Phosphatase PTPN22 acts as a helper protein in the mTORC2 cell signaling complex

While PTPN22 is known to be expressed in hematopoietic cells and involved in immune signaling (Bottini and Peterson, 2014; Cloutier and Veillette, 1996; Cohen et al, 1999; Matthews et al, 1992; Stanford and Bottini, 2014; Zhang et al, 2020), its expression and role in non-hematopoietic cells remain less explored. To address this, we screened epithelial cell lines (HCT116, HeLa, HEK293T, and U-2OS) for PTPN22 expression using RT-PCR. As a positive control, Jurkat T cells, known for their high expression of PTPN22, were included. We found detectable levels of PTPN22 expression in all epithelial cell lines tested (Fig. 1F). Additionally, we detected PTPN22 expression at protein level in the same panel of cell lines by immunoprecipitating PTPN22 from cell extracts by using its antibody (Fig. 1G). Next, we confirmed the association of endogenous PTPN22, as well as transiently expressing exogenous SFB-PTPN22 with components of mTORC2 in multiple epithelial cell lines (Figs. 1H,I and EV1C). To assess whether the interaction of PTPN22 with mTORC2 is specific to this phosphatase, we performed a co-IP with PTPN5, another tyrosine phosphatase. Unlike PTPN22, PTPN5 did not co-immunoprecipitate with mTORC2 (Fig. 1I), suggesting that PTPN22's interaction with mTORC2 is specific. Consistent with these findings, PTPN22 co-fractionate along with mTORC2 complex in our size fractionation experiments (Fig. 1J). Together, our data indicate that PTPN22 interacts with mTORC2 in a wide range of cells derived from haematopoietic as well as non-haematopoietic origin.

In conclusion, our study identifies phosphatase PTPN22 as a non-catalytic scaffolding protein that regulates mTORC2 activity by facilitating the mSIN-RICTOR interaction (model shown in Fig. 5I). This work expands the functional repertoire of PTPN22 beyond its well-established role in immune signaling and suggests its involvement in regulating key cellular processes through mTORC2. We show that PTPN22 promotes AKT activation in both hematopoietic and non-hematopoietic cells, independent of its phosphatase activity, and contributes to oncogenesis in vitro and in vivo. In cells, mTOR forms the catalytic core of two distinct multi-subunit complexes, mTORC1 and mTORC2 (Kim and Guan, 2019; Saxton and Sabatini, 2017). Unlike the well-established regulatory mechanisms of mTORC1, only a few protein regulators have been reported to modulate mTORC2. For instance, oncogenic Ras activates mTORC2 kinase activity by directly interacting with its components at the plasma membrane (Kovalski et al, 2019). In another study, hyper-phosphorylated Rb (Retinoblastoma) protein was shown to suppress mTORC2 function by directly binding mTORC2, and inhibiting its activity (Zhang et al, 2016). However, the functional interactions between mTORC2 and phosphatases are not known. Interestingly, here we observed that a non-receptor tyrosine phosphatase, PTPN22 acts as a molecular bridge to facilitate mSIN and RICTOR binding, in enzyme independent fashion to promote mTORC2 activity and functions. Thus, our work fills an important gap of phosphatase connection with mTORC2 biology.

Previous work has suggested that PTPN22 regulates AKT activity in haematopoietic cells through indirect mechanisms, such as modulation of lipid phosphatases (Baghbani et al, 2017; Bai et al, 2023; Negro et al, 2012; Pogue et al, 2000). Our study extends these findings by providing direct evidence for a scaffolding role of PTPN22 in mTORC2, offering a new mechanistic insight into the regulation of AKT activation. While PTPN22 phosphatase activity is well studied in the context of immune regulation, its function outside of the immune system remains poorly understood. Recently, in the nematode worm C. elegans, PTPN22 was shown to associate with DNBP-1, a Cdc42 guanine nucleotide exchange factor, to promote epithelial cell integrity and regulate cytoskeletal machinery (Binti et al, 2024). Additionally, increased PTPN22 levels have been linked with several cancer types including esophageal carcinoma, stomach adenocarcinoma, and lymphoid and myeloid leukemias (Bottini and Peterson, 2014; Chen et al, 2020; Hebbring et al, 2013). In addition to this, various PTPN22 mutations listed in the COSMIC (Catalogue of Somatic Mutations in Cancer) database are distributed throughout the protein (Sondka et al, 2024). However, the mechanisms underlying its role in cancer are poorly understood. Given that mTOR pathway is often dysregulated in cancer, it is tempting to speculate that hyperactivation of mTORC2/AKT axis by PTPN22 might be one of the possible underlying mechanisms dysregulated in these cancer types. Collectively, our data suggest that PTPN22 may act as a potential oncogene by regulating mTORC2 assembly and functions.

