Gemcitabine Resistance In Pancreatic Adenocarcinoma Research Articles

Abstract 4613: Tumor-derived interleukin 35 facilitates the gemcitabine resistance in pancreatic adenocarcinoma by mediating the dissemination of chemoresistance among PDAC cells

Abstract Rapid drug resistance after chemotherapy is a key cause of treatment failure in human pancreatic ductal adenocarcinoma (PDAC). Here, we found that tumor-derived interleukin 35 (IL-35) mediated the rapid resistance of PDAC to gemcitabine (GEM). The role of IL-35 in GEM resistance was determined by molecular and cell biology approaches in PDAC cells and in patient-derived tumor xenograft (PDX) models. We used whole genome RNA sequencing, molecular cloning and other molecular biological methods to study the molecular mechanism of IL-35 mediated GEM resistance. An anti-IL-35 neutralizing antibody was used in mouse models to investigate the feasibility of targeting IL-35 to overcome drug resistance in pancreatic cancer. We observed the phenomenon that GEM resistance could spread from GEM resistant PDAC cells to GEM sensitive ones. The results of sequencing and experiments confirmed that the IL-35 secretion is responsible for the dissemination of chemoresistance. IL-35 secreted from GEM resistant cells activated the IL35 expression in GEM sensitive cells and upregulated SOD2 expression via GP130-STAT1 signaling, which scavenges reactive oxygen species (ROS) and then leads to GEM resistance.Furthermore, we found that a neutralizing antibody against IL-35 significantly enhanced the tumor suppressive effect of GEM. These findings collectively reveal that IL-35 plays crucial roles in mediating GEM resistance in pancreatic cancer. Moreover, IL-35 might serve as a promising therapeutic target for combating PDAC chemoresistance. Citation Format: Chongbiao Huang, Huizhi Sun, Antao Chang, Jihui Hao. Tumor-derived interleukin 35 facilitates the gemcitabine resistance in pancreatic adenocarcinoma by mediating the dissemination of chemoresistance among PDAC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4613.

Tumor-derived interleukin 35 mediates the dissemination of gemcitabine resistance in pancreatic adenocarcinoma.

Rapid development of drug resistance after chemotherapy is a major cause of treatment failure in individuals with pancreatic ductal adenocarcinoma (PDAC). In this study, we illustrate that tumor-derived interleukin 35 (IL-35) mediates the accelerated resistance of PDAC to gemcitabine (GEM). We observe that GEM resistance can spread from GEM-resistant PDAC cells to GEM-sensitive cells, and that IL-35 is responsible for the propagation of chemoresistance, which is supported by sequencing and experimental data. Additionally, we discover that GEM-resistant cells have significantly higher levels of IL-35 expression. Mechanistically, aberrantly expressed IL-35 triggers transcriptional activation of SOD2 expression via GP130-STAT1 signaling, scavenging reactive oxygen species (ROS) and leading to GEM resistance. