Paclitaxel Suppressed N6-Methyladenosine of PUM1 offsets cetuximab resistance in colorectal cancer

doi:10.21203/rs.3.rs-3128434/v1

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Research Article

Paclitaxel Suppressed N⁶-Methyladenosine of PUM1 offsets cetuximab resistance in colorectal cancer

https://doi.org/10.21203/rs.3.rs-3128434/v1

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Background

We have previously demonstrated that RNA-binding protein Pumilio-1 (PUM1) is ubiquitous in cetuximab-resistant colorectal cancer (CRC) cells. The role of the N⁶-methyladenosine modification of PUM1 influenced by paclitaxel (PTX) in modulating cetuximab-resistance in CRC cells was investigated in the current work.

Methods

PUM1 mRNA expression in CRC tissues and cells was measured by quantitative real-time PCR (qRT-PCR), and PUM1 protein expression was measured by immunohistochemistry (IHC). The involvement of PUM1 expression in CRC prognosis was evaluated by survival analysis. RNA immunoprecipitation (RIP) was performed to evaluate combination capability of PUM1 and YTHDF1. RNA m⁶A dot blot assays were conducted to investigate variations in the degree of the METTL3-induced N⁶-methyladenosine modification of PUM1. Patient derived tumor xenograft (PDX) models to investigate the regulatory role of paclitaxel inhibited N⁶-demethyladenosine modification of PUM1 and diminished PUM1 mRNA level.

Results

The N⁶-methyladenosine content of PUM1 was increased in CRC by cetuximab treatment and increased PUM1 mRNA stability (P < 0.05). PUM1 induced monocyte-to-macrophage differentiation of CRC cells during in vitro functional assays and activated the WNT axis by enhancing DDX5 expression (P < 0.05). Paclitaxel increased PUM1 N⁶-demethyladenosine levels and decreased PUM1 expression (P < 0.05), resulting in suppression of the WNT pathway. Paclitaxel also reduced PUM1-induced cetuximab-resistance in CRC cells. CRC xenografts from human patients had enhanced m⁶A modification of PUM1 in paclitaxel-treated samples (P < 0.05) shown by ex vivo studies. In clinic, high PUM1 levels in CRC patients correlated with elevated TAM content and poor survival (P < 0.05).

Conclusion

Inhibition of N⁶-methyladenosine modification of PUM1 is proposed as a novel therapeutic target to overcome cetuximab-resistance in CRC.

colorectal cancer

cetuximab

PUM1

Paclitaxel

Cetuximab resistance

Colorectal cancer (CRC) ranks 3rd for global cancer incidence and second for cancer-associated mortality due to pervasive metastasis and drug resistance ^[1]. The 5-year overall survival (OS) rate for metastatic CRC (mCRC) patients is barely 13%, as opposed to 91.1% for those with localized disease ^[2].

Cetuximab (CTX) is recommended as a first-line drug for mCRC by the National Comprehensive Cancer Network (NCCN) ^[3].However, about 70% of CTX-responsive patients experience disease progression within 3–12 months of initiating treatment ^[4]. Paclitaxel (PTX) has been shown to elicit a response rate of over 20% ^{[5, 6]} in endometrial cancer and combined therapy with PTX suppressed proliferation of human endometrial carcinoma cells and promoted apoptosis ^[7]. Moreover, PTX alone was shown to be as effective as PTX and carboplatin combination therapy and more effective than carboplatin alone in suppressing endometrial cancer cell proliferation ^[8].However, little is known about the impact of PTX on signaling pathways that alleviates the cetuximab-mediated chemoresistance of CRC cells.

N⁶-methyladenosine (m⁶A) modification describes methylation of the N6-position adenosine, a common modification of non-coding RNAs (ncRNAs) and messenger RNA (mRNA)^[9–11]. N⁶-methyladenosine has been linked to chemoresistance ^[12] and knockdown of the m⁶A modified METTL3 RNA was shown to regulate HIF-1α and suppress the Warburg effect in CRC ^[13]. Our previous studies have demonstrated that PUM1 is ubiquitous in cetuximab-resistant CRC cells ^[14]. The current work investigated the PTX-induced m⁶A modification of PUM1 and the mechanisms whereby cetuximab resistance is modulated.

Patients and tissue samples

Patients undergoing treatment for CRC at the Shanghai Fourth People's Hospital were enrolled. Ethical approval was granted by the Ethical Committee of the Shanghai Fourth People's Hospital (No.2022018-001) and investigations were conducted in accordance with the Declaration of Helsinki (as revised in 2013). Written informed consent was given by all participants.