Immunoprecipitation (IP) and western blotting (WB) were performed by using the following antibodies: PTPN22 (CST 14693S; WB 1:1000 or IP 1:100 or 1:1000), mTOR (CST 2972S; WB 1:1000), mTOR (CST 2983S; WB 1:1000), RICTOR (CST 9476S; WB 1:1000), RAPTOR (CST 2280S; WB 1:1000), mLST8/GβL (CST 3274S; WB 1:1000), SIN1 (abcam ab71152; WB 1:3000 or IP 2.5 µg), AKT (CST 9272S; WB 1:2000), pS473-AKT (CST 9271L; WB 1:1500), pS473-AKT (CST 9018S; WB 1:1500), pT308-AKT (CST 9275L; WB 1:1000), GSK-3β (CST 12456T; WB 1:2000), pS9-GSK-3β (CST 5558T; WB 1:1000), FoxO1 (CST 2880T; WB 1:1000), pT24-FoxO1/pT32-FoxO3a (CST 9464T; WB 1:1000), p70S6K (CST 2708S; WB 1:2000), pT389-p70S6K (CST 9205S; WB 1:1000), PKCα (CST 2056T; WB 1:1000), phospho-PKCα/βII [pT638/641] (CST 9375T; WB 1:1000), SGK1 (ab59337; WB 1:1000), pS422-SGK1 (ab55281; WB 1:500), pS10-Histone H3 (CST 9701L; IF 1:200), β-actin (Sigma-Aldrich A5441; WB 1:10,000), FLAG (Sigma-Aldrich F3165; WB 1:10,000 or IP 2 µg), c-Myc (Santa Cruz Biotechnologies clone 9E10 (sc-40); WB 1:1000), c-Myc (Sigma-Aldrich M4439; IP 1 µg), MBP (Sigma-Aldrich M1321; WB 1:5000), anti-6-His (Bethyl A190-114A; 1:800) and GST (Santa Cruz Biotechnologies sc-138; WB 1:2500). Secondary antibodies: HRP-conjugated anti-mouse (315-035-048) and anti-rabbit (111-035-144) antibodies were obtained from Jackson Immunologicals. TrueBlot secondary antibodies: HRP-conjugated anti-mouse TrueBlot (18-8817-33) and anti-rabbit TrueBlot (18-8816-33) antibodies were purchased from Rockland Immunochemicals. Alexa Fluor^TM 488 (A-11034) was purchased from Invitrogen. Phorbol 12-myristate 13-acetate (PMA) (HY-18739) and PTPN22 inhibitor, PTPN22-IN-1 (HY-139693) were purchased from MedChemExpress. Torin1 (4247) was purchased from Tocris.

