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Cancer-related CD15/FUT4 overexpression decreases benefit to agents targeting EGFR or VEGF acting as a novel RAF-MEK-ERK kinase downstream regulator in metastatic colorectal cancer.

Giordano G, Febbraro A, Tomaselli E, Sarnicola ML, Parcesepe P, Parente D, Forte N, Fabozzi A, Remo A, Bonetti A, Manfrin E, Ghasemi S, Ceccarelli M, Cerulo L, Bazzoni F, Pancione M - J. Exp. Clin. Cancer Res. (2015)

Bottom Line: CD15/FUT4-high expressing colon cancer cells with primary resistance to cetuximab or bevacizumab are significantly more sensitive to MEK inhibitors than CD15/FUT4-low counterparts.Cancer-related CD15/FUT4 overexpression participates in cetuximab or bevacizumab mechanisms of resistance in mCRC patients.CD15/FUT4 as a potential target of the antitumor immune response requires further evaluation in clinical studies.

View Article: PubMed Central - PubMed

Affiliation: Medical Oncology Unit, Fatebenefratelli Hospital, 82100, Benevento, Italy.

ABSTRACT

Background: Cancer-related immune antigens in the tumor microenvironment could represent an obstacle to agents targeting EGFR "cetuximab" or VEGF "bevacizumab" in metastatic colorectal cancer (mCRC) patients.

Methods: Infiltrating immune cells into tumor tissues, cancer-related expression of immune antigens (CD3, CD8, CD68, CD73, MPO, CD15/FUT4) from 102 mCRC patients receiving first-line Cetuximab or Bevacizumab plus chemotherapy were assessed by immunohistochemistry and validated in an independent tissue microarrays of 140 patients. Genome-wide expression profiles from 436 patients and 60 colon cancer cell lines were investigated using bioinformatics analysis. In vitro kinase assays of target genes activated by chemokines or growth factors were performed.

Results: Here, we report that cancer-related CD15/FUT4 is overexpressed in most of mCRCs patients (43 %) and associates with lower intratumoral CD3+ and CD8+ T cells, higher systemic inflammation (NLR at diagnosis >5) and poorer outcomes, in terms of response and progression-free survival than those CD15/FUT4-low or negative ones (adjusted hazard ratio (HR) = 2.92; 95 % CI = 1.86-4.41; P < 0.001). Overexpression of CD15/FUT4 is induced through RAF-MEK-ERK kinase cascade, suppressed by MEK inhibitors and exhibits a close connection with constitutive oncogenic signalling pathways that respond to ERBB3 or FGFR4 activation (P < 0.001). CD15/FUT4-high expressing colon cancer cells with primary resistance to cetuximab or bevacizumab are significantly more sensitive to MEK inhibitors than CD15/FUT4-low counterparts.

Conclusion: Cancer-related CD15/FUT4 overexpression participates in cetuximab or bevacizumab mechanisms of resistance in mCRC patients. CD15/FUT4 as a potential target of the antitumor immune response requires further evaluation in clinical studies.

No MeSH data available.


