The microRNA-210-Stathmin1 Axis Decreases Cell Stiffness to Facilitate the Invasiveness of Colorectal Cancer Stem Cells

Simple Summary Metastasis of tumor cells is the leading cause of death in cancer patients. Concurrent therapy with surgical removal of primary and metastatic lesions is the main approach for cancer therapy. Currently, therapeutic resistant properties of cancer stem cells (CSCs) are known to drive malignant cancer progression, including metastasis. Our study aimed to identify molecular tools dedicated to the detection and treatment of CSCs. We confirmed that microRNA-210-3p (miR-210) was upregulated in colorectal stem-like cancer cells, which targeted stathmin1 (STMN1), to decrease cell elasticity for increasing mobility. We envision that strategies for softening cellular elasticity will reduce the onset of CSC-orientated metastasis. Abstract Cell migration is critical for regional dissemination and distal metastasis of cancer cells, which remain the major causes of poor prognosis and death in patients with colorectal cancer (CRC). Although cytoskeletal dynamics and cellular deformability contribute to the migration of cancer cells and metastasis, the mechanisms governing the migratory ability of cancer stem cells (CSCs), a nongenetic source of tumor heterogeneity, are unclear. Here, we expanded colorectal CSCs (CRCSCs) as colonospheres and showed that CRCSCs exhibited higher cell motility in transwell migration assays and 3D invasion assays and greater deformability in particle tracking microrheology than did their parental CRC cells. Mechanistically, in CRCSCs, microRNA-210-3p (miR-210) targeted stathmin1 (STMN1), which is known for inducing microtubule destabilization, to decrease cell elasticity in order to facilitate cell motility without affecting the epithelial–mesenchymal transition (EMT) status. Clinically, the miR-210-STMN1 axis was activated in CRC patients with liver metastasis and correlated with a worse clinical outcome. This study elucidates a miRNA-oriented mechanism regulating the deformability of CRCSCs beyond the EMT process.


Cell Culture
Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium (Gibco/Life Technologies, New York, NY, USA). Human colorectal carcinoma HT29, HCT15 and Colo205 cells were cultured in RPMI-1640 medium (Gibco/Life Technologies). SW1116 human colorectal carcinoma cells were cultured in L-15 medium (Gibco/Life Technologies). The above media were supplemented with 10% fetal bovine serum (FBS, Gibco/Life Technologies). HEK293, HT29, HCT15 and Colo205 cells were cultured in a humidified incubator at 37 • C with 5% CO 2 . SW1116 cells were cultured in a humidified incubator at 37 • C with atmospheric air. HCT15-vec and HCT15-Snail stable clones were generated previously [24]. The authenticity of cell lines was verified by examining their DNA-short tandom repeat (STR) profiles.

Expansion of Colorectal Cancer Stem Cells
To expand sphere-derived cancer stem cells (SDCSCs), a single-cell suspension was prepared, and cells were cultured in stem cell medium (SCM; DMEM/F12 supplemented with N2 Plus Supplement (Invitrogen, New York, NY, USA), 10 ng mL −1 recombinant bFGF (PeproTech Asia, Suzhou, China), 10 ng mL −1 EGF (PeproTech Asia) and 1% penicillinstreptomycin (Gibco/Life Technologies) for 20 days to form tumor spheres. TryPLE express (Gibco/Life Technologies) was used to dissociate cells and SDCSCs to prepare single-cell suspensions for experiments. Cells were cultured in a humidified incubator at 37 • C with 5% CO 2 .

Transwell Migration Assay
RPMI medium (600 µL) supplemented with 10% FBS was added to the bottom wells, and 2 × 10 5 cells suspended in basal RPMI medium were then seeded in the 6.5 mm diameter upper chamber with an 8 µm pore size membrane (Corning, New York, NY, USA) and incubated for 20 h. Cell suspensions in the upper inserts were discarded, and the remaining cells were removed with cotton swabs. Cells adhering to the underside of the membranes were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and stained with 1% crystal violet reagent (Sigma-Aldrich) for 30 min at room temperature. Images were acquired with an inverted microscope (Eclipse Ts-2, Nikon Instruments Inc., Tokyo, Japan), and the migrated cells in a 10x low-power field (LPF) were counted for quantification.