HEK293T, Jurkat, HCT116, HeLa, U-2 OS and BOSC23 cell lines were used in this study. HEK293T, U-2 OS, HeLa and Jurkat cell lines were maintained in RPMI 1640 medium (HiMedia, AL028A) supplemented with 10% fetal bovine serum (FBS; Gibco, 10270-106) and 1% penicillin/streptomycin (GeneDireX, CC502-0100). HCT116 and BOSC23 cell lines were grown in DMEM (Gibco, 10313-021) supplemented with 10% fetal bovine serum, 2 mM L-glutamine (Sigma-Aldrich, 25030149) and 1% penicillin and streptomycin. All cell lines were continuously monitored by microscopy for their original morphology and also regularly tested for mycoplasma contamination by using 4', 6-diamidino-2-phenylindole (DAPI) staining. For transfection of various plasmids in HEK293T or BOSC23 cells polyethylenimine (PEI; Polysciences, 23966) was used according to the manufacturer's protocol. Plasmids were transfected into HCT116 and HeLa cells using TurboFect transfection reagent (Invitrogen, R0531) according to the manufacturer's protocol. Briefly, the plasmids were diluted in serum-free medium was mixed with PEI (1 µg/µl)/TurboFect at a ratio of 1:3 (DNA amount: Transfection reagent volume), followed by incubation for 15–20 min at room temperature and then added to cells. Jurkat cells were transfected using Lipofectamine LTX reagent with Plus reagent (Invitrogen, 15338030) according to the manufacturer's protocol. At 6-8 h post-transfection, the culture medium was replaced with fresh medium, and cells were harvested 24–36 h after transfection for further analyses.

For serum starvation and re-stimulation with growth factors to assess mTORC2 activation, subconfluent cells were incubated with serum-free media for 16 h, followed by stimulation with 100 ng/ml EGF (Invitrogen, 53003-018) for indicated time points.

pRK5-myc-mTOR (Addgene, #1861) plasmid was purchased from Addgene. All cDNAs (RICTOR, RAPTOR, mSIN1, mLST8, PRAS40, PTPN22, AKT, PTPN5) were amplified by PCR and cloned into donor vector pDONOR201 (Invitrogen) using gateway cloning, and then moved to triple-tagged (S-protein/Flag/streptavidin-binding protein) SFB, Myc, glutathione S-transferase (GST), 6X-His, and maltose-binding protein (MBP)-tagged destination vectors (Invitrogen) for expression. All clones were verified by Sanger sequencing. Point mutants, PTPN22, mSIN1 and RICTOR domain deletions and the shRNA-resistant mutants were generated by PCR-based site-directed mutagenesis in pDONOR201, and then cloned into SFB, GST and Myc-tagged destination vectors. Lentiviral-based shRNAs for PTPN22 (shRNA#1: 5'-CTAGTGCTCTTGGTGTATATT-3' [TRCN0000003037], shRNA#2: 5'-GAATTGATACAGCAGAGAGAA-3' [TRCN0000003038]) were purchased from Open Biosystems. Lentiviral-based shRNA plasmids for mSIN (shRNA#1: 5'-GTTGGGACTTTGGTATTAGAA-3' [TRCN0000003152], shRNA#2: 5'-GCCACAGTACAGGATATGCTT-3' [TRCN0000003153]), and RICTOR (shRNA#1: 5'-CGTCGGAGTAACCAAAGATTA-3' [TRCN0000074291], shRNA#2: 5'-GCACCCTCTATTGCTACAATT-3' [TRCN0000074290], shRNA#3: 5'-CGGAGGTTCATACAAGAATTA-3' [TRCN0000074289]) were purchased from Dharmacon. CRISPR sgRNA oligos for PTPN22 were designed using CHOP-CHOP web tool. The sequences for the guide RNAs were as follows: (sgRNA#1: 5'-GGAGTTTGCCAATGAATTTC-3', sgRNA#2: 5'-GGATGTACGTTGTTACCAAG-3', sgRNA#3: 5'-CAAAACCTATCCTACAACTG-3'). Double-stranded DNA oligos that encode single sgRNAs were annealed and then cloned into BsmBI restriction sites of the LentiCRISPRv2 vector. All clones were verified by Sanger sequencing.

Lentivirus-based pLKO.1 vectors encoding PTPN22, mSIN, RICTOR or a scrambled shRNA were co-transfected along with the psPAX2 packaging and pMD2.G envelope plasmids (Addgene, 12260 and 12259) in BOSC23 or HEK293T packaging cells using PEI. Viral supernatants were collected 48 and 72 h post-transfection, filtered through 0.45 µm filters (Millipore, SLHV033RS). Target cells were infected twice with viral medium along with polybrene (8 µg/ml; Sigma-Aldrich, H9268) for 36 h. After transduction, cells were collected and processed for various assays, and immunoblotting was performed with specific antibodies to check the efficiency of knockdown. Stable cell lines were generated by replacing culture medium with fresh medium containing puromycin (2 µg/ml; Gibco, A11138-02), 36 h after the second transduction, followed by clonal selection (5–10 days). The positive clones were confirmed by immunoblotting with specific antibodies. Lentiviral-based transduction in Jurkat cells was performed as described in (Kory et al, 2020). Briefly, cells were cultured in RPMI 1640 medium containing polybrene (8 µg/ml), and then transduced with lentiviral particles by centrifugation at 2200 rpm for 1 h at 37 °C. After incubation for 18 h, viral media was removed and cell pellets were washed twice in phosphate-buffered saline (PBS) (HyClone, SH30256.01), then re-seeded into fresh RPMI 1640 medium having puromycin (1 µg/ml). After 3–7 days post-infection, cells were collected and processed for various assays, and immunoblotting. For mSIN and RICTOR knockdown experiments, multiple shRNAs viral medium was pooled to obtain better knockdown efficiency.

LentiCRISPRv2 vector encoding PTPN22 guide RNAs or a control sgRNA were co-transfected along with the psPAX2 packaging and pMD2.G envelope plasmids (Addgene, 12260 and 12259) in BOSC23 packaging cells using PEI. Viral supernatants were collected 48 h post-transfection, filtered through 0.45 µm filters (Millipore, SLHV033RS). Target cells were infected twice with viral medium along with polybrene (8 µg/ml; Sigma-Aldrich, H9268) for 36 h. Stable cell lines were generated by replacing culture medium with fresh medium containing puromycin (2 µg/ml; Gibco, A11138-02), 36 h after the second transduction, followed by clonal selection. The positive clones were confirmed by immunoblotting for the absence of protein expression with specific antibodies.

To analyze various protein levels in the whole cell lysates, cells were lysed with NETN buffer as described above. The soluble fractions were isolated by centrifugation at 15,871× g for 15 min at 4 °C. Total protein concentration of the samples was measured using Pierce^TM Bradford plus protein assay kit (Thermo Fisher Scientific, 23236), and were boiled after addition of 6X SDS sample buffer at 100 °C for 5 min. For immunoprecipitation and pulldown assays, cells were rinsed once in ice-cold PBS and lysed either in 0.3% CHAPS lysis buffer [40 mM HEPES pH 7.4, 120 mM NaCl, 1 mM EDTA and 0.3% (w/v) CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)] or in 1% Triton X-100 lysis buffer (20 mM Tris/HCl pH 8.0, 100 mM NaCl, 1 mM EDTA and 1% (v/v) Triton X-100) supplemented with 1 µg/ml pepstatin A, 1 µg/ml aprotinin and 100 mM PMSF (phenylmethylsulfonyl fluoride) on ice for 20 min. Cell debris was removed by centrifugation at 15,871× g for 15 min at 4 °C, and protein concentrations were determined as mentioned above. Equal amount of total protein from each lysate was incubated with 1–2.5 µg of specified antibody bound to either protein A/G sepharose beads (Cytiva, 17096303/17061801), or streptavidin sepharose beads (Cytiva, 17511301), or S-protein agarose beads (Millipore, 69704) for 3–6 h at 4 °C. The bound protein complexes were washed five times with lysis buffer and eluted by boiling in 2 × SDS sample buffer at 100 °C for 5 min. The eluates were then resolved by SDS-PAGE, transferred on PVDF membranes (Millipore, IPVH00010) and immunoblotting was performed following standard protocols. Protein bands were visualized using enhanced chemiluminescence (ECL) based detection (Amersham, RPN2236). Densitometry based quantitative analyses were done by Fiji ImageJ software (NIH, Bethesda, MD, USA) when needed.

