent failure of circumferential actomyosin cable formation. Given the decreased cell-cell adhesion of KAP3 KO cells, we postulated that AJC components might be altered in mutant cells. Although immunofluorescence for ZO-1, E-cadherin, and β-catenin showed no significant differences in fluorescence intensity and distribution between KAP3 WT and KO cells (Fig. 3A,B), rhodamine-phalloidin staining indicated a different distribution of F-actin in the two cell lines (Fig. 3C, upper). WT cells exhibited dense staining of F-actin around the inside of the cell membrane, indicating the formation of circumferential actomyosin cables along the cell-cell junction. In contrast, KAP3 KO cells exhibited a patchy distribution of F-actin in the cytoplasm, suggesting the failure of circumferential actomyosin cable formation. These findings were confirmed by quantification of immunosignals for F-actin across the cell junctions by densitometric scanning (Fig. 3D). www.nature.com/scientificreports/ Because actomyosin contractility, which strengthens cell-cell adhesion, is regulated by active RhoA at the cell membrane, we examined expression of RhoA in WT and KAP3 KO cells. Immunofluorescence analysis showed decreased localization of RhoA at the cell membrane in KAP3 KO cells compared to WT cells (Fig. 3C, lower).
Another KAP3 KO clone (KO #2), established independently, showed a similar phenotype for the subcellular mislocalization of F-actin and RhoA (Supplementary Fig. 2A, B). The SRCC cell line, NUGC4, completely lacks cell-cell adhesion ( Supplementary Fig. 3A). We showed that NUGC4 cells also do not show circumferential actomyosin cable formation or the localization of RhoA at the cell membrane ( Supplementary Fig. 3B).  www.nature.com/scientificreports/ RhoA activation was quantitated in KAP3 WT and KO cells using a pull-down assay. The amount of total RhoA was significantly higher in KO cells than in WT cells (Fig. 3E,F), although there was no difference in the level of RHOA mRNA (data not shown) or in the amount of active RhoA (Fig. 3E,F) between the two cell lines. As a result, the ratio of active RhoA to total RhoA in KAP3 KO cells was approximately three-fold lower than that in WT cells (Fig. 3E,F). These findings indicated that RhoA is inactivated in KAP3 KO cells compared to WT cells.
To confirm these findings, we conducted genetic rescue experiments by introducing a construct encoding HaloTag-fused KAP3 (HaloTag-KAP3) into KAP3 KO cells (Fig. 3G). The dense staining of F-actin around the inside of the cell membrane was restored in cells expressing HaloTag-KAP3 (Fig. 3H, upper). Furthermore, RhoA was re-localized to the cell membrane in cells that expressed HaloTag-KAP3 (Fig. 3H, lower).

KAP3 depletion inhibits post-Golgi transport of laminin.
Because the inactivation of RhoA also decreases microtubule stability, and microtubule disruption causes basement membrane breakdown 28 , we investigated expression of laminin, a key component of the basement membrane, in WT and KAP3 KO cells. Immunofluorescence analysis revealed an accumulation of laminin, which stained as spots with a small, rounded appearance or crescent shape, adjacent to the nucleus in KAP3 KO cells; in contrast, laminin was uniformly distributed throughout the cytoplasm in WT cells (Fig. 4A). Double staining of laminin and giantin, a Golgi marker,  www.nature.com/scientificreports/ indicated the localization of laminin primarily in the peri-Golgi area in KAP3 KO cells (Fig. 4B). Mander's colocalization coefficient analysis confirmed that the fraction of laminin colocalized with giantin was significantly higher in KO cells than in WT cells (Fig. 4C). Furthermore, the rescue experiments showed that laminin was re-distributed uniformly throughout the cytoplasm in cells that expressed HaloTag-KAP3 (Fig. 4D). Another KAP3 KO clone (KO #2) also showed the colocalization of laminin with giantin ( Supplementary Fig. 2C). These findings indicated pooling of laminin in the peri-Golgi area in KAP3 KO cells, suggesting inhibition of the post-Golgi transport of laminin in the mutant cell lines.
In the SRCC cell line NUGC4, a portion of the laminin appeared to co-localize with giantin (Supplementary Fig. 3C). Rescue experiments in NUGC4 cells suggested that laminin is distributed more uniformly in the cytoplasm in KAP3 KO cells that express HaloTag-KAP3, compared to KAP3 KO cells that do not express the fusion protein (Supplementary Fig. 3D).
WT cells were treated with nocodazole, an inhibitor of microtubule polymerization. The treatment resulted in co-localization of laminin with giantin (Fig. 4E), suggesting that the post-Golgi transport of laminin depends on microtubules.
Moreover, the expression of laminin protein was decreased in KAP3 KO cells compared to WT cells (Fig. 4F). Expression of laminin also was significantly decreased in two SRCC cell lines, NUGC4 and KATOIII, compared with MKN74 cells (i.e., KAP3 WT cells), the tubular adenocarcinoma cell line (Supplementary Fig. 3E).