Two-and-a-Half Dimentional (2.5D) Time-Lapse Trajectory
For measuring 2.5D cell motility, a mixture of 0.85 mL of 3 mg mL −1 PureCor bovine collagen solution (Advance Biomatrix, Carlsbad, CA, USA), 0.3 mL of 5× RPMI basal medium, 6 µL of 1 M NaOH and 0.35 mL of water to a total volume of 1.5 mL was prepared as the collagen solution. Then, 250 µL of the collagen solution was added to wells in a 24-well plate for solidification at 37 • C for 30 min. A total of 3 × 10 4 cells were suspended in RPMI basal medium and seeded on top of the thick collagen layer for 6 h prior to time-lapse recording using an IX83 inverted microscope (Olympus Corporation, Tokyo, Japan). Images were acquired every 10 min for up to 5 h, and a video was exported using cellSens software (Olympus Corporation).

Three-Dimensional (3D) Invasion Assay
A total of 1 × 10 6 cells were suspended in 500 µL of basal RPMI medium and plated in one well of a four-well chambered borosilicate coverglass slide (Lab-tek, New York, NY, USA) overnight for attachment. Then, the supernatant was removed, and the surface of the well was covered with 350 µL of the collagen solution described above for 30 min to allow solidification. Then, 700 µL of basal RPMI medium was added, and the slide was incubated at 37 • C and 5% CO 2 for 72 h. After incubation, cells were fixed with 4% formaldehyde (Sigma-Aldrich) at 4 • C overnight and mounted with Fluoroshield with DAPI (Sigma-Aldrich). Confocal images (49 layers) of each well were acquired at 1.5 µm steps from the bottom to a height of 73.5 µm with an Olympus FV1000 laser confocal microscope (Olympus Corporation).

Paired Cell Assay
For BrdU labeling, cells were cultured in medium containing 0.5 µM BrdU (Sigma-Aldrich) for 2 weeks to ensure BrdU incorporation in cells. Cells were then synchronized with 40 ng mL −1 nocodazole (Sigma-Aldrich) overnight in the presence of 0.5 µM BrdU. For the BrdU chase, cells were washed intensively, trypsinized and seeded on poly-L lysine (Sigma-Aldrich)-coated coverslips placed in wells of 6-well plates in BrdU-free medium and synchronized through sequential exposure to thymidine, nocodazole, and blebbistatin [25] to control cell division and entry into the second mitosis. Briefly, cells were treated with 5 mM thymidine (Sigma-Aldrich) for 24 h and released for 10 h prior to additional thymidine treatment for 24 h (the first round of division). The thymidine was then removed for 6 h prior to nocodazole treatment for 16 h to enrich mitotic-phase cells. The nocodazole was then removed by washing for 15 min prior to 50 µM blebbistatin (Sigma-Aldrich) treatment for up to two hours (the second round of division). Paired cells were observed at this stage. Cells were fixed with 4% paraformaldehyde for 30 min at 4 • C and permeabilized with 0.1% Triton X-100 for 5 min. Then, cells were immersed first in 1 N HCl for 10 min on ice and then in 2 N HCl/1% Triton X-100 for 45 min in a 37 • C incubator to open the DNA helix. Immediately after one acid wash with PBS, borate buffer (0.1 M, pH = 8) was used to buffer cells for 12 min at room temperature. Cells were washed again with PBS three times and incubated overnight with an antibody against BrdU (1:200, 14-5071-82, eBioscience, San Diego, CA, USA) and a fluorescein-conjugated goat anti-mouse antibody (1:200, F2761, Invitrogen). Cells were mounted with Fluoroshield with DAPI (Sigma-Aldrich), and images were acquired with a Leica DM600B fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Elasticity Measurements
Video particle tracking microrheology (VPTM) was used to measure the elastic modulus of cells [4,26]. A total of 2 × 10 6 cells were suspended in basal RPMI medium in a 10-ch dish, and 20 µL of 200 nM fluorescent carboxylated polystyrene particles (F8810, Invitrogen, fluorescence excitation/emission peaks: 580 nm/605 nm) was then injected into the cells with a biolistic particle delivery system (PDS-100; pressure, Bio-Rad, 450 psi, Hercules, CA, USA). Three hours after particle injection, the cells were washed twice with PBS and transferred to 35 mm glass bottom culture dishes (Alpha Plus, Saitama, Japan). After incubation for 4 h, the two-dimensional Brownian motion of intracellular fluorescent beads was recorded with an inverted epifluorescence microscope (Eclipse Ti, Nikon Instruments Inc.), equipped with an oil immersion objective (100×, NA = 1.45), a sCMOS camera (Hamamatsu, Hamamatsu-shi, Japan), and a cell incubation chamber (INUB-GSI-F1, TOKAI HIT, Fujinomiya City, Japan). The two-dimensional projection of the trajectories of the intracellular fluorescent beads was recorded for 10 s at a frame rate of 100 Hz and analyzed via customized MATLAB software (MathWorks, Natic, MA, USA) [27]. From the two-dimensional (2-D) position (x(t), y(t)) of each particle as a function of time, we calculated the ensemble-averaged mean square displacement (MSD, Kenilworth, NJ, USA), the effective creep compliance, and the elastic modulus G'(ω) [28]. The subcellular locations of injected particles were observed using confocal microscopy (LSM 880, ZEISS, Oberkochen, Germany), and 3D images were generated using ZEISS Zen software.