Gel-filtration experiments were adapted as described previously (Liu et al, 2013). In brief, Jurkat cells were cultured in 10 cm dishes (four dishes per gel filtration experiment). Cells were pellet down, and washed with phosphate-buffered saline (PBS), lysed in 0.3% CHAPS buffer (as mentioned above) on ice for 30 min, and insoluble fractions were removed by centrifugation at 15,871 × g for 15 min at 4 °C. Supernatant was filtered through a 0.22 µm filter and concentrated to 1 ml ( ~ 8 mg) using microsep centrifugal filters (MWCO#10 K; MCP010C41, Pall Life Sciences). Before run, the Superdex 200 column (GE HealthCare) was calibrated using gel filtration HMW calibration kit (Cytiva, 28-4038-42) that contains: Blue dextran 2000 (to determine void volume) ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa) and thyroglobulin (669 kDa). Lysates were injected onto the column and chromatography was performed on the AKTA-FPLC system (GE HealthCare) with CHAPS buffer. In total, 500 µl of fractions were collected and samples were concentrated to a final volume of 100 µl, and indicated fractions were resolved by SDS-PAGE followed by immunoblotting with specific antibodies.

Total RNA was isolated from control and PTPN22 knockdown cell lines using Nucleospin RNA isolation kit (Macherey-Nagel, 740955) following the manufacturer's instructions. 1 µg of total RNA was reverse transcribed using PrimeScript^TM cDNA synthesis kit (Takara Bio, 6110B). The synthesized cDNA products were amplified through PCR using respective primers.

PTPN22 forward: 5'-AGCTAGTTTTGCACCCTGCTA-3'

PTPN22 reverse: 5'-TTGGTCCGTGTTATTGGCACC-3'

GAPDH forward: 5'-CGACCACTTTGTCAAGCTCA-3'

GAPDH reverse: 5'-AGGGGAGATTCAGTGTGGTG-3'.

The PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide (Sigma-Aldrich, E7637). The gels were visualized by Gel Doc^TM XR system with Image Lab^TM software (Bio-Rad). GAPDH was used as a control to normalize the PTPN22 expression levels.

GST, GST-mSIN, GST-AKT (K179D), GST-PTPN22, His-tagged PTPN22, MBP or MBP-mSIN, MBP-PTPN22 full-length proteins were expressed in Escherichia coli BL21 (DE3) cells, and the proteins were affinity purified using either glutathione sepharose^TM 4B beads (GE Healthcare, GE17-0756-01) [for GST-tagged proteins] or dextran sepharose^TM beads (GE Healthcare, GE28-9355-97) [for MBP-tagged proteins] or Nickel NTA agarose beads (Qiagen, 30210) for 2 h at 4 °C. Then, beads were washed five times with wash buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.01% IGEPAL, 1 mM DTT, and protease inhibitors) and bound proteins were eluted with the elution buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, and 10 mM reduced glutathione (GST-tagged proteins), 20 mM Tris (pH 7.5), 200 mM NaCl, 1 mM EDTA, and 10 mM maltose (MBP-tagged proteins) or 50 mM Tris (pH 7.5), 150 mM NaCl, 300 mM imidazole with 0.1% IGEPAL (His-tagged proteins) for 60 min at 4 °C.

For purification of RICTOR, either GST-RICTOR full-length protein were expressed in Escherichia coli BL21 (DE3) cells, after sonication the cell lysates were cleared by centrifugation at 15,871 × g for 45 min at 4 °C. Cell lysates were then concentrated using centrifugal filters (MWCO#10 K; Millipore) or HEK293T cells having stable knockdown of mSIN were transfected with SFB-RICTOR. On the day after transfection, cells were collected and lysed in 1% Triton X-100 lysis buffer as described above. Cell lysates were clarified by centrifugation at 15,871 × g for 20 min at 4 °C, and incubated with streptavidin sepharose beads (Cytiva, 17511301) for 1 h at 4 °C followed by washing with high salt buffer [NETN lysis buffer having 200 mM NaCl] (10 times), and then with low salt buffer [NETN lysis buffer having 50 mM NaCl] (five times). The purified SFB-RICTOR was eluted with 2 mg/ml biotin (Sigma-Aldrich, B4501) for 90 min at 4 °C.