Related in: MedlinePlus

Genome-wide expression analysis identifies CD15/FUT4 as a novel RAF-MEK-ERK kinase downstream target. a The most enriched network of CD15/FUT4 regulators is depicted comprising 20 regulons and two closely connected upstream genes involving EGFR and FGFR pathways (see Additional file 2 and Additional file 3: Figure S5 for more information). The gene expression datasets GSE17536/GSE17537 series (n = 226) were analyzed simultaneously with the ARACNe algorithm to infer transcriptional regulatory network fromgenome-wide expression profiles CD15/FUT4-connected. bCD15/FUT4 expression in CRC using patient-matched tumor-normal data available from TCGA. The P value refers to Mann–Whitney test. Expression profiles of ERBB1, ERBB2, ERBB3, FGFR4, CD15/FUT4 and KRAS mutant across a series of CRC cell lines (n = 60) by applying a fold change of gene expression microarray data of 1.5 and subdivided on the basis of chromosomal instability calculated as fraction of copy-number alteration pattern (form 0 to 0.922). Significant correlation between CD15/FUT4, ERBB3 and FGFR4 expression levels. c Linear correlation between ERBB3, FGFR4 and CD15/FUT4 transcript levels is validated in our independent series of (n = 12) CRC cell lines. Immunofluorescence labeling “green” of nonpermeabilize cancer cells shows differential expression of CD15/FUT4 “arrows,” on the cell plasma membrane of RKO and HT29, respectively. To visualize nuclei, the merged images were stained with 4’6-diamidino-2-phenylindole (Dapi), “blue”. d CD15/FUT4-dependent transcript induction by EGF (10 nM) or IL1b (20U/ml) by using 0.1 % of DMSO as vehicle, in two representative CRC cell lines SW480 and RKO, respectively. Cells were treated for 8 h and the ratio anti-p-ERK/ERK1/2 was quantified by western-blot analysis. The treatment with IL-10 (200U/ml) or IL-6 (50 ng/ml) for 30 min revealed a significant induction of IL6-STAT1/STAT3 dependent phosphorylation in a panel of 4 CRC cell lines as detected by western-blot analysis. Mononuclear cells (Mono) purified from buffy coats of healthy donors were used as positive control for IL10-STAT3 dependent phosphorylation. CD15/FUT4 transcript did not reveal any significant induction following IL10 or IL6 at 48 h of treatment. The P value were obtained by Mann–Whitney test; *P ≤ .05; **P ≤ .01
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Fig3: Genome-wide expression analysis identifies CD15/FUT4 as a novel RAF-MEK-ERK kinase downstream target. a The most enriched network of CD15/FUT4 regulators is depicted comprising 20 regulons and two closely connected upstream genes involving EGFR and FGFR pathways (see Additional file 2 and Additional file 3: Figure S5 for more information). The gene expression datasets GSE17536/GSE17537 series (n = 226) were analyzed simultaneously with the ARACNe algorithm to infer transcriptional regulatory network fromgenome-wide expression profiles CD15/FUT4-connected. bCD15/FUT4 expression in CRC using patient-matched tumor-normal data available from TCGA. The P value refers to Mann–Whitney test. Expression profiles of ERBB1, ERBB2, ERBB3, FGFR4, CD15/FUT4 and KRAS mutant across a series of CRC cell lines (n = 60) by applying a fold change of gene expression microarray data of 1.5 and subdivided on the basis of chromosomal instability calculated as fraction of copy-number alteration pattern (form 0 to 0.922). Significant correlation between CD15/FUT4, ERBB3 and FGFR4 expression levels. c Linear correlation between ERBB3, FGFR4 and CD15/FUT4 transcript levels is validated in our independent series of (n = 12) CRC cell lines. Immunofluorescence labeling “green” of nonpermeabilize cancer cells shows differential expression of CD15/FUT4 “arrows,” on the cell plasma membrane of RKO and HT29, respectively. To visualize nuclei, the merged images were stained with 4’6-diamidino-2-phenylindole (Dapi), “blue”. d CD15/FUT4-dependent transcript induction by EGF (10 nM) or IL1b (20U/ml) by using 0.1 % of DMSO as vehicle, in two representative CRC cell lines SW480 and RKO, respectively. Cells were treated for 8 h and the ratio anti-p-ERK/ERK1/2 was quantified by western-blot analysis. The treatment with IL-10 (200U/ml) or IL-6 (50 ng/ml) for 30 min revealed a significant induction of IL6-STAT1/STAT3 dependent phosphorylation in a panel of 4 CRC cell lines as detected by western-blot analysis. Mononuclear cells (Mono) purified from buffy coats of healthy donors were used as positive control for IL10-STAT3 dependent phosphorylation. CD15/FUT4 transcript did not reveal any significant induction following IL10 or IL6 at 48 h of treatment. The P value were obtained by Mann–Whitney test; *P ≤ .05; **P ≤ .01

Mentions: To gain insights into the mechanisms that regulate CD15/FUT4 expression, we collected two independent publicly available gene-expression datasets involving a total of 436 colorectal cancer and 60 colon cancer cell lines [7, 23, 24]. Strikingly, in-silico analysis, inferred a number of 1350 interactions and 20 regulons CD15/FUT4-associated, and revealed a series of disease-relevant pathways: a) prosurvival; b) immune-evasion; c) protein kinase cascade and viral infectious response closely connected to the efficacy of drugs (Fig. 3a and Additional file 3: Figure S5A-D) [28–30]. CD15/FUT4 transcript was significantly higher in tumor tissues than normal control in TCGA series comprising 210 CRCs and 22 and normal colorectal mucosa specimens, respectively (Fig. 3b). We further confirmed the association between CD15/FUT4 overexpression and cancer recurrence in the GSE17536/GSE17537 series (n = 226), for which reported follow-up data were available (Additional file 3: Figure S6A, B) [23]. In this cohort, CD15/FUT4 overexpression was associated with short time-to-recurrence (TTR) but not with OS, independently from initial tumor stage (Additional file 3: Figure S6B). CD15/FUT4 transcripts were significantly higher in chromosomal instability (CIN) positive than CIN-negative tumors. Its upregulation was associated to relevant genetic aberrations such as: ERBB2, ERBB3 and FGFR4 overexpression, a finding confirmed in a large series of 60 CRC cell lines (Fig. 3b and Additional file 3: Figure S6A-C).Fig. 3


Cancer-related CD15/FUT4 overexpression decreases benefit to agents targeting EGFR or VEGF acting as a novel RAF-MEK-ERK kinase downstream regulator in metastatic colorectal cancer.

Giordano G, Febbraro A, Tomaselli E, Sarnicola ML, Parcesepe P, Parente D, Forte N, Fabozzi A, Remo A, Bonetti A, Manfrin E, Ghasemi S, Ceccarelli M, Cerulo L, Bazzoni F, Pancione M - J. Exp. Clin. Cancer Res. (2015)

Genome-wide expression analysis identifies CD15/FUT4 as a novel RAF-MEK-ERK kinase downstream target. a The most enriched network of CD15/FUT4 regulators is depicted comprising 20 regulons and two closely connected upstream genes involving EGFR and FGFR pathways (see Additional file 2 and Additional file 3: Figure S5 for more information). The gene expression datasets GSE17536/GSE17537 series (n = 226) were analyzed simultaneously with the ARACNe algorithm to infer transcriptional regulatory network fromgenome-wide expression profiles CD15/FUT4-connected. bCD15/FUT4 expression in CRC using patient-matched tumor-normal data available from TCGA. The P value refers to Mann–Whitney test. Expression profiles of ERBB1, ERBB2, ERBB3, FGFR4, CD15/FUT4 and KRAS mutant across a series of CRC cell lines (n = 60) by applying a fold change of gene expression microarray data of 1.5 and subdivided on the basis of chromosomal instability calculated as fraction of copy-number alteration pattern (form 0 to 0.922). Significant correlation between CD15/FUT4, ERBB3 and FGFR4 expression levels. c Linear correlation between ERBB3, FGFR4 and CD15/FUT4 transcript levels is validated in our independent series of (n = 12) CRC cell lines. Immunofluorescence labeling “green” of nonpermeabilize cancer cells shows differential expression of CD15/FUT4 “arrows,” on the cell plasma membrane of RKO and HT29, respectively. To visualize nuclei, the merged images were stained with 4’6-diamidino-2-phenylindole (Dapi), “blue”. d CD15/FUT4-dependent transcript induction by EGF (10 nM) or IL1b (20U/ml) by using 0.1 % of DMSO as vehicle, in two representative CRC cell lines SW480 and RKO, respectively. Cells were treated for 8 h and the ratio anti-p-ERK/ERK1/2 was quantified by western-blot analysis. The treatment with IL-10 (200U/ml) or IL-6 (50 ng/ml) for 30 min revealed a significant induction of IL6-STAT1/STAT3 dependent phosphorylation in a panel of 4 CRC cell lines as detected by western-blot analysis. Mononuclear cells (Mono) purified from buffy coats of healthy donors were used as positive control for IL10-STAT3 dependent phosphorylation. CD15/FUT4 transcript did not reveal any significant induction following IL10 or IL6 at 48 h of treatment. The P value were obtained by Mann–Whitney test; *P ≤ .05; **P ≤ .01
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Fig3: Genome-wide expression analysis identifies CD15/FUT4 as a novel RAF-MEK-ERK kinase downstream target. a The most enriched network of CD15/FUT4 regulators is depicted comprising 20 regulons and two closely connected upstream genes involving EGFR and FGFR pathways (see Additional file 2 and Additional file 3: Figure S5 for more information). The gene expression datasets GSE17536/GSE17537 series (n = 226) were analyzed simultaneously with the ARACNe algorithm to infer transcriptional regulatory network fromgenome-wide expression profiles CD15/FUT4-connected. bCD15/FUT4 expression in CRC using patient-matched tumor-normal data available from TCGA. The P value refers to Mann–Whitney test. Expression profiles of ERBB1, ERBB2, ERBB3, FGFR4, CD15/FUT4 and KRAS mutant across a series of CRC cell lines (n = 60) by applying a fold change of gene expression microarray data of 1.5 and subdivided on the basis of chromosomal instability calculated as fraction of copy-number alteration pattern (form 0 to 0.922). Significant correlation between CD15/FUT4, ERBB3 and FGFR4 expression levels. c Linear correlation between ERBB3, FGFR4 and CD15/FUT4 transcript levels is validated in our independent series of (n = 12) CRC cell lines. Immunofluorescence labeling “green” of nonpermeabilize cancer cells shows differential expression of CD15/FUT4 “arrows,” on the cell plasma membrane of RKO and HT29, respectively. To visualize nuclei, the merged images were stained with 4’6-diamidino-2-phenylindole (Dapi), “blue”. d CD15/FUT4-dependent transcript induction by EGF (10 nM) or IL1b (20U/ml) by using 0.1 % of DMSO as vehicle, in two representative CRC cell lines SW480 and RKO, respectively. Cells were treated for 8 h and the ratio anti-p-ERK/ERK1/2 was quantified by western-blot analysis. The treatment with IL-10 (200U/ml) or IL-6 (50 ng/ml) for 30 min revealed a significant induction of IL6-STAT1/STAT3 dependent phosphorylation in a panel of 4 CRC cell lines as detected by western-blot analysis. Mononuclear cells (Mono) purified from buffy coats of healthy donors were used as positive control for IL10-STAT3 dependent phosphorylation. CD15/FUT4 transcript did not reveal any significant induction following IL10 or IL6 at 48 h of treatment. The P value were obtained by Mann–Whitney test; *P ≤ .05; **P ≤ .01
Mentions: To gain insights into the mechanisms that regulate CD15/FUT4 expression, we collected two independent publicly available gene-expression datasets involving a total of 436 colorectal cancer and 60 colon cancer cell lines [7, 23, 24]. Strikingly, in-silico analysis, inferred a number of 1350 interactions and 20 regulons CD15/FUT4-associated, and revealed a series of disease-relevant pathways: a) prosurvival; b) immune-evasion; c) protein kinase cascade and viral infectious response closely connected to the efficacy of drugs (Fig. 3a and Additional file 3: Figure S5A-D) [28–30]. CD15/FUT4 transcript was significantly higher in tumor tissues than normal control in TCGA series comprising 210 CRCs and 22 and normal colorectal mucosa specimens, respectively (Fig. 3b). We further confirmed the association between CD15/FUT4 overexpression and cancer recurrence in the GSE17536/GSE17537 series (n = 226), for which reported follow-up data were available (Additional file 3: Figure S6A, B) [23]. In this cohort, CD15/FUT4 overexpression was associated with short time-to-recurrence (TTR) but not with OS, independently from initial tumor stage (Additional file 3: Figure S6B). CD15/FUT4 transcripts were significantly higher in chromosomal instability (CIN) positive than CIN-negative tumors. Its upregulation was associated to relevant genetic aberrations such as: ERBB2, ERBB3 and FGFR4 overexpression, a finding confirmed in a large series of 60 CRC cell lines (Fig. 3b and Additional file 3: Figure S6A-C).Fig. 3

Bottom Line: CD15/FUT4-high expressing colon cancer cells with primary resistance to cetuximab or bevacizumab are significantly more sensitive to MEK inhibitors than CD15/FUT4-low counterparts.Cancer-related CD15/FUT4 overexpression participates in cetuximab or bevacizumab mechanisms of resistance in mCRC patients.CD15/FUT4 as a potential target of the antitumor immune response requires further evaluation in clinical studies.

View Article: PubMed Central - PubMed

Affiliation: Medical Oncology Unit, Fatebenefratelli Hospital, 82100, Benevento, Italy.

ABSTRACT

Background: Cancer-related immune antigens in the tumor microenvironment could represent an obstacle to agents targeting EGFR "cetuximab" or VEGF "bevacizumab" in metastatic colorectal cancer (mCRC) patients.

Methods: Infiltrating immune cells into tumor tissues, cancer-related expression of immune antigens (CD3, CD8, CD68, CD73, MPO, CD15/FUT4) from 102 mCRC patients receiving first-line Cetuximab or Bevacizumab plus chemotherapy were assessed by immunohistochemistry and validated in an independent tissue microarrays of 140 patients. Genome-wide expression profiles from 436 patients and 60 colon cancer cell lines were investigated using bioinformatics analysis. In vitro kinase assays of target genes activated by chemokines or growth factors were performed.

Results: Here, we report that cancer-related CD15/FUT4 is overexpressed in most of mCRCs patients (43 %) and associates with lower intratumoral CD3+ and CD8+ T cells, higher systemic inflammation (NLR at diagnosis >5) and poorer outcomes, in terms of response and progression-free survival than those CD15/FUT4-low or negative ones (adjusted hazard ratio (HR) = 2.92; 95 % CI = 1.86-4.41; P < 0.001). Overexpression of CD15/FUT4 is induced through RAF-MEK-ERK kinase cascade, suppressed by MEK inhibitors and exhibits a close connection with constitutive oncogenic signalling pathways that respond to ERBB3 or FGFR4 activation (P < 0.001). CD15/FUT4-high expressing colon cancer cells with primary resistance to cetuximab or bevacizumab are significantly more sensitive to MEK inhibitors than CD15/FUT4-low counterparts.

Conclusion: Cancer-related CD15/FUT4 overexpression participates in cetuximab or bevacizumab mechanisms of resistance in mCRC patients. CD15/FUT4 as a potential target of the antitumor immune response requires further evaluation in clinical studies.

No MeSH data available.


Related in: MedlinePlus