S100A4 contributes to colorectal carcinoma aggressive behavior and to chemoradiotherapy resistance in locally advanced rectal carcinoma

doi:10.21203/rs.3.rs-4870710/v1

Download PDF

Article

S100A4 contributes to colorectal carcinoma aggressive behavior and to chemoradiotherapy resistance in locally advanced rectal carcinoma

https://doi.org/10.21203/rs.3.rs-4870710/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

To investigate the functional role of S100A4 in advanced colorectal carcinoma (Ad-CRC) and locally advanced rectal carcinoma (LAd-RC) receiving neoadjuvant chemoradiotherapy (NCRT). We analyzed histopathological and immunohistochemical sections from 150 patients with Ad-CRC and 177 LAd-RC patients treated with NCRT. S100A4 knockout (KO) HCT116 cells were also used. S100A4 expression was absent in normal mucosa but increased progressively from colorectal adenoma to carcinoma, suggesting that S100A4 regulation is in an early event in colorectal carcinogenesis. In Ad-CRC, high S100A4 expression correlated with high tumor budding and nuclear b-catenin, deep invasion, lymph-vascular involvement, and unfavorable prognosis. In NCRT-treated LAd-RC, high S100A4 expression was associated with poor treatment response and short progression-free survival. S100A4 KO decreased the proliferation of HCT116 cells through activation of the p53/p21^waf1 axis, and sensitized cells to adriamycin-induced apoptosis. Levels of the apoptotic marker, cleaved poly (ADP-ribose) polymerase 1, were significantly higher in samples with low S100A4 and wild type p53. Finally, we observed a direct interaction between S100A4 and p53. In conclusion, S100A4 expression engenders aggressive behavior in Ad-CRC through association with b-catenin-driven tumor buddings. S100A4 exerts anti-apoptotic and proliferative effects via inhibition of p53 in LAd-RC patients receiving NCRT, which leads to chemoradioresistance and poor prognosis.

Biological sciences/Cancer

Biological sciences/Cell biology

S100A4

p53

apoptosis

colorectal carcinoma

neoadjuvant chemoradiotherapy

Colorectal carcinoma (CRC) is one of the most common malignancies worldwide and a major cause of carcinoma death [1]. Approximately 70% of CRC are sporadic cases which are due to environmental factors, with genetic predisposition and familial mutations accounting for the remaining 30% [2, 3]. The incidence of CRC in Asia (especially in Japan, China, Korea, and Taiwan) is rising steadily, with a two- to four-fold increase over the last few decades [4].

Five-year overall survival for patients with stage I CRC is approximately 90%; this decreases to about 10% for stage IV patients [5]. The stage IV patient group contains individuals with advanced CRC, and locally advanced rectal carcinoma (Ad-CRC and LAd-RC hereafter). Prior to total mesorectal excision in these patients, neoadjuvant chemoradiotherapy (NCRT) is performed to induce downstaging, downsizing, and significant changes in histological characteristics [6, 7]. Although this increases the response rate to systemic NCRT to 50%, nearly all patients with LAd-RC subsequently develop resistance [8–10]. Understanding the molecular mechanisms that drive NCRT resistance may therefore elucidate novel therapeutic strategies to prolong patient life.

S100A4 belongs to the S100 family and is a calcium-binding protein with two EF-hands [11–13]. The S100 proteins do not have enzymatic activity and instead exert their biological activities through interaction with target proteins [11–13]. In particular, S100A4 contributes to tumor progression, through modulation of cell motility, adhesion, proliferation, and invasion. Although aberrant S100A4 expression is a useful biomarker of chemo- or radio-resistance in several human malignancies [14, 15], the molecular mechanisms underpinning S100A4-dependent resistance to NCRT remain unclear.

To address this gap in knowledge, we profiled the expression of S100A4 during Ad-CRC progression, and examined its relationship with b-catenin, a known S100A4 regulator [16]. We also examined the role of S100A4 in NCRT-treated LAd-RC and CRC cell lines using CRISPR knockout (KO) technology.

Clinical cases

We evaluated pathological specimens of 150 Ad-CRC cases, surgically resected between 2009 and 2021, at Kitasato University Hospital, according to the criteria of the Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma and the TMN classification [17,18]. We also selected 177 LAd-RC cases, which were defined as clinical T3 or T4, N0 to N2, or M0 tumors, that underwent NCRT [19], followed by total mesorectal excision at Kitasato University Hospital between 2006 and 2021. Pretreatment-biopsied samples from 106 NCRT-treated LAd-RC patients were also investigated. In addition, 20 samples of normal colorectal epithelium adjacent to the tumors, low-grade adenoma, high-grade adenoma, carcinoma in adenoma, and non-invasive CRC, respectively, were also examined. This study was approved by the Kitasato University Medical Ethics Committee (B19-155).