Laminin deposition at basement membrane is impaired in SRCC of the stomach. Based on
the finding that the post-Golgi transport of laminin is inhibited in KAP3 KO cells, we postulated that laminin secretion might be reduced, leading to a defect of the basement membrane, in SRCC lacking KAP3 expression. To test this hypothesis, we performed immunohistochemistry for laminin in specimens of primary GC. Laminin was present at the basement membrane on which tubular adenocarcinoma cells resided, judging from the sheetlike deposition of stained material at the basal surface of those cells (Fig. 5A). However, laminin deposition was not observed at the surface of SRCC cells (Fig. 5A). Additionally, cytoplasmic staining of laminin in SRCC cells was weaker than that in tubular adenocarcinoma cells. Expression levels of laminin were significantly decreased in primary SRCC (sig) compared to tubular adenocarcinoma (tub) and poorly differentiated adenocarcinoma (por) of the stomach (Fig. 5B).
We further examined expression of KAP3 and laminin by immunohistochemistry in the normal gastric mucosa and in an early GC composed of a pure histological type of SRCC that is limited to the mucosa (Fig. 5C-E). KAP3 and laminin were co-expressed in the normal gastric mucosa (Fig. 5C). The staining intensities of KAP3 and laminin at the base of gastric glands were stronger than in the superficial portion. However, the expression of KAP3 and laminin was absent in SRCC (Fig. 5D, shown in dotted circles). Intriguingly, expression of KAP3 and laminin also was sparse or absent in non-tumor cells that were immediately adjacent to SRCC (Fig. 5D).
Taken together, these findings suggested that loss of KAP3 may impair the formation of the basement membrane through inhibition of the post-Golgi transport of laminin in SRCC of the stomach, and that expression of KAP3 and laminin may be decreased in the background gastric mucosa of SRCC.

Discussion
In the present study, we observed decreased expression of KAP3 in SRCC of the stomach. KAP3 is a component of KIF3, a multi-subunit protein that is ubiquitously expressed in mammalian cells 29 . KIF3 regulates microtubulebased transport that is critical for membrane organelle transport, anterograde fast axonal transport, and early embryonic and neuronal development [30][31][32][33][34] ; KIF3 also regulates microtubule organization 35 , as well as intraflagellar transport that is indispensable for the formation and maintenance of cilia and flagella 36 . Dysregulation of KIF3 contributes to ciliopathies such as polycystic kidney disease 37 , retinitis pigmentosa 38 , situs inversus 39 , and schizophrenia 40 . KIF3 also has been implicated in the tumorigenesis of several types of cancer, including brain tumor 32 , medulloblastoma 41 , breast cancer 42 , non-small cell lung cancer 43 , and prostate cancer 44 , primarily via dysregulation of Wnt signaling and ciliary function. KIF3A restrains canonical Wnt signaling through ciliary and non-ciliary mechanisms 32,45 . KIF3 regulates cell migration by transporting the tumor suppressor adenomatous polyposis coli (APC) to membrane protrusions 46 . KAP3 is known to interact with APC 46 , small GTP-binding protein GDP dissociation stimulator (Smg GDS) 47 , PAR-3 48 , and fodrin 49 . KAP3 deficiency in mouse neuroepithelium leads to malignant transformation due to impaired post-Golgi transport of N-cadherin 32 , suggesting a potential tumor-suppressing activity for KAP3. Although the mechanism by which KAP3 expression is decreased in SRCC of the stomach is unknown, we hypothesize that loss of KAP3 may contribute to the development and progression of SRCC.
Our results suggested that loss of KAP3 decreased the localization of RhoA at the cell membrane and consequently reduced RhoA activity. The inactivation of RhoA caused the failure of circumferential actomyosin cable formation, leading to decreased cell-cell adhesion in KAP3 KO cells. Although the mechanism by which KAP3 depletion led to impaired localization of RhoA at the cell membrane was not elucidated, Smg GDS may be involved in the mechanism. Smg GDS interacts with small GTPases possessing a C-terminal polybasic region (PBR), including proteins such as Rap1, RhoA, Rac1, and K-Ras. The PBR controls various functions of these small GTPases, including their interaction with other proteins and association with membranes 50 . Smg GDS is known to have direct interactions with both KAP3 and RhoA 47 . In addition to its function as guanine-nucleotide exchange factor (GEF) that activates RhoA, Smg GDS acts as a chaperone controlling RhoA prenylation, a modification that is essential for membrane localization 51 . KAP3 depletion might alter the localization and functions of Smg GDS, thereby inhibiting RhoA activity and localization at the cell membrane. www.nature.com/scientificreports/ small fraction of all RhoA protein is activated, primarily at the plasma membrane 52 . Activated RhoA at cellular protrusions is targeted for degradation by E3 ubiquitin ligases such as Smurf-1 53 . Therefore, the total levels of RhoA protein are determined primarily by the amount bound to RhoGDIs in the cell 52 . Given these previous findings, impaired RhoA localization at the cell membrane as a result of KAP3 depletion may increase the amount bound to cytosolic RhoGDIs, leading to the observed increase in total RhoA protein.