Cell culture and shRNA transfection

Human colon cancer cell lines, Caco-2(BFN60800651), HCT116 (BFN607200665), SW620 (BFN60800643), SW480 (BFN60800644), and LOVO (BFN60800645) were obtained from ATCC Cell Bank (Shanghai, China) and cultured in either RPMI-1640 media (1×) or DMEM with 10% FBS at 37°C with 5% CO₂ and all cells tested negative for mycoplasma contamination verified by short tandem repeat fingerprinting after initial recovery of cells. For Caco-2-cetuximab resistance (Caco-2-Cr), HCT116-cetuximab resistance (HCT116-Cr), SW620-cetuximab resistance (SW620-Cr), SW480-cetuximab resistance (SW480-Cr) and LOVO-cetuximab resistance (LOVO-Cr), were obtained as our previously described ^[14]. Kits for transfection of siRNA and NC siRNA were used, in accordance with the manufacturer’s instructions (Shanghai Yuanzhi Biotechnology), using Lipofectamine 2000 (Invitrogen). siRNA sequences are given in Supplement Table 1.

Table 1

Correlation between PUM1 expression and clinicopathologic characteristics of CRC patients
Characteristics	PUM1 protein level		p-value*	χ2value
Characteristics	Low	High	p-value*	χ2value
Age			1	0.000
≤ 60 years	45	45
> 60 years	105	105
Gender			0.294	1.102
Female	60	69
Male	90	81
Tumor diameter			0.005	7.957
≤ 5 cm	73	49
> 5 cm	77	101
Local invasion			0.139	2.191
Absent	119	108
Present	31	42
Lymph node metastasis			0.001	11.092
Absent	107	79
Present	43	71
TNM Stage			< 0.001	138.204
I + II	142	43
III + IV	8	107
*p-value by χ2 test.

Lentiviral transfection

Lentivirus was purchased from Shanghai Yuanzhi Biotechnology (Shanghai, China) and infection performed according to the manufacturer’s instructions. Cells were treated with 2–3 µg/mL puromycin for 2 weeks and knockdown confirmed by qPCR.

qRT-PCR

Total RNA was extracted from CRC tissues and cells using Trizol reagent (Invitrogen, 15596026) according to the manufacturer’s instructions, and quantified by NanoDrop 2000c (Thermo Fisher). RNA was reverse transcribed into cDNA by HiScript cDNA synthesis kit (Vazyme, R323–01) and Universal SYBR qPCR Master Mix (Vazyme, Q511–02) was used for qPCR. Primer (Shanghai Yuanzhi Biotechnology) sequences are given in Supplement Table 2. Fold change in mRNA expression relative to GAPDH was calculated by the 2^−ΔΔct technique.

Analysis of mRNA stability

The stability of mRNA was measured as described previously ^[15]. Cells were grown to 50% confluence in 6-well plates before being treated with 5 µg/mL actinomycin D (Sigma-Aldrich, Shanghai, China), as specified, and harvested for RNA extraction and qRT-PCR analysis.

MeRIP-PCR

mRNA enrichment for m⁶A modification was assessed as described previously^[16]. The m⁶A modification status of a single gene was determined by Magna methylated RNA immunoprecipitation m⁶A Kit (Millipore), according to the manufacturer’s instructions. In brief, 5 µg anti-m⁶A antibody (CS220007, Millipore) or normal mouse IgG (CS200621, Millipore) prewashed and treated with Magna ChIP protein A/G magnetic Beads (CS203152, Millipore) was incubated with pure poly-(A) RNA for half an hour at RT. Enrichment of m⁶A modified mRNA was determined by RT-qPCR with a positive control using primers specific to a PUM1 region without m⁶A and a negative control using primers specific to a PUM1 region with m⁶A. m⁶A enrichment was normalized to the input. Supplement Table 2 lists the primer sequences.

Animal models of PDX

Xenograft tumors were created in the PDX model, as previously reported^[17]. Ethical approval for animal experiments was granted by the Shanghai Tongji University Ethics Committee (No. TJBH0122101) and experiments were conducted in accordance with the regulations of the Shanghai Fourth People's Hospital.