Cell Viability, Clonogenicity and Sphere Formation Assays
A total of 1 × 10 4 cells were suspended in 100 µL of complete RPMI medium and seeded in wells in 96-well plates for 48 h. The medium was discarded, and MTT reagent (Sigma-Aldrich) was added to the cells for 45 min at 37 • C. Mitochondrial MTT crystals were dissolved with DMSO (J.T. Baker, Phillipsburg, NJ, USA), and the optical density values were then read in a microplate reader (SpectraMax 250, Molecular Devices Corp., San Jose, CA, USA). To evaluate clonogenicity, 1 × 10 4 cells were resuspended in complete RPMI medium and seeded in wells of a 6-well plate for 10 days. Colonies were visualized by crystal violet staining prior to counting. For sphere formation assays, 1000 cells were suspended in SCM, and the spheroids were counted after 10 days. Images were acquired with an inverted microscope (Eclipse Ts-2, Nikon Instruments Inc.), and spheroids in a 4× LPF were counted for quantification.

Bioinformatic Analysis
The small RNA-seq (smRNA-seq) data of HT29 cells, HCT15 cells and expanded SDCSCs were collected from GSE43793. The smRNA-seq and RNA-seq data for the TCGA COAD dataset were retrieved from established databases: DriverDB [29] and YM500 [30]. In-house pipelines were used to estimate the expression levels of miRNAs (30) as reads per Human microRNA-V2 chip platforms, respectively, were downloaded from the CellMiner database [31]. We used Gene Set Enrichment Analysis (GSEA) (http://www.broadinstitute. org/gsea (accessed on 20 June 2020) to assess degree of association defined signature and expression profiles of CRC patients downloaded from GSE17538. The clinical phenotypes were used for permutation.