Evaluation of histopathological findings

Tumor budding (BD) [20] and the BD score was evaluated according to the criteria of the Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma [17]. Ad-CRC lesions were subcategorized into three groups based on the depth of tumor invasion as follows: mucosal (m), submucosal (sm), and muscularis propria/subserosal (mp/ss) layers. NCRT-treated LAd-RC cases were also subclassified into three groups on the basis of the therapeutic efficacy (TE) of NCRT, as follows: grade (G) 1, mild efficacy; G2, moderate; and G3, no tumor elements, according to the criteria of the Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma [17].

Immunohistochemistry (IHC)

IHC was performed using a combination of microwave -oven heating and immuno-enzyme polymer (Nichirei Bioscience Inc, Tokyo, Japan) and the evaluation for S100A4 score and nuclear b-catenin and Ki-67 immunopositive labeling indices (LIs) was performed as described previously [21]. The S100A4 scores and nuclear b-catenin LIs were subdivided into two categories based on the mean – standard deviation (SD) or mean values as cutoff (Supplementary Figure S1). p53 immunopositivity was subdivided into mutant (mt, 0% or 60–100%) and wild type (wt, 1–59%) classes [22]. The cleaved PARP1 IHC score was derived from counting cells in five randomly selected high-power fields (HPFs).

Cell line and plasmids

An S100A4 KO CRC cell line was generated using HCT-116 (ATCC, Manassas, VA, USA). Briefly, the guide RNA sequence (gRNA: 5’-CCACAAGTACTCGGGCAAAG-3’) was designed using CRISPRdirect (https://crispr.dbcls.jp) and cloned into pSpCas9n(BB)-2A-Puro (PX459) V2.0 (Addgene #62988) to establish the S100A4 KO cell line as described previously [21]. We also used the following plasmids: pcDNA-b-catenin^△S45, pCI-p300, pGL3B-(-1976/+1012) S100A4-luc, and pCMVp53wt as described previously [23, 24].

Antibodies and reagents

Antibodies are listed in Supplementary Table S1. Adriamycin (ADR) was obtained from Sigma-Aldrich Chemicals (St. Louis, MO, USA).

Western blot and co-immunoprecipitation (co-IP) assays

Isolation of total cellular proteins and western blot and co-IP assays were carried out as described previously [21, 23–25]. The signals were analyzed using ImageJ software version 1.41 (NIH, Bethesda, MD, USA; http//imgaeJ.nih.gov/ij) as described previously [25]. The reconstructed images of all blots with membrane edges visible are accessible in the Supplementary Materials, because some of the original full-length blots were cut prior to hybridization with antibodies.

Flow cytometry

Cell cycle analysis using a BD FACS Calibur instrument (BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest Pro software v3.3 (BD Biosciences) as described previously [21, 25].

Proximity ligation assay (PLA)

Mouse b-catenin or mouse p53 were used alone (negative controls) or in combination with rabbit S100A4 antibody. The slides were treated using the Duolink Detection kit with PLA PLUS and MINUS probes for mouse and rabbit antibodies, respectively (Olink Bioscience, Uppsala, Sweden). Average number of intranuclear and intracytoplasmic PLA signals were expressed as an signals per cell as described previously [26].

Cell Counting Kit-8 assay

Cell viability after ADR treatment was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo Lab, Kumamoto, Japan), according to the manufacture’s instructions.

Apoptotic index

The number of apoptotic cells identified in HE-stained sections was calculated by counting the mean number of apoptotic figures per field as described previously [27].

Transfection and luciferase assay

Transfection was carried out using LipofectAMINE PLUS (Invitrogen, Carlsbad, CA, USA) and luciferase activity was assayed 24 h after transfection using the Dual-luciferase reporter assay system (Promega) as described previously [24, 25].

Statistical analysis

Comparative data were analyzed using the Mann-Whitney U-test and Chi-square test, as appropriate. Overall survival (OS) and progression-free survival (PFS) were evaluated as described previously [21, 25]. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. The cutoff for statistical significance was set as P < 0.05.