Dot blot of m⁶A

Dot blot experiments using the m⁶A probe were performed as described previously ^[15]. Poly(A) + RNAs were heated to 65°C for 5 min and transferred to a nitrocellulose membrane (GE Healthcare) using Bio-Dot device (Bio-Rad, Hercules). Membranes were probed with goat anti-mouse IgG conjugated with HRP (1:3000, Proteintech, Hercules), followed by UV cross-linking, blocking and overnight 4°C incubation with anti-m⁶A antibody (1:1000, Abcam). Modified RNAs were visualized by chemiluminescence (Bio-Rad). The membranes used were dyed with 0.02% methylene blue (MB) in 0.3 M sodium acetate (pH 5.2).

RNA immunoprecipitation (RIP)

Magna RIP Kit (17–700, Millipore, MA, USA) was used, following the manufacturer’s instructions, as previously reported ^[15]. Cells were washed twice with ice-cold PBS, scraped, resuspended in 1 mL PBS, centrifuged and resuspended in RIP lysis buffer (Merck Millipore, MA, USA). 5 µg antibodies was conjugated to Protein A/G magnetic beads in immunoprecipitation buffer (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 0.05% TritonX-100) for 2h and 100 µL cell lysates incubated overnight at 4°C in the same buffer. Beads were incubated in 400 µL elution buffer for 2h to elute RNA and the RNA precipitated with ethanol and dissolved in RNase-free water. Enriched fragments were identified by RT-PCR.

Immunohistochemistry (IHC)

IHC procedures were performed as described previously ^[15]. Deparaffinized sections were heated in 10 mM sodium citrate buffer (pH 6.0) to expose antigen and inactivate endogenous peroxidase and incubated with primary antibody (1:50; cat. no. 703275) for 30 min and with secondary antibody (1:200; cat. no. 31423) for 1h at RT. Diaminobenzidine staining and light microscopy image capture (Olympus)was performed before incubation with secondary antibody conjugated with peroxidase. Two independent observers rated each set of IHC slides.

H-score was assessed at ×200 magnification and staining intensity scored as 0 (negative), 1 (weak), 2 (intermediate) or 3 (strong brown). Numbers of cells stained at each intensity were counted in each field. The mean percentage of positive cells was calculated, as follows: H-score = (% of cells stained at intensity category 1 × 1) + (% of cells stained at 2 × 2) + (% of cells stained at 3 × 3). H-scores between 0 and 300 were obtained, where 0described 100% cells with negative staining and 300 described 100% cells with strong staining. Median H-score was identified as the cutoff between low and high expression.

Analytical statistics

Unless otherwise noted, all data are presented as mean ± SD. GraphPad Prism8 was used for all statistical analyses. Intergroup comparisons were performed by unpaired or paired Student’s t-test for two groups and log-rank test for multiple groups. Survival was estimated by Kaplan-Meier (K-M) analysis. A two-tailed p-value of < 0.05was considered to be statistically significant.

Cetuximab-induced N⁶-methyladenosine modification of PUM1 mRNA enhanced stability in CRC cells

PUM1 mRNA content was markedly elevated in cetuximab-resistant CRC cells, relative to controls (Fig. 1A, DMSO vs. Cetuximab resistance, p < 0.05). Results of MeRIP-qPCR experiments showed thatm⁶A content of PUM1 was also enhanced in the cetuximab-resistant CRC cells (Fig. 1B, DMSO vs. Cetuximab resistance, p < 0.05). Knockdown experiments using siRNA showed that inhibition of METTL3 methyltransferase, but not KIAA1429 or METTL14 downregulation, was mediated by PUM1. Knockdown of the m⁶A demethylases, ALKBH5 and FTO, did not alter PUM1 expression in CRC cells (Figs. 1C and S1A, B). METTL3 knockdown was shown to greatly reduce the m⁶A modification of PUM1 mRNA in CRC cells (Figs. 1D and E, si-NC vs. si-METTL3, p < 0.05) and the METTL3-mediated m⁶A modification enhanced PUM1 levels. The YT521-B homology domain family (YTH) proteins have been shown to regulate mRNA stability via m⁶A ^[9]. YTHDF1 was physically associated with PUM1 mRNA in HCC cells (Figs. 1F, G, anti-IgG vs. anti-YTHDF1, p < 0.05) and YTHDF1 knockdown reduced PUM1 mRNA stability (Figs.S1C, 1H, I, si-NC vs. si-YTHDF1, p < 0.05). In addition, PUM1 transcript methylation was recognized by the reader “m⁶A” and YTHDF1 which maintained mRNA stability and enhanced expression through an m⁶A-YTHDF1-dependent mechanism.

PUM1 Promoted Monocyte-to-Macrophage Differentiation

Monocyte differentiation into macrophages or dendritic cells (DCs) is known to influence CRC resistance^[18]. Low levels of the macrophage markers, CD14, CD71 and CD68, and DC markers, CD1a and CD83, in si-PUM1 CRC cells confirmed that PUM1 stimulated the differentiation of THP-1 cells into macrophages and not DCs (Figs. 2A, B, S2A: si-NC vs. si-PUM1, p < 0.05). Macrophages may be classified into M1 or M2 subtypes, depending on activation state and tumor-associated macrophages (TAMs) are known to be M2-like ^[19]. THP-1 cells exposed to si-PUM1 CRC cells had patterns of expression of the M1 marker genes, NOS2 and human leukocyte antigen DR alpha [HLA-DRA], which indicated an improved M1 phenotype (Figs. 2C and D, si-NC vs. si-PUM1, p < 0.05).

LV-PUM1 CRC cells showed upregulation of the macrophage markers, CD14, CD71 and CD68, and DC markers, CD1a and CD83 (Figs. 3A, B, S2B: LV-NC vs. LV-PUM1, p < 0.05). LV-PUM1 CRC cell lines prevented differentiation toward an M2 phenotype. TAMs showed enhanced M2 macrophage-specific marker genes, CD163 and MRC1, and genes associated with TAM polarization, CCL5 and interleukin-10 [IL-10] (Figs. 3C and D, LV-NC vs. LV-PUM1, p < 0.05). By contrast, when THP-1 cells were exposed to si-PUM1 CRC cells, NOS2 and HLA-DRA decreased (Figs. 3C and D, LV-NC vs. LV-PUM1, p < 0.05).

PUM1 activated the WNT pathway via modulation of DDX5 expression

DDX5 has been shown to promote CRC progression by stimulating the Wnt pathway ^[20]. Results of the TOP/FOP-flash luciferase assay showed enhanced Wnt signaling activation in PUM1 overexpressing cells whereas DDX5 knockdown suppressed Wnt signaling (Figs. 4A, S.2C, LV-PUM1 + si-NC vs.LV-PUM1 + si-DDX5, p < 0.05). PUM1 knockdown was demonstrated to suppress the Wnt pathway and DDX5 overexpression to prevent the suppression (Figs. 4B, S.2D, si-PUM1 + LV-Control vs. si-NC + LV-DDX5, p < 0.05). qRT-PCR results showed that elevation of PUM1 caused increasedAxin2, c-Myc and cyclin D1 content and DDX5 knockdown prevented these increases (Figs. 4C, E, G, LV-PUM1 + si-NC vs.LV-PUM1 + si-DDX5, p < 0.05). By contrast, reduction of PUM1 reduced Axin2, c-Myc and cyclin D1 content and DDX5 overexpression prevented this decrease (Figs. 4D, F, H, si-PUM1 + LV-Control vs. si-NC + LV-DDX5, p < 0.05). Hence, PUM1 modulated DDX5 expression to activate the WNT pathway.

Paclitaxel inhibited N⁶-demethyladenosine modification of PUM1 and suppressed the WNT pathway by diminishing PUM1 levels

Paclitaxel in combination with microRNA-34A has been shown to inhibit CRC progression ^[21]. Dot blot assays showed that paclitaxel treatment reduced the content of m⁶A in CRC cells (Fig. 5A) and PUM1 m⁶A content was shown to be reduced by MeRIP-PCR (Fig. 5B, DMSO vs. paclitaxel, p < 0.05). Paclitaxel also reduced levels of PUM1 and DDX5 mRNA transcripts in CRC cells (Figs. 5C and D, DMSO vs. paclitaxel, p < 0.05).

Paclitaxelreducedm⁶A modification of PUM1 in cetuximab-resistant CRC cells

A PDX model was developed from cetuximab-resistant CRC patients with varying levels of m⁶A modification of PUM1. Two CRC patients had a high level ofm⁶A modification and 2 had a low level (Fig. 6A, IgG vs. anti-m⁶A, p < 0.05). Paclitaxel treatment inhibited the m⁶A modification of PUM1in the PDX models and reduced PUM1 mRNA levels were seen (#1 and #2: DMSO vs. paclitaxel, p > 0.05; #3 and #4: DMSO vs. paclitaxel, p < 0.05). It is noteworthy that the PDXs with reduced m⁶A modification of PUM1 had marked growth inhibition on treatment with paclitaxel relative to controls (Fig. 6D, #1 and #2: DMSO vs. paclitaxel, p > 0.05; #3 and #4: DMSO vs. paclitaxel, p < 0.05). In summary, paclitaxel reduced the m⁶A modification of PUM1 in cetuximab-resistant CRC cells.

Increased PUM1 in human CRC was linked to elevated TAM levels and poor outcome

Levels of PUM1, CD86 and CD163 were measured in 30 pairs of CRC and adjacent non-cancer tissues (ANT) by RT-qPCR. PUM1 and CD163 mRNA levels were found to be elevated in CRC tissues relative to ANTs (Fig. 7A, Tumor vs. ANT, p < 0.05). Correlation analysis demonstrated that PUM1 transcript levels were inversely related to CD86 mRNA (R² = 0.656, p < 0.001) and directly proportional to CD163 mRNA (R² = 0.619, p < 0.001, Fig. 7B). PUM1 protein and macrophage invasion status were assessed in CRC tissues from 300 postoperative patients using IHC. PUM1 protein was elevated in CRC versus ANTs and normal tissues (Fig. 7C, Tumor Tissues vs. Adjacent normal, p < 0.05; Tumor Tissues vs. normal, p < 0.05). Numbers of CD206 + and CD163⁺ TAMs were also increased in tumor tissues with positive PUM1 expression (N = 150) relative to tumor tissues with negative PUM1 expression (N = 150) (Fig. 7D, p < 0.05). PUM1 levels were analyzed with respect to clinicopathological profile of 300 CRC patients (Table 1) and positive PUM1 expression correlated with tumor size > 5 cm (p = 0.005), presence of lymph node metastasis (p = 0.001) and advanced TNM stage (p < 0.001). K-M analysis showed that reduced PUM1 expression was associated with increased survival of CRC patients with TNM stage I + II disease (Figs. 7E, 7F, p < 0.05). Moreover, reduced PUM1 expression was associated with increased survival of CRC patients receiving cetuximab treatment (Figs. 7G and H, p < 0.05).

Cetuximab is an anti-EGFR antibody that enhances overall survival of CRC patients but resistance to this drug is linked to tumor recurrence and metastasis ^[22], resulting in poor patient outcomes. Analysis of signaling pathways related to cetuximab-resistance has shown that mutations of the EGFR ^[23] or of the signaling network, such as KRAS-MAPK, phosphoinositide 3-kinase (PI3K)-PTEN-AKT and Janus kinase (JAK)/signal transducer and activator of transcription (STAT), are contributory factors ^[24]. Additional dysregulation of miRNAs and proteins have also been found to be involved in cetuximab-resistance in CRC ^[25].

m⁶A modifications have long been known to determine RNA fate^{[12, 26, 27]} and have been linked to cancer pathology. m⁶A modifications are mediated by the METTL3-METTL14 methyltransferase complex and a targeted METTL3 inhibitor has shown promise as a therapy for acute myeloid leukemia^[28]. PUM1 has been shown by previous work to be involved in cetuximab-resistance among CRC patients and DDX5 has been linked to this process ^[14]. However, little was known about the association between PUM1 and m⁶A modifications. The current work has demonstrated that the METTL3-induced m⁶A methylation enhanced PUM1 mRNA stability via modulation of YTHDF1 and the DDX5-WNT network, promoting CRC cetuximab resistance.

Downstream targets of PUM1 in CRC, including c-myc, cyclinD1 and AXIN2, were identified and PUM1 shown to activate the WNT pathway via DDX5 regulation. Paclitaxel-induced demethylation of PUM1 mRNA at the m⁶A position suppressed the WNT axis by diminishing PUM1 content. CRC PDXs showed paclitaxel-induced cetuximab-resistance via reduction of m⁶A-modified PUM1 levels. PUM1 protein expression in CRC tissues is a robust indicator of the potential benefits of cetuximab therapy for CRC patients. Thus, m⁶A-modified PUM1 may be a new bioindicator for patient selection for targeted immunotherapy. Further clinical research involving m⁶A-modified PUM1 and its significance in CRC therapy is required to confirm these findings.

In conclusion, a novel therapeutic target, inhibition of the N6-Methyladenosine modification of PUM1 was identified as a therapy for overcoming cetuximab-resistance in CRC.