Immunohistochemical (IHC) Assay
Sections of tissues (4 µm thick) from microarrays were deparaffinized and rehydrated before staining. Tissue antigens were retrieved by autoclaving in 10 mM (pH 6) citrate buffer for 10 min. Sections were cooled on ice for 30 min before treatment with 3% H 2 O 2 . Samples were permeabilized with 0.2% Triton X-100 (Sigma) in DPBS and reacted with a diluted primary STMN1 antibody (1:200, 13655S, Cell Signaling). Signals were amplified and detected with a Super Sensitive TM Link-Label IHC Detection System (BioGenex, Fremont, CA, USA) according to the instructions and counterstained with hematoxylin QS (Vector, Burlingame, CA, USA) for 20 s. The H-scores represent the percentage of STMN1 immunoreactivity-positive regions multiplied by the STMN1 staining intensity. Images were acquired with a BX43 light microscope equipped with a DP22 CCD camera (Olympus).

Preparation of Patient-Derived Xenografts (PDXs)
The experimental animal procedure was approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Veterans General Hospital (2018-191). The CRC specimens were first rinsed twice with DPBS and immersed in Matrigel (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37 • C. The tumors were then cut into L mm 3 pieces and implanted subcutaneously into 4-week-old female nude mice to establish patient-derived xenografts (PDXs). The mice were sacrificed, and tumors were homogenized in TRIzol ® reagent (Life Technologies) and subjected to total RNA isolation.

Biological Samples
This study was approved by the Institutional Ethics Committee/Institutional Board of Taipei Veterans General Hospital (2016-03-001BC, 2018-11-002C). Two sets of human specimens were used. First, 2 CRC specimens (one primary tumor and one unpaired liver metastatic tumor) were collected to prepare PDXs with informed consents. Second, 11 paraffin-embedded sections from the paired primary and metastatic CRC specimens collected from the tissue biobank were subjected to IHC staining.

Statistical Analysis
Unless indicated otherwise, Student's t-test was used to assess the significance of differences. The Pearson correlation analysis was used to analyze correlations between two factors described by continuous data. The log-rank test was used for survival analysis. The x 2 test was applied for comparisons of dichotomous variables. Two-tailed p-values of <0.05 were considered to indicate significant differences.

Small RNA Sequencing (smRNA-seq) Reveals Enhanced miR-210-3p Expression in CRCSCs
In an attempt to discover mechanisms regulating the motility of CSCs, we initiated this study by expanding CRCSCs from two CRC cell lines, HT29 and HCT15, using a serum-free cultivation platform, because stem-like cancer populations were enriched as cancer spheroids [32]. We found that the expanded HT29-and HCT15-CRCSCs grew as suspended colonospheres ( Figure 1a) and showed increased expression of stemness genes, including NANOG, POU5F1, LGR5, CD44, and SNAI1 ( Figure S1a).
The resultant spheroids are referred to as sphere-derived cancer stem cells (SDCSCs) hereafter. Both HT29-and HCT15-SDCSCs exhibited higher transwell migration capacity ( Figure 1b) and enhanced three-dimensional (3D) vertical invasiveness ( Figure 1c) than their parental cells, and cell viability was not affected (Figure 1d). The top 500 upregulated gene signature analyzed in HT29-SDCSCs (GSE14773) was positively associated with the expression profiles of recurrent ( Figure S1b, upper) and late stage ( Figure S1b, lower) CRC patients deposited at GSE17538, suggesting the expanded SDCSCs are migrating CRCSCs.
Next, we sought to identify the primary microRNA(s) (miRNAs) responsible for CRCSC motility, because miRNA deregulation is critically involved in cancer progression [33]. Global miRNA expression patterns of HT29-SDCSCs, HCT15-SDCSCs, and their parental cells (GSE43793) were explored by small RNA sequencing (smRNA-seq). The miRNAs with log counts per million (logCPM) values of >1 and fold changes of ≥2 were selected for examination of their clinical relevance. The Venn diagram shows the nine differentially expressed miRNAs in HT29-SDCSCs and HCT15-SDCSCs ( Figure 1e); four miRNAs were significantly upregulated and three were downregulated in both SDCSC datasets ( Figure 1f). To explore the clinical relevance of these seven dysregulated miR-NAs in CRC, we evaluated these miRNAs according to the patient survival data in the TCGA COAD dataset (n = 425 patients) and found that miR-210-3p (miR-210) was the only miRNA both enriched in SDCSCs and associated with poor overall survival ( Figure 1g