Upregulation of S100A4 in development and progression of CRC

Nuclear/cytoplasmic S100A4 immunoreactivity was observed in colorectal tumors but not normal mucosal lesions (Fig. 1A), with a significant stepwise increase in expression from adenomas to non-invasive CRC (Fig. 1B).

S100A4 scores were significantly higher in Ad-CRC tumors localized at mp/ss lesions (including the tumor invasive front) as compared to m and sm lesions (Fig. 1C). High S100A4 expression was significantly associated with highly invasive tumors, high tumor budding, lymphovascular invasion (Table 1), and the poorest OS (Fig. 1D). Comparison of univariate and multivariate Cox analyses revealed that S100A was a significant (but not independent) prognostic indicator for OS (Table 2).

High nuclear b-catenin was also significantly associated with the tumor invasive front and correlated with poor PFS in Ad-CRC (Supplementary Figure S2). The PLA assay, which can be used to quantify protein-protein interactions in tissue sections [28], revealed significantly higher number of nuclear S100A4/b-catenin foci in the tumor invasive front, including at site of tumor budding (Supplementary Figure S3A); this contrasted with a lack of signal following co-IP experiments (Supplementary Figure S3B). In addition, the S100A4 promoter was activated by co-transfection of b-catenin and p300 (Supplementary Figure S3C).

Relationship between S100A4 and NCRT resistance in LAd-RC

The results of S100A4 and Ki-67 immunoreactivity in resected LAd-RCs after NCRT are shown in Fig. 2A. The S100A4 scores and Ki-67 LIs were significantly higher in patients who responded poorly to NCRT (TE: G1) compared to those patients with TE: G2. High S100A4 expression was associated with the shortest PFS in NCRT-treated LAd-RC patients (Fig. 2B). Comparison of univariate and multivariate Cox analyses revealed that S100A4 expression was a significant (but not independent) prognostic indicator of PFS (Table 3). Nuclear b-catenin status was not a prognostic indicator of PFS (Supplementary Figure S4). In addition, S100A4 scores in pretreatment-biopsied samples from LAd-RC patients did not correlate with TE grade in surgical specimens (Supplementary Figure S5).

Knockout of S100A4 decreases proliferation and sensitizes to apoptosis

To examine the functional role of S100A4 in NCRT resistance in LAd-RC, we generated two independent S100A4 KO HCT116 cell line clones (KO#3 and KO#4). In response to ADR treatment, S100A4 KO significantly decreased cell viability when compared to parental cells (Fig. 3A). Consistent with this, we observed increased apoptosis (Fig. 3B) and sub-G1 fraction (Fig. 3C), as well as increased expression of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP1), and BCL-2 (Fig. 3D and Supplementary Figure S6).

S100A4-KO cells also proliferated more slowly than parental cells; consistent with this, there were proportionally less cells in S phase (Fig. 4A). We next examined the impact of S100A4 loss on several cell cycle-related markers. First, S100A4 KO cells were rendered quiescent by serum starvation. We then added back serum to trigger cell cycle entry, and took samples for western bot at 7 and 24 h post-serum stimulation. Levels of cyclin A2 and cyclin B1 were lower in S100A4 KO cells when compared to parental cells, whereas p21^waf1 expression was higher (Fig. 4B and Supplementary Figure S7).

Relationship between S100A4 expression and p53-dependent apoptosis

Since p53 interacts directly with S100A4 [29–31], we examined whether there was a functional interaction between these two proteins in NCRT-treated LAd-RC. A PLA assay revealed distinct nuclear dots containing S100A4 and p53 in LAd-RC (Fig. 5A). This indication that they interact in LAd-RC was supported by the results of co-IP assay (Fig. 5B and Supplementary Figure S8A). There was no significant difference in p53 protein stability between S100A4 KO and parental cells (Fig. 5C and Supplementary Figure S8B). In addition, S100A4 promoter activity was repressed by transfection of p53 (Supplementary Figure S9). Finally, the percentage of cleaved PARP1-positive cells was significantly higher in S100A4-low as compared to S100A4-high NCRT-treated-LAd-RC cases (Fig. 6A), with the highest percentage being found in the “S100A4-low, p53wt” group (Fig. 6B).

In this report, we clearly show that S100A4 expression progressively increases as cells transition from normal tissue to adenoma and then to non-invasive CRC. Moreover, loss of S100A4 robustly reduces molecular markers of the cell cycle, which ultimately leads to decreased proliferation. These observations are consistent with early reports that S100A4 is highly expressed in both growth factor-stimulated normal cells [32, 33], and in metastatic tumor cell lines [34]. It has been suggested that S100A4 overexpression is an early event during colorectal carcinogenesis, and that it enhances the malignant potential of early tumor cells [35, 36].

Tumor budding at the invasive front is a reliable histopathological marker for estimating CRC aggressiveness [37, 38]. We found that high S100A4 expression was also significantly associated with tumor budding in Ad-CRC. Although nuclear foci containing S100A4 and b-catenin (which is also associated with tumor budding [25]) were more abundant at the tumor invasive front, these proteins did not co-immunoprecipitation. Given that the S100A4 promoter was activated by a b-catenin/p300 complex, we suggest that S100A4 may indirectly cooperate to establish and maintain b-catenin-dependent tumor budding in Ad-CRC. Further studies are required to address this hypothesis.

The impact of S100A4 levels on the response to chemotherapy appears context-dependent. For example, a CRC cell line resistant to doxorubicin had moderate S100A4 expression [39], whereas sensitivity to 5-fluorouracil was independent of S100A4 in other CRC cell lines [40]. In our current study, we found that high S100A4 expression was associated with chemoradioresistance and poor prognosis in LAd-RC. By contrast, the lack of association between S100A4 levels in pretreatment-biopsied samples and NCRT efficacy in the corresponding resected RC samples may be due to the heterogenous expression of S100A4 in LAd-RC tissues.

In tissues from Aad-RC patients who received NCRT, we found that low S100A4 expression was significantly associated with a high score for cleaved PARP1, which is a major product of caspase-3 activity in vivo [41]. Moreover, S100A4 KO enhanced susceptibility to ADR-induced apoptosis, suggesting that S100A4 acts as a pro-survival factor by suppressing the apoptotic pathway in response to NCRT in LAd-RC. Similar findings have also been observed in pancreatic ductal adenocarcinoma and osteosarcoma [42, 43].

Finally, we found that p53 wild type cells with little or no S100A4 were much more sensitive to chemoradiotherapy-induced apoptosis than their p53 mutant counterparts. Given that S100A4 directly interacted with p53 in both LAd-RC tissue and CRC cell line, we suggest that S100A4 effectively blocks p53 activation and DNA binding, thereby facilitating cell survival and NCRT resistance in LAd-RC. A corollary is that a negative feedback loop may also operate, in which p53 represses S100A4 promoter.

Our result show that elevated S100A4 expression engenders aggressive behavior in Ad-CRC through indirect modulation of nuclear b-catenin-mediated tumor budding. S100A4 also inhibits apoptosis and favors proliferation effects, at least partly due to attenuation of p53; this is in turn associated with chemoradioresistance and poor prognosis in LAd-RC (Fig. 7).

Acknowledgements

Not applicable

Authors’ contributions statement

YH, SI, YK, HT, and MS carried out the majority of the experiments, analyzed the data, and wrote the manuscript. They were helped by YO, Miki H, AY, AS, NF, MH, MO, and CK. All authors reviewed and approved the final manuscript.

Funding statement

This study was supported by a grant from JSPS KAKENHI Grant Number 22K06967.

Ethics approval statement

This study was conducted in accordance with the principles expressed in the Declaration of Helsinki 1964 and later versions. This study was approved by the Kitasato University Medical Ethics Committee (B19-155). This article does not contain any studies with animals performed by any of the authors.

Consent for publication

Not applicable

Data availability statement

The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest

Potential conflicts do not exist.

Informed consent statement

Informed consent was obtained from all subjects involved in the study.