For in vitro binding of mSIN and PTPN22, equal amounts of the purified GST or GST-mSIN proteins immobilized to glutathione sepharose^TM 4B beads were incubated with bacterially purified MBP-PTPN22 (2–2.5 µg) for 2 h at 4 °C. For RICTOR and PTPN22 binding, equal amounts of the purified MBP or MBP-PTPN22 proteins immobilized to dextran sepharose^TM 4B beads were incubated with concentrated bacterial cell lysates expressing GST-RICTOR for 1 h at 4 °C. The beads were washed five times with wash buffer and bound proteins were eluted by boiling in 2× SDS sample buffer at 100 °C for 5 min. The proteins were resolved by SDS-PAGE, and the interactions were analyzed by western blotting. For in vitro binding experiments of mSIN with AKT, equal amounts of the purified MBP or MBP-mSIN proteins immobilized to dextran sepharose^TM 4B beads were incubated with bacterially purified GST-AKT (0.5 µg) either in the presence of recombinant His-PTPN22 or equal volume of corresponding buffer for 1 h at 4 °C. The beads were washed five times with wash buffer and bound proteins were eluted by boiling in 2× SDS sample buffer at 100 °C for 5 min. The proteins were resolved by SDS-PAGE, and the interactions were analyzed by western blotting. For in vitro binding of mSIN with RICTOR, equal amounts of the purified GST or GST-mSIN proteins immobilized to glutathione sepharose^TM 4B beads were incubated with purified SFB-RICTOR either in the presence of recombinant His-PTPN22 or equal volume of corresponding buffer for 1 h at 4 °C. The beads were washed five times with wash buffer and bound proteins were eluted by boiling in 2× SDS sample buffer at 100 °C for 5 min. The proteins were resolved by SDS-PAGE, and the interactions were analyzed by western blotting with anti-Flag antibody.

For the mTORC2 in vitro kinase assays, HCT116 cells stably expressing control shRNA and PTPN22 shRNA were transfected with SFB-RICTOR. Forty-eight hours post-transfection, RICTOR was affinity purified with streptavidin sepharose beads (Cytiva, 17511301) in 0.3% CHAPS lysis buffer, followed by elution with 2 mg/ml biotin (Sigma-Aldrich, B4501), and used as a kinase source for the assays. Bacterially purified 1 µg of GST-AKT (K179D) was incubated with SFB-RICTOR immunoprecipitates and 200 µM ATP in the kinase assay buffer (25 mM HEPES pH 7.4, 100 mM potassium acetate, 1 mM MgCl₂) at 30 °C for 1 h. The reactions were stopped by the addition of 2× SDS sample buffer, and the samples were resolved by SDS-PAGE. Levels of AKT phosphorylation were detected by immunoblotting using the pS473-AKT antibody.

Cells were seeded in six-well plates at a density of 1000 cells/well [for knockdown experiments] or 500 cells/well [for rescue experiments] in 2 ml complete DMEM medium (in duplicates) and cultured for 10–14 days until the colonies were formed. Cells were washed twice with phosphate-buffered saline (PBS) (HyClone, SH30256.01), and then fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich, 158127) for 15 min at room temperature. After washing with distilled water, cells were stained with 0.5% (w/v) crystal violet (Sigma-Aldrich, C3886) for 15 min and then again washed with water twice to remove unbound dye. The cells were air-dried and the number of colonies were counted and imaged under Gel Doc^TM XR system with Image Lab^TM software (Bio-Rad). All assays were performed independently at least three times.

Cells were transfected with indicated constructs. Seventy-two hours post-transfection, equal numbers of cells were seeded in four different plates (of each type) in complete DMEM medium. After day 1, cells were trypsinzed and counted regularly for the indicated durations using Neubauer chamber (Hausser Scientific) according to the manufacturer's instructions. Quantification of cell number was calculated from three independent experiments.

