Oncogenic ADAM28 induces gemcitabine resistance and predicts a poor prognosis in pancreatic cancer

Figure 1 ADAM28 expression is elevated in gemcitabine-resistant pancreatic cancer cells. A: GSE35141 was used to evaluate DEGs, and the significant genes are showed by a volcano plot with a threshold of fold change ≥ 1.5 and P-value < 0.01. The figure was prepared with NetworkAnalyst 3.0[28]; B: The mRNA levels of ADAM28 detected by RT-PCR in SW1990 cells treated with different doses of gemcitabine as indicated for 24 h. ^aP < 0.05, ^cP < 0.001 vs without gemcitabine treatment; C: The mRNA levels of ADAM28 examined by RT-PCR in gemcitabine-resistant SW1990 cells (SW1990-R) and sensitive parallel SW1990 (SW1990-C) cells. ^cP < 0.001 vs SW1990 sensitive cells (SW1990-C); D: The protein expression of ADAM28 was examined in gemcitabine-resistant and sensitive parallel SW1990 cells by Western blot, and Quantity One software was applied to quantify the protein levels. ^cP < 0.001 vs with SW1990 sensitive parallel cells (SW1990-C). DEGs: Differentially expressed genes; FC: Fold change; RT-PCR: Reverse transcription-polymerase chain reaction; SW1990-C: SW1990 sensitive parallel cells; SW1990-R: Gemcitabine-resistant SW1990 cells.

Figure 2 ADAM28 is overexpressed in pancreatic cancer tissues. A: The TCGA database was used to evaluate the transcript of ADAM28 in pancreatic tumor tissues and adjacent normal pancreatic tissues. ^cP < 0.001 vs normal group; B: GSE16515 was included to analyze the ADAM28 gene levels in 36 pancreatic cancer tissues and 16 adjacent normal tissues. ^cP < 0.001 vs normal group; C: GSE15471 was included to analyze the ADAM28 gene levels in 36 pancreatic cancer tissues and 16 adjacent normal tissues. ^cP < 0.001 vs normal group; D: Difference of ADAM28 expression between basal and classical types of pancreatic cancer. ^cP < 0.001 vs normal group; ^aP < 0.05 vs basal group. TCGA: The Cancer Genome Atlas; PDAC: Pancreatic ductal adenocarcinoma.

Figure 3 Expression of ADAM28 in collected clinical pancreatic cancer tissues. A: A total of 16 pancreatic cancer tissues and the corresponding adjacent normal tissues were analyzed. ^cP < 0.001 vs normal group; B: The respective ratio between tumor and adjacent normal tissues (T/N) showed as a bubble chart. T: Tumor tissue; N: Normal tissue; PDAC: Pancreatic ductal adenocarcinoma.

Figure 4 Overall survival and relapse-free survival evaluated between pancreatic cancer patients with high and low expression of ADAM28. A: OS of pancreatic cancer patients from TCGA; B: RFS of pancreatic cancer patients from TCGA; C: OS of pancreatic cancer patients with high TMB between the high and low ADAM28 expression cohorts; D: OS of pancreatic cancer patients with low TMB between the high and low ADAM28 expression cohorts. OS: Overall survival; RFS: Relapse-free survival; TMB: Tumor mutation burden; TCGA: The Cancer Genome Atlas.

Figure 5 Effect of ADAM28 on cell viability inhibition by gemcitabine. A: Western blot analysis of overexpression of ADAM28 in SW1990 cells. The right panel is the semi-quantitative result for ADAM28 protein expression. ^cP < 0.001 vs vector group; B: The mRNA expression of ADAM28 in ADAM28 overexpressing and vector control cells. ^cP < 0.001 vs vector group; C: Western blot analysis of ADAM28 protein in SW1990 cells treated with shRNA against ADAM28 and scramble control. The right panel is the semi-quantitative result for ADAM28 protein. ^cP < 0.001 vs scramble group; D: The mRNA expression of ADAM28 in ADAM28 knock-down and scramble control cells. ^cP < 0.001 vs scramble group; E: Relative cell viability evaluated according to absorbance value in ADAM28 overexpressing and vector control SW1990 cells treated with gemcitabine. ^cP < 0.001 vs vector group; F: Cell viability of ADAM28 knock-down and scramble SW1990 cells treated with different doses of gemcitabine for 24h. ^cP < 0.001 vs scramble group. shADAM28: shRNA against ADAM28; Scramble: Knockdown control.

Figure 6 ADAM28 is closely related to drug resistance-related signaling pathways. A: Enrichment analysis of ADAM28 and its co-expression network in pancreatic cancer; B: GSEA analysis showed that ADAM28 was closely associated with drug metabolism; C: Correlations of ADAM28 with GSTP1, ABCC1, GSTM4, and BCL2 evaluated by the Spearman method. GSEA: Gene set enrichment analysis. ADAM28: AMAM metallopeptidase domain 28; NES: Normalized enrichment score; GSTP1: Glutathione S-transferase pi 1; ABCC1: ATP binding cassette subfamily C member 1; GSTM4: Glutathione S-transferase mu 4; TPM: Transcripts per million.