Onyoh EF, et al. The rise of colorectal cancer in Asia: epidemiology, screening, and management. Curr. Gastroenterol Rep. 2019;21;36.
Jasperson KW, Tuohy TM, Neklason DW, Burt RW. Hereditary and familial colon cancer. Gastroenterology. 2010;138;2044-58.
Migliore L, Migheli E, Spisni R, Coppede E. Genetics, cytogenetics, and epigenetics of colorectal cancer. J Biomed Biotechnol. 2011;2011;792362.
Wong MC, et al. Prevalence and risk factors of colorectal cancer in Asia. Intest Res. 2019;17;317-29.
O’Connell JB, Maggard MA, Ko CY. Colon cancer survival rates with the new American joint committee on cancer sixth edition staging. J Nalt Cancer Inst. 2004;96;1420-5.
Medich D, et al. Preoperative chemoradiotherapy and radical surgery for locally advanced distal rectal adenocarcinoma: pathologic findings and clinical implications. Dis. Colon Rectum. 2001;44;1123-8.
Ruo L, et al. Long-term prognostic significance of extent of rectal cancer response to preoperative radiation and chemotherapy. Ann Surg. 2002;236;75-81.
Sauer R, et al. Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med. 2004;351;1731-40.
Valentini V, et al. Doses downstaging predict improved outcome after preoperative chemoradiation for extraperitoneal locally advanced rectal cancer? A long-term analysis of 165 patients. Int J Radiat Oncol Biol Phys. 2002;53;664-74.
Reerink O, et al. Molecular prognosis factors in locally irresectable rectal cancer treated preoperatively by chemo-radiotherapy. Anticancer Res. 2004;24;1217-21.
Helfman DM, Kim EJ, Lukanidin E, Grigorian M. The metastasis associated protein S100A4: role in tumor progression and metastasis. Br J Cancer. 2005;92;1955-8.
Garrett SC, Varney KM, Weber DJ, Bresnick AR. S100A4, a mediator of metastasis. J Biol Cem. 2006;281;677-80.
Bresnick AR, Weber DJ, Zimmer DB. S100 proteins in cancer. Nat Rev Cancer. 2015;15;96-109.
Mencia N, et al. Overexpression of S100A4 in human cancer cell lines resistant to methotrexate. BMC Cancer. 2010;10;250.
Qi R, Qiao T, Zhuang X. Small interfering RNA targeting S100A4 sensitizes non-small-cell lung cancer cells (A549) to radiation treatment. Onco Targets Ther. 2016;9;3753-62.
Dahlmann M, et al. Combination of Wnt/b-catenin targets S100A4 and DKK1 improves prognosis of human colorectal cancer. Cancers. 2022;14;37.
Japanese Society for Cancer of the Colon and Rectum, ed. General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus, 3rd English edition. Tokyo, Japan: Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma, 2019.
Brierley JD, Gospodarowicz MK, and Wittekind C eds. TNM Classification of Malignant Tumours. Wiley-Blackwell: Oxford, UK, 2016.
Sato T, et al. A multicenter phase I study of preoperative chemoradiotherapy with S-1 and irinotecan for locally advanced lower rectal cancer (SAMRAI-1). Radiother Oncol. 2016;120;222-7.
Zengin M, Cifci A. Tumour budding in preoperative biopsy specimens is a useful for identifying high-risk patients in early-stage (pN0) colon cancer. Turk J Med Sci. 2020;50;375-85.
Ishibashi Y, et al. Nucleobindin 2 inhibits senescence in gastric cancer. Sci Rep. 2024;14; 11261.
Yemelyanova A, et al. Immunohistochemical staining patterns of p53 can serve as a surrogate marker for TP53 mutations in ovarian carcinomas: an immunohistochemical and nucleotide sequencing analysis. Mod Pathol. 2011;24;1248-53.
Saegusa M, et al. Upregulation of TCF4 expression as a transcriptional target of b-catenin/p300 complexes during trans-differentiation of endometrial carcinoma cells. Lab Invest. 2005;85;768-79.
Tochimoto M, et al. S100A4/non-muscle myosin II signaling regulates epithelial-mesenchymal transition and stemness in uterine carcinosarcoma. Lab Invest. 2020;100;682-95.
Itou T, et al. EBP50 depletion and nuclear b-catenin accumulation engender aggressive behavior of colorectal carcinoma through induction of tumor budding. Cancers. 2024;16;183.
Nakagawa M, et al. Interaction between membranous EBP50 and myosin 9 as a favorable prognostic factor in ovarian clear cell carcinoma. Mol Oncol. 2023;17;2168-82.
Akiya M, et al. Identification of LEFTY as a molecular marker for ovarian clear cell carcinoma. Oncotarget. 2017;8:63646-63664.
Fredriksson S, et al. Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol. 2002;20;473-7.
Kwak JM , et al. Expression of protein S100A4 is a predictor of recurrence in colorectal cancer. World J Gastroenterol. 2010;16;3897-904.
Orre LM, et al. S100A4 interacts with p53 in the nucleus and promotes p53 degradation. Oncogene. 2013;32;5531-40.
Grigorian M, et al. Tumor suppressor p53 protein is a new target for the metastasis-associated Mts1/S100A4 protein. J Biol Chem. 2001;276;22699-708.
Goto K, Endo H, Fijiyoshi T. Cloning of the sequences expressed abundantly in established cell lines: identification of a cDNA clone highly homologous to S-100, a calcium binding protein. J Biochem. 1988;103;48-53.
Linzer DI, Nathans D. Growth-related changes in specific mRNAs of cultured mouse cells. Proc Natl Acad Sci USA. 1983;80;4271-75.
Ebralidze A, et al. Isolation and characterization of a gene specifically expressed in different metastatic cells and whose deduced gene product has a high degree of homology to a Ca2+-binding protein family. Genes Dev. 1989;3;1086-93.
De Vouge MW, Mukherjee BB. Transformation of normal rat kidney cells by v-K-ras enhances expression of transin 2 and an S-100-related calcium-binding protein. Oncogene. 1992;7;109-19.
Takenaga K, Nakamura Y, Endo H, Sakiyama S. Involvement of S100-related calcium-binding protein pEL98 (or mts1) in cell motility and tumor cell invasion. Jpn J Cancer Res. 1994;85;831-9.
Ueno H, et al. Tumour ‘budding’ as an index to estimate the potential of aggressiveness in rectal cancer. Histopathology. 2002;40;127-32.
Shinto E, et al. A novel classification of tumour budding in colorectal cancer based on the presence of cytoplasmic pseudo-fragments around budding foci. Histopathology. 2005;47;25-31.
Bertram J, Palfner K, Hiddemann W, Kneba M. Elevated expression of S100P, CAPL and MAGE 3 in doxorubicin-resistance cell lines: comparison of mRNA differential display reverse transcription-polymerase chain reaction and subtractive suppressive hybridization for the analysis of differential gene expression. Anticancer Drugs. 1998;9;311-7.
Boye K, et al. Prognostic significance of S100A4 expression in stage II and III colorectal cancer: results from a population-based series and a randomized phase III study on adjuvant chemotherapy. Cancer Med. 2016;5;1840-9.
Boulares AH, et al. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. J Biol Cem. 1999;274;22932-40.
Mahon PC, et al. S100A4 contributes to the suppression of BNIP3 expression, chemoresistance, and inhibition of apoiptosis in pancreatic cancer. Cancer Res. 2007;67;6786-95.
Pedersen KB, Andersen K, Fodstad O, Maelandsmo GM. Sensitization of interferon-gamma induced apoptosis in human osteosarcoma cells by extracellular S100A4. BMC Cancer. 2004;4;52.

Tables 1-3 are available in the Supplementary Files section.

No competing interests reported.

Table1.xlsx
Table2.xlsx
Table3.xlsx
SupTableS1.xlsx
SupFigureS1.tif
Supplementary Figure S1. (A,B) Dot plot analyses for S100A4 scores (A) and nuclear b-catenin LIs (B) in 150 Ad-CRC cases (left) and 150 NCRT-treated LAd-RC cases (right). The data are shown as mean ± SD. Based on the mean – standard deviation (SD) or mean values as cutoff, cases are divided into two (high versus low expression) categories.
SupFigureS2.tif
Supplementary Figure 2. Nuclear b-catenin accumulation in Ad-CRC. (A) Upper: staining with HE and IHC for b-catenin in Ad-CRC. Note the high levels of nuclear b-catenin at the invasive front in Ad-CRC. The closed boxes in the lower panels are magnified in the insets. Original magnification, x40 and x400 (insets). Lower: nuclear b-catenin LIs between mucosal (m), submucosal (sm), and muscularis propria (mp)/subserosal (ss) lesions of Ad-CRC. The data are presented shown as mean ± SD. Statistical analyses were carried out using the Mann-Whitney U-test. (B) OS (upper) and PFS (lower) relative to nuclear b-catenin LI values (low versus high accumulation) in Ad-CRC. n, number of cases.
SupFigureS3.tif
Supplementary Figure S3. Interaction between S100A4 and b-catenin in CRC cells. (A) Left: PLA assay for the S100A4/b-catenin interaction, as well as IHC for S100A4 and b-catenin, in Ad-CRC. Note the small aggregated dots in nuclear/cytoplasmic compartments of the tumor cells. The closed box in the PLA panel is magnified in the inset. Original magnification, x100 and x400 (inset). Right: numbers of PLA scores for S100A4/b-catenin combination between mucosal (m), submucosal (sm), and muscularis propria (mp)/subserosal (ss) lesions of Ad-CRC. The data are shown as mean ± SD. Statistical analyses were carried out using the Mann-Whitney U-test. (B) After immunoprecipitation (IP) with the indicated antibodies using HCT116 cell lysates, western blot assay (WB) with anti-b-catenin (upper panel) and anti-S100A4 antibodies (lower panel) was carried out. Input was 5% of the total cell extract. Normal rabbit IgG was used as a negative control. The reconstructed images of all blots with membrane edges visible are shown because some of the original full-length bots were cut prior to hybridization with antibodies. (C) HCT116 cells were transfected S100A4 promoter luciferase (luc), together with b-catenin delS45 and p300, using LipofectAMINE PLUS. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiments were performed in triplicate.
SupFigureS4.tif
Supplementary Figure S4. Relation between nuclear b-catenin accumulation and NCRT resistance in LAd-RC. (A) Upper: staining with HE and IHC for b-catenin in samples of LAd-RC cases that respond poorly to NCRT (TE: Grade 1) or respond moderately (TE: Grade 2). The closed boxes in the IHC panels are magnified in the insets. Original magnification, x100 and x400 (insets). Lower: nuclear b-catenin LIs in samples from NCRT-treated LAd-RC patients. The LIs are shown as mean ± SD. (B) OS (upper) and PFS (lower) relative to nuclear b-catenin (low versus high LIs) in LAd-RC receiving NCRT. N, number of cases.
SupFigureS5.tif
Supplementary Figure S5. Relationship between S100A4 expression in pretreatment-biopsied samples and TE grade in surgical specimens of NCRT-treated LAd-RC. (A) Staining with HE and IHC for S100A4 in pretreatment LAd-RC patient biopsies from TE: Grade 1 (left), TE: Grade 2 (middle), and TE: Grade 3 (right). Original magnification, x200. (B) IHC scores for S100A4 in pretreatment biopsied samples from TE grade 1, 2, and 3. The scores are shown as mean ± SD.
SupFigureS6.tif
Supplementary Figure S6. Original images of western blot analysis for the indicated proteins in total lysates from HCT116-S100A4 KO and parental cells after 0.5 mg/mL ADR treatment. The reconstructed images of all blots with membrane edges visible are shown because some of the original full-length bots were cut prior to hybridization with antibodies.
SupFigureS7.tif
Supplementary Figure S7. Original images of western blot analysis for the indicated proteins in total lysates from HCT116-S100A4 KO and parental cells following re-stimulation of serum-starved (24 h) cells with 10% serum for the indicated times. The reconstructed images of all blots with membrane edges visible are shown because some of the original full-length bots were cut prior to hybridization with antibodies.
SupFigureS8.tif
Supplementary Figure S8. Original images of western blot analysis for the indicated proteins in co-IP-western blot (A) and in total lysates from HCT116-S100A4 KO and parental cells treated with 25 mg/mL cycloheximide (CHX) at the indicated timepoints. The reconstructed images of all blots with membrane edges visible are shown because some of the original full-length bots were cut prior to hybridization with antibodies.
SupFigureS9.tif
Supplementary Figure S9. p53-dependent repression of the S100A4 promoter. HCT116 cells were transfected with S100A4 promoter luciferase (luc), together with p53 using LipofectAMINE PLUS. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiments were performed in triplicate.

Download PDF

Editorial decision: Revision requested
21 Oct, 2024
Reviewers agreed at journal
22 Sep, 2024
Reviews received at journal
12 Sep, 2024
Reviewers agreed at journal
01 Sep, 2024
Reviews received at journal
27 Aug, 2024
Reviewers agreed at journal
17 Aug, 2024
Reviews received at journal
17 Aug, 2024
Reviewers agreed at journal
17 Aug, 2024
Reviewers invited by journal
17 Aug, 2024
Editor assigned by journal
13 Aug, 2024
Editor invited by journal
13 Aug, 2024
Submission checks completed at journal
13 Aug, 2024
First submitted to journal
06 Aug, 2024

You are reading this latest preprint version

S100A4 contributes to colorectal carcinoma aggressive behavior and to chemoradiotherapy resistance in locally advanced rectal carcinoma

Status:

Version 1

Abstract

Figures

Introduction

Methods

Clinical cases

Immunohistochemistry (IHC)

Statistical analysis

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1