MiR-210 Is Required for the Migration and Invasiveness of CRCSCs
We investigated the functional roles of miR-210 in SDCSCs. In human cancers, accumulated evidence suggests that defects in asymmetric cell division (ACD) and increased symmetric cell division (SCD) of somatic stem cells expand stem cell pools and fuel tumor growth [34,35]. As stem cells tend to retain the mother strand (old) DNA template in one daughter stem cell and segregate newly synthesized DNA strands to differentiating daughter cells [36], we investigated the segregation of mother strand DNA in SDCSCs by pulse-chase BrdU labeling and paired cell assays ( Figure S2a). Upon knockdown of miR-210 in HT29 SDCSCs with a specific antagomir ( Figure S2b), predominantly symmetrical BrdU segregation (i.e., SCD) was observed ( Figure S2c We investigated the functional roles of miR-210 in SDCSCs. In human cancers, accumulated evidence suggests that defects in asymmetric cell division (ACD) and increased symmetric cell division (SCD) of somatic stem cells expand stem cell pools and fuel tumor growth [34,35]. As stem cells tend to retain the mother strand (old) DNA template in one daughter stem cell and segregate newly synthesized DNA strands to differentiating daughter cells [36], we investigated the segregation of mother strand DNA in SDCSCs by pulse-chase BrdU labeling and paired cell assays ( Figure S2a). Upon knockdown of miR-210 in HT29 SDCSCs with a specific antagomir ( Figure S2b), predominantly symmetrical BrdU segregation (i.e., SCD) was observed ( Figure S2c,d), indicating that silencing miR-210 did not promote early differentiation of SDCSCs. The observation of the reduced sphereforming capacity of HT29-SDCSCs with stable miR-210 knockdown (Zip-210) (Figure 2a

Stathmin1 Expression Attenuates the Motility of CRCSCs
STMN1 is a phosphoprotein regulated by extracellular signals and can bind to α/βtubulin to modulate microtubule dynamics [40,41]. We next evaluated whether STMN1 is involved in regulating the motility CRCSCs. We found that restoration of STMN1 expression in HT29-and HCT15-SDCSCs (Figure 4a) did not alter the viability (Figure 4b), sphere-forming capacity (Figure 4c), or clonogenicity (Figure 4d,e) of these cells. Although STMN1 was shown to regulate EMT [42], restoration of STMN1 expression decreased the transwell migration ability ( Figure 4f) and invasiveness (Figure 4g,h) of both HT29-and HCT15-SDCSCs without affecting a complete EMT program ( Figure S3a-c).
In an attempt to verify the participation of the miR-210-STMN1 axis in mediating SDCSC motility, we silenced STMN1 expression in miR-210 knockdown HCT15-SDCSCs. The reduced expression of STMN1 was first confirmed (Figure 5a). We found that silencing STMN1 expression had no effects on the viability (Figure 5b), sphere-forming capacity (Figure 5c

The miR-210-STMN1 Axis Determines the Stiffness of CRCSCs
As SDCSCs must change their shapes while migrating through 8-μm pores in the transwell migration assay and through the collagen matrix in the 3D invasion assay, and