Cells were harvested by centrifugation at 200 × g for 5 min at 4 °C and the cell pellets were resuspended in 300 µl of phosphate-buffered saline (PBS) followed by addition of 700 µl of ice-cold ethanol (100%) dropwise. The cells were fixed on ice for 30 min. DNA fragments were extracted with 0.2 M phosphate-citrate buffer (pH 7.8) and 100 µg/ml RNase A (Sigma-Aldrich, R4875) at 37 °C for 30 min, then stained with 200 µl Propidium Iodide (50 µg/ml; Sigma-Aldrich, P4864) at 37 °C for 30 min. Cells were gated to exclude the debris and the sub-G1 population was analyzed by flow cytometry (BD Accuri^TM C6, BD Biosciences or BD LSRFortessa^TM X-20 Cell Analyzer, CA, USA). Assays were performed independently either two or three times.

Transwell migration assays were performed as described previously (Kumar et al, 2019). Briefly, cells from different experimental conditions were seeded in the upper chamber of transwell inserts (24-well, 8 µm polyethylene terephthalate membrane filters; Falcon, BD Biosciences, 353097) at 1.0 × 10⁵ cells per well, in 200 µl of serum-free DMEM. The bottom chamber contained DMEM with 10% FBS as a chemoattractant. Cells were allowed to migrate for 24 h. After the incubation period, non-migrant cells on the upper side of the filter were removed by a cotton swab. Filters were then washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature, and inserts were stained with 0.1% crystal violet for 20 min. The migrated cells were imaged using an inverted microscope (Olympus, IX73). Quantification of migrated cells per field was calculated from two independent experiments (derived from average of four different fields).

Cells from different experimental conditions were grown on glass coverslips and fixed with 2% paraformaldehyde solution in PBS for 15 min at room temperature. Then cells were washed with 1× PBS three times, and permeabilized with 0.2% Triton X-100 buffer at room temperature for 5 min. After this step, cells were washed thrice with 1X PBS, and were incubated with 5% BSA for blocking at room temperature for 60 min. After incubation, cells were incubated with primary antibody overnight at 4 °C followed by washing thrice with 1× PBS. Cells were then incubated with Alexa Fluor^TM 488 secondary antibody at 37 °C for 60 min followed by washing thrice with 1× PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), followed by washing with 1× PBS three times, and coverslips were mounted with glycerol containing paraphenylenediamine and imaged using Carl Zeiss ELYRA Super-Resolution Microscope with Plan apochromat 63×/1.4 DIC M27 oil immersion lens. Raw images were acquired with Zen Black software using Z-stacks with a step size of 0.5 µm and processed through Structured Illumination Microscopy (SIM) module using Zen Blue software.

All animal xenograft experiments were conducted following approval from CDFD institutional animal ethics committee (approved by CPCSEA, GOI), and performed at the institutional experimental animal facility (EAF). Briefly, HCT116 cells (5.0 × 10⁶) of each subline were trypsinized and washed with phosphate-buffered saline (PBS). The cell suspensions were mixed with Matrigel (1:1 ratio, v/v) (Corning, 356234) and injected subcutaneously into athymic nude female mice (Foxn1^−/−) (5–6 weeks old). Tumor size was measured every 3 days with a digital caliper, and the tumor volumes were calculated with formula V = 0.5 x Length (L) x Width² (W), where L is the longest diameter and W is the shortest diameter. On the last day (45 day), the mice were euthanized, and the xenograft tumors were dissected and the weights were noted.

Statistical analyses were performed by two-tailed unpaired Student's t tests for two samples and one-way analysis of variance (ANOVA) for multiple samples and further corrected by Bonferroni's multiple comparisons test using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). For cell proliferation assays (Figs. 5A and EV5E), two-way analysis of variance (ANOVA) corrected with Tukey's multiple comparisons test was performed. Error bars either represented as the mean ± standard error of mean (SEM) or mean ± standard deviation (SD) as mentioned in the figure legends. The significance levels are indicated by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns: not significant. The exact P values are provided in the respective figure legends.