RHOA mutations recurrently occur in non-SRCC diffuse-type GC (i.e., poorly differentiated adenocarcinoma). Several lines of evidence have indicated that mutant RhoA works in a gain-of-function manner 54 . In contrast, RHOA mutation is rare in SRCC 14 . Our findings showed that loss of KAP3, which occurs in SRCC, causes RhoA inactivation. The clinicopathological significance of altered RhoA signaling may differ between SRCC and non-SRCC diffuse-type GC.
The post-Golgi transport of laminin was inhibited in KAP3 KO cells. ECM proteins, including laminin, are synthesized on ribosomes bound to the endoplasmic reticulum (ER) membrane, post-translationally modified in the ER, transported through the Golgi complex in vesicles, and then secreted to the cell surface. KAP3 is known to be concentrated around the ER 47 , implicating KAP3 in the secretory pathway. While one previous study showed the involvement of KAP3 in shuttle transport between the ER and the Golgi 55 , another demonstrated the contribution of KAP3 to the post-Golgi transport of N-cadherin to the cell-surface 32 . Thus, the precise function of KAP3 in intracellular transport remains elusive.
Teng et al. 32 have shown that post-Golgi transport of N-cadherin by the KIF3 molecular motor complex is crucial for maintaining a balance between proliferation and cell-cell adhesion of neural progenitor cells, and that the subcellular localization of N-cadherin is disrupted and cell aggregation activity is decreased in KAP3deficient cells.
Although the mechanism by which KAP3 depletion impaired the post-Golgi transport of laminin still needs to be elucidated, this effect may be mediated in part by the inactivation of RhoA. Notably, RhoA and its effector mDia1 regulate the stabilization of microtubules 56 , which are used as tracks for fast and directed transport, and the RhoA-mDia1 pathway is involved in regulation of the Golgi structure 57 .
The in vitro findings that the post-Golgi transport of laminin was inhibited in KAP3 KO cells were supported by immunohistochemical analysis using clinical specimens of GC. While laminin was present at the basement membrane of tubular adenocarcinoma cells, this protein was not observed at the surface of SRCC cells, suggesting the impairment of basement membrane formation in SRCC. Microscopically, SRCC exhibits discohesive cells infiltrating the lamina propria even in its early stage 58 . One possible explanation for this observation is a defect of the basement membrane that separates epithelial cell sheets from the lamina propria; in normal tissues, the basement membrane acts as a mechanical barrier that prevents malignant cells from invading the deeper tissues.
In the specimens of early SRCC, expression of KAP3 and laminin was absent in tumor cells. Notably, not only tumor cells themselves but also immediately adjacent non-tumor cells showed decreased expression of KAP3. Although the mechanism and significance of this finding are unknown, we hypothesize that the decreased expression of KAP3 may occur in the background gastric mucosa of SRCC prior to the development of SRCC, and so may serve as an early marker of carcinogenesis in SRCC.
Given that the gastric epithelium is continually exposed to the harsh luminal environment, discohesive single cells of SRCC should readily be cleared from the epithelium unless such cells are somehow retained in the lamina propria. Several studies have suggested that the development of discohesive gastric cells into cancer is difficult 59,60 . According to Hayakawa et al. 60 , conditional knockout of CDH1 in mouse gastric isthmus stem cells results in the development of atypical cells similar to SRCC. However, these atypical cells are gradually depleted before subsequently disappearing, and are incapable of developing into SRCC. Since CDH1 and RHOA mutations often co-occur in diffuse-type GC, most studies have assumed that anoikis inhibition by an altered RhoA pathway enables discohesive gastric cells to develop into SRCC 60,61 . Nonetheless, based on our observation, we assume that changes in the basement membrane resulting from the loss of KAP3 enable tumor cells to easily invade the lamina propria and thereby facilitate the initiation of SRCC. KAP3 deficiency decreased cell-cell adhesion, and impaired basement membrane formation. These traits may enable tumor cells to deviate from epithelial cell sheets as single cells, and to persist in the lamina propria, leading to the initiation and progression of SRCC.
In conclusion, loss of KAP3 was specifically detected in SRCC of the stomach, and was shown to result in decreased RhoA-mediated cell-cell adhesion and impaired basement membrane formation through inhibition of the post-Golgi transport of laminin. This study provides new insights into the carcinogenesis of SRCC by uncovering new functions of KAP3 in the regulation of RhoA signaling and laminin transport. The precise molecular and physical mechanisms underlying KAP3 function in these processes are challenging subjects for future study. Loss of KAP3 expression could be a useful marker that predicts onset risk of SRCC, and could serve as a molecular target for treatment of this cancer.

Data availability
The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.