CRC, colorectal cancer; PTX, paclitaxel; qRT-PCR, quantitative real-time PCR; IHC, immunohistochemistry; RIP, RNA immunoprecipitation; OS, overall survival; CTX, Cetuximab; NCCN, National Comprehensive Cancer Network; m⁶A, N⁶-methyladenosine; ncRNAs, non-coding RNAs; cetuximab resistance, Cr; K-M, Kaplan-Meier; DCs, dendritic cells; TAMs, tumor-associated macrophages; PI3K, phosphoinositide 3-kinase; STAT, activator of transcription; PDX, Patient derived tumor xenograft.

Data and Material Availability

The raw data that supported the conclusions made in the article can be made available by the authors unreservedly.

Author Contributions

XH Tu designed the research study. QZ Liu, HR Yu, and MJ Zhou performed the research. QZ Liu and HR Yu analyzed the data. All authors made contribution to the editorial changes. The final manuscript has been read by and obtained the approval of all authors. All authors, with sufficient participation, agreed to be in charge of all aspects of the work.

Ethics Approval and Consent to Participate

The study was completed in line with the Declaration of Helsinki (as revised in 2013). We obtained all participants’ informed consents. Animal experiments were carried out under a project license (No. TJBH0122101) granted by the Ethics Committee of Shanghai Tongji University, as per Shanghai Fourth People’s Hospital guidelines for the care and use of animals.

Acknowledgment

We sincerely thank Edit Springs (https://www.editsprings.com/) for their professional linguistic services provided.

Funding

This study was completed with the partial support from the “Special fund for scientific research startup” of Shanghai Fourth People's Hospital (sykyqd04401); Shanghai Hongkou District Science and Technology Committee (hongwei2202-19).

Conflict of Interest

The authors declare that no potential conflict of interest exist in the study.

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No competing interests reported.

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Version 1

posted

You are reading this latest preprint version

Paclitaxel Suppressed N⁶-Methyladenosine of PUM1 offsets cetuximab resistance in colorectal cancer

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and methods

Patients and tissue samples

Cell culture and shRNA transfection

Lentiviral transfection

qRT-PCR

Analysis of mRNA stability

MeRIP-PCR

Animal models of PDX

Dot blot of m⁶A

RNA immunoprecipitation (RIP)

Immunohistochemistry (IHC)

Analytical statistics

Results

Cetuximab-induced N⁶-methyladenosine modification of PUM1 mRNA enhanced stability in CRC cells

PUM1 Promoted Monocyte-to-Macrophage Differentiation

PUM1 activated the WNT pathway via modulation of DDX5 expression

Paclitaxel inhibited N⁶-demethyladenosine modification of PUM1 and suppressed the WNT pathway by diminishing PUM1 levels

Paclitaxelreducedm⁶A modification of PUM1 in cetuximab-resistant CRC cells

Increased PUM1 in human CRC was linked to elevated TAM levels and poor outcome

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Paclitaxel Suppressed N6-Methyladenosine of PUM1 offsets cetuximab resistance in colorectal cancer

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and methods

Patients and tissue samples

Cell culture and shRNA transfection

Lentiviral transfection

qRT-PCR

Analysis of mRNA stability

MeRIP-PCR

Animal models of PDX

Dot blot of m6A

RNA immunoprecipitation (RIP)

Immunohistochemistry (IHC)

Analytical statistics

Results

Cetuximab-induced N6-methyladenosine modification of PUM1 mRNA enhanced stability in CRC cells

PUM1 Promoted Monocyte-to-Macrophage Differentiation

PUM1 activated the WNT pathway via modulation of DDX5 expression

Paclitaxel inhibited N6-demethyladenosine modification of PUM1 and suppressed the WNT pathway by diminishing PUM1 levels

Paclitaxelreducedm6A modification of PUM1 in cetuximab-resistant CRC cells

Increased PUM1 in human CRC was linked to elevated TAM levels and poor outcome

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Paclitaxel Suppressed N⁶-Methyladenosine of PUM1 offsets cetuximab resistance in colorectal cancer

Dot blot of m⁶A

Cetuximab-induced N⁶-methyladenosine modification of PUM1 mRNA enhanced stability in CRC cells

Paclitaxel inhibited N⁶-demethyladenosine modification of PUM1 and suppressed the WNT pathway by diminishing PUM1 levels

Paclitaxelreducedm⁶A modification of PUM1 in cetuximab-resistant CRC cells