The miR-210-STMN1 Axis Determines the Stiffness of CRCSCs
As SDCSCs must change their shapes while migrating through 8-µm pores in the transwell migration assay and through the collagen matrix in the 3D invasion assay, and the deformability of cancer cells is associated with their metastatic competence [43], we next examined the deformability of SDCSCs. To this end, we performed elasticity measurements by monitoring the time-lapse trajectory of injected fluorescent carboxylated polystyrene beads in dissociated single CRC cells (Figure 6a). the deformability of cancer cells is associated with their metastatic competence [43], we next examined the deformability of SDCSCs. To this end, we performed elasticity measurements by monitoring the time-lapse trajectory of injected fluorescent carboxylated polystyrene beads in dissociated single CRC cells (Figure 6a).  The intracellular fluorescent particles were mainly distributed in the cytoplasm (Figure 6b,c). The enhanced movement of the intracellular carboxylated polystyrene beads in HCT15-SDCSCs (Video S1) suggested the reduced intracellular elasticity (i.e., enhanced deformability) of SDCSCs (Figure 6d, bars 1-2). Furthermore, knockdown of miR-210 (Zip-210) in HCT15-SDCSCs restored the elastic modulus (stiffness), and silencing STMN1 expression reversed the reduction in elasticity in miR-210 knockdown HCT15-SDCSCs (Figure 6d, bars 3-4). In contrast, interference with the miR-210-STMN1 axis had limited impact on the 2.5D horizontal movement of HCT15-SDCSCs on the collagen gel (Figure 6e). Collectively, these results indicated that the miR-210-STMN1 axis determined the deformability of SDCSCs to facilitate their motility.

The miR-210-STMN1 Axis Promotes the Stiffness of CRC Cells
To validate impacts of the miR-210-STMN1 axis on parental CRC cells, we ectopically express STMN1 in HT29 parental cells receiving miR-210 agomiRs. The expression of miR-210 ( Figure S4a) and Myc-DDK-tagged STMN1 ( Figure S4b) were conformed. It was found that the miR-210-STMN1 axis had limited impacts on the viability ( Figure S4c) and clonogenicity ( Figure S4d,e) of HT29 parental cells. On the contrary, the enhanced motility ( Figure S4f) and reduced elasticity ( Figure S4g) of HT29 cells receiving miR-210 agomiR could be reverted upon expressing STMN1 without modulating the EMT markers (CDH1. CDH2 and VIM) ( Figure S4h). Consistently, we found that expression of STMN1 in parental HT29 cells ( Figure S5a) had no effect on cell viability ( Figure S5b), clonogenicity ( Figure S5c,d) or EMT marker expression ( Figure S5e). However, the transwell migration ability was reduced ( Figure S5f) and the cellular elasticity was elevated ( Figure S5g) upon STMN1 expression. This results indicated the miR-210-STMN1 axis identified in CRCSCs also contributes to deformability and motility of CRC cells.

The miR-210 High /STMN1 Low Expression Signature Is Associated with Liver Metastasis and Predicts a Poor Clinical Outcome in CRC Patients
As enhanced cellular deformability benefits local dissemination in the extracellular matrix (ECM), along with intravasation and extravasation, the miR-210 High /STMN1 Low expression signature may predict distal metastasis in CRC patients. To this end, we examined the expression of miR-210-STMN1 axis components in primary and paired liver metastatic CRC specimens from our collection and databases in the public domain. Increased expression of miR-210 and decreased expression of STMN1 were observed in liver metastatic CRC samples from GSE54088 and GSE3964 datasets, respectively (Figure 6f). Additionally, decreased STMN expression in liver metastastic CRC patients was verified in paired, paraffin-embedded tissues by IHC staining (Figure S6a,b). However, the expression of STMN1 were not changed in lymph node metastatic ( Figure S6c) or lung metastatic CRC specimens ( Figure S6d) comparing to liver metastasis of CRC. As miR-210 High /STMN1 Low expression signature was associated with CRC metastasis that contributes to poor patient outcomes, we verified the clinical significance of the miR-210 High /STMN1 Low expression pattern. In analysis of the TCGA data sets, the miR-210 High /STMN1 Low expression signature predicted worse CRC patient survival (Figure 6g). Taken together, our results suggested the elevated expression of miR-210 attenuated STMN1 expression to engender deformability of CRCSCs for facilitating invasiveness, resulting in poor prognosis of CRC patients (Figure 6h).

Discussion
STMN1, also called oncoprotein 18 (Op18), metablastin (p19) and prosolin, is identified as a cytosolic microtubule-destabilizing phosphoprotein [44]. Unphosphorylated STMN1 promotes microtubule depolymerization by sequestering soluble tubulin and promotes microtubule catastrophe [45,46]. STMN1 contains four serine phosphorylation sites (Ser16, Ser25, Ser38, and Ser63), and the microtubule-destabilizing ability of STMN1 is regulated by its phosphorylation [47,48]. Phosphorylation of STMN1 in early mitosis abolishes its microtubule-destabilizing ability, allowing the formation of mitotic spindles, and it becomes dephosphorylated when cells exit mitosis and undergo cytokinesis [40]. Overexpression or inhibition of STMN1 expression in K562 cells resulted in accumulation of mitotic cells that were arrested in early and late mitotic stages, respectively [40,49], suggesting a threshold level of STMN1 is required for mitosis progression. Aside from cell cycle regulation, roles of STMN1 in hematopoiesis have been addressed in leukemia cells. STMN1 is abundant in acute leukemia blasts [50] and its expression was decreased when inducing differentiation by exposing an acute promyelocytic leukemia cell line HL60 to Me 2 SO or exposing erythroleukemia cells K562 to hemin [51]. Inhibition of STMN1 promoted higher megakaryocytic differentiation and polyploidization of phorbol ester-induced K562 cells [52]. On the contrary, overexpression of STMN1 in human primary CD34(+) cells reduced the megakaryocyte maturation and platelet production [53]. The megaloblastic anemia and thrombocytosis phenotypes observed in aged Stmn1 knockout mice further support STMN1 s roles in hematopoiesis [54]. Additionally, aged Stmn1 deficient mice also developed a progressive axonopathy [55]. Under social defeat stress, Stmn1 deficient mice showed anxious hyperactivity, impaired object recognition, and decreased levels of social investigating behaviors [56]. Thus, pleiotropic roles of STMN1 are highlighted.
In cancers, STMN1 expression correlates with a malignant phenotypes and has been suggested as a therapeutic target [57]. Silencing STMN1 expression inhibited the metastatic ability of a CRC cell line HCT-116 [58]. STMN1 expression was associated with aggressive phenotypes in breast cancer [59]. The oncogenic Stathmin1 is also regulated by a tumor suppressor miRNA-223 in gastric cancer [60] and liver cancer [61] or a tumor suppressor miR-34a in prostate cancer [62]. Overexpression of the somatic STMN1 Q18E mutation identified in esophageal adenocarcinoma promoted the malignant transformation of 3T3 fibroblast cells [63] and chromosomal instability in K562 cells [64]. A S31Y STMN1 missense mutation was noted in colorectal cancer patients analyzed with Tu-morPortal (http://www.tumorportal.org (accessed on 4 May 2020)) without functional annotation [65]. Nevertheless, D'Andrea et al. showed that Stmn1 knockout mice showed no impact on the onset of the p53-dependent nor RAS-driven tumorigenesis in bladder and fibrosarcomas or skin carcinomas in mice, respectively [66], suggesting cellular context may contribute to diverse functions of STMN1 during oncogenesis.
As local inactivation of STMN1 at the leading edge of the migrating Xenopus A6 cells potentiated localized microtubule growth, STMN1 may function as a negative regulator in cell movement [67]. Consistently, tumor suppressive roles of STMN1 were identified in prostate cancer cells [42]. Williams et al. showed that the highly invasive, EMT-like prostate cancer cells isolated from undifferentiated adenocarcinoma exhibited low STMN1 expression. Inhibition of STMN1 expression in a prostate cancer cell line DU-145, a standard prostate cancer cell line used for CSC enrichment [68], accelerated the metastatic process by initiating an EMT program via activation of p38 and cooperation of TGF-β signaling [42]. In this study, we identified an increased expression of an oncomiR miR-210 ( Figure 1h) in both HT29-and HCT15-CRCSCs characterized previously [24] and showed the miR-210 mediated STMN1 suppression in CRCSCs (Figure 3i). The tumor suppressive roles of STMN1 were demonstrated by observing the reduced invasiveness of STMN1restored CRCSCs (Figure 4g,h) and decreased motility of STMN1-overexpressed HT29 cells ( Figure S5f), a CRC cell line exhibits higher stem-like properties [24]. The EMT program was found to be disconnected from the miR-210-STMN1 activated invasiveness of both CRCSCs (Figure 5h) and HT29 cells ( Figure S4h). Here, we unraveled metastatic inhibition effects of STMN1 in our CRC cell models. Reduced STMN1 expression was also observed in paired, liver metastatic CRC specimens ( Figure S6a,b). Taken together, these findings indicate that STMN1 tends to function as a metastatic suppressor in stem-like tumor cells and suggest that understanding the stemness profiles and numbers of stem-like cells in cancer patients are crucial for utilizing STMN1 as a therapeutic target.
According to the present results and our previous findings about CRCSCs, we propose a model in which CRCSCs trigger different signaling pathways to maintain cancer stemness and subsequent metastasis: CRCSCs are Snail-dominant cells that undergo EMT [24]. In CRCSCs, Snail suppresses E-cadherin, leading to EMT and cellular disaggregation. Decreased E-cadherin expression results in nuclear translocation of β-catenin and activation of the Wnt pathway, which induces miR-146a expression in CRCSCs. During seruminduced differentiation, mIR-146a could be segregated non-randomly into CD44(+), Snail(+) daughter colorectal stem cells to initiate a feedforward β-catenin/TCF signaling to maintain stem cell pools without promoting CRCSC migration by targeting NUMB [35]. Here, we identified one additional oncomiR, namely miR-210, that suppressed STMN1 expression to facilitate invasiveness of CRCSCs. The ectopic Snail expression was not found to activate the miR-210-STMN1 axis in CRC cells ( Figure S7a,b), indicating the miR-210-STMN1 axis was disconnected from an EMT program. Our findings suggest that Snaildominant CRCSCs uncouple cancer cell division mode and deformability by utilizing distinct miRNAs for maintaining aggressive CSC phenotypes. The sequential activation of miR-210 and miR-146 and the collaboration of these miRNAs with other coding and noncoding genes in the TME require further investigation.
Our study has some limitations. First, our findings mainly rely on CRC cell linederived cell models, primary cells or CSCs isolated from different tumor types may help to delineate dual roles of STMN1 under diverse context of cells or tissues. Second, the molecular mediators driving dual roles of STMN1 and STMN1-driven metastasis need further exploration.

Conclusions
The significance of our study is double-edged. Scientifically, the miR-210-STMN1 axis determines the invasiveness of CRCSCs without affecting cancer stemness. As the low STMN1 expression is essential for migrating of CRCSCs, using STMN1 as a therapeutic target might accelerate metastasis of CRCSCs. Clinically, this study proposes a miR-210 High /STMN1 Low expression pattern as a potential indicator for monitoring the liver metastasis longitudinally along with therapeutic regimes.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cancers13081833/s1, Figure S1: Characterization of sphere-derived cancer stem cells (SDC-SCs), Figure S2: Silencing miR-210-3p expression does not affect the symmetric cell division (SCD) of HT29-SDCSCs, Figure S3: Restoration of STMN1 does not consistently alter the expression of epithelial and mesenchymal markers in HT29-and HCT15-SDCSCs, Figure S4: Ectopically expression of STMN1 reverses the miR-210-regulated trans well migration ability and elasticity of HT29 parental cells, Figure S5: Overexpression of STMN1 inhibits the transwell migration potential and promotes intracellular elasticity of HT29 parental cells, Figure S6:Decreased expression of STMN1 in liver metastatic CRC specimens, Figure S7: Examining the expression of miR-210-3p-STMN1 axis components in Snail-expressing HCT15 cells, Figure S8: Uncropped blots, Video S1: A video showing the Brownian motion of intracellular fluorescent beads in HCT15 cells and HCT15-SDCSCs. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.