Cancer Res Treat.  2019 Jan;51(1):194-210. 10.4143/crt.2018.031.

Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

• Ma J
• Song X
• Xu X
• Mou Y

• KMID: 2437613
• DOI: http://doi.org/10.4143/crt.2018.031

Abstract

PURPOSE
Our aim was to detect the potential role of interleukin 11 (IL-11) in the development of chemo-resistance in gastric cancer and to reveal the mechanism involved in the process.
MATERIALS AND METHODS
Here, we used flow cytometry to examine the percentage of cancer-associated-fibroblasts in tumor samples from chemo-resistant and -sensitive gastric cancer patients. Using MTT assay, we detected the cell viability under different conditions. Using quantitative real-time polymerase chain reaction and Western blotting, we determined the target expressions in mRNA and protein levels. We also performed immunohistochemistry and immunofluorescence to detect the target proteins under different conditions. Animal models were constructed to verify the potential role of IL-11 in chemo-resistant develop in vivo.
RESULTS
Herein, we observed enriched cancer associated fibroblasts in drug resistant tumor tissues from gastric patients. Those fibroblasts facilitate the chemotherapeutic drugs resistance development through the secretion of IL-11, which activates the IL-11/IL-11R/gp130/JAK/STAT3 anti-apoptosis signaling pathway in gastric cancer cells. We found that the combination of chemotherapeutic drugs and JAK inhibitor overcomes the resistance and increases the survival of mice with gastric cancer xenografts.
CONCLUSION
Ourresults demonstrated that IL-11 contributed to the obtain ofresistance to chemotherapy drugs through gp130/JAK/STAT3/Bcl2 pathway, and targeting the IL-11 signaling pathway induced by fibroblasts might be a promising strategy to overcome the multi-drugs resistant cancer in clinic.

Keyword

Cancer-associated fibroblasts; Stomach noplasms; Drug resistance; Interleukin-11; JAK/STAT3

MeSH Terms

Animals
Blotting, Western
Cell Survival
Drug Resistance
Drug Therapy
Fibroblasts*
Flow Cytometry
Fluorescent Antibody Technique
Heterografts
Humans
Immunohistochemistry
Interleukin-11*
Mice
Models, Animal
Real-Time Polymerase Chain Reaction
RNA, Messenger
Stomach Neoplasms*
Interleukin-11
RNA, Messenger

Figure

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  Fig. 1. Enriched cancer-associated-fibroblasts (CAFs) enhanced the resistance of gastric cancer cells to chemotherapy. Accumulated CAFs in the chemotherapy-resistant gastric tumor sites and facilitate the resistance to chemotherapy drugs in gastric cancer cells. (A) The percentage of the cancer-associated fibroblasts in samples from chemo-sensitive (CS) and chemo-resistance (CR) gastric cancer patients was detected by flow cytometry. (B) The expression of the cancer-associated fibroblasts in samples from CS and CR gastric cancer patients was detected by immunohistochemistry. (C-H) The cell viability of SGC7901was detected after treated by different concentration of DDP (C), doxorubicin (D), and etoposide (E) pre-co-cultured with or without CAFs by using MTT assay. The cell viability of BGC823 was detected after treated by different concentration of DDP (F), doxorubicin (G), and etoposide (H) pre-co-cultured with or without CAFs by using MTT assay. The data was presented as mean±standard error of mean from three independent experiments. ***p < 0.001.

• 

  Fig. 2. Cancer-associated-fibroblasts (CAFs) regulated chemo-resistance through secreting interleukin 11 (IL-11). The effect of CAFs on the sensitivity of gastric cancer cells to chemotherapy drugs was examined by using MTT assay. (A) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs medium (CM) pretreated. Dox, doxorubicin; Eto, etoposide. (B) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin. respectively with or without CM pretreated. (C) The mRNA expression of IL-11, SDF-1, HGF, FGF, PDGF, VEGF, and IL-1F9 in normal fibroblast and CAFs. (D) The expression of IL-11R in normal fibroblast and CAFs was detected by using enzyme-linked immunosorbent assay. (E) The mRNA expression of IL-11R, VEGFR, PDGFR, HGFR, CXCR4, SDF-1R, and IL-1F9R in SGC7901 cells with or without CAFs co-cultured. (F) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (G) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 200 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (H) The tumor volume of NOD-SCID mice bearing SGC7901 cells co-injected with CAFs-CM (50 μL 10× CM) or IL-11 (2.5 μg/kg) treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (I) The survival curve of NOD-SCID mice bearing SGC7901 cells co-injected with CAFsCM (50 μL 10× CM) or IL-11 treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (J) Expression of IL-11 in gastric cancer tissues from chemo-sensitive and chemo-resistant patients. The data was presented as the mean±standard error of mean from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 3. Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through JAK/STAT3 pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. **p < 0.01.

• 

  Fig. 4. Interleukin 11 (IL-11) triggers the JAK/STAT3 pathway to elevate Bcl2 expression. (A) The expression of Bcl2 in BGC823 and SGC7901 cells treated with cultured medium of cancer-associated-fibroblasts (CAFs-CM) or rhIL-11 in the presence or absence of ruxolitinib (5 μM) in mRNA level. (B) The expression of Bcl2 in BGC823 and SGC7901 cells treated with CAFs-CM or rhIL-11 (10 ng/mL) in the presence or absence of ruxolitinib (5 μM) in protein level. (C) The cell viability of BGC823 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. (D) The cell viability of SGC7901 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. The data was presented as the mean±standard error of mean from three independent experiments. **p < 0.01.

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  Fig. 5. Blockade interlukin (IL)-11/IL-11R signal relieves chemotherapy drug resistance in gastric cancer. (A) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in phosphate buffered saline (PBS), doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). Eto, etoposide; Dox, doxorubicin. (B) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (C) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (D) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). (E) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (F) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (G) The schematic diagram of drug resistance development induced by IL-11 in gastric cancer cells. The data was presented as the mean±standard error of mean from three independent experiments. **p < 0.01, ***p < 0.001.

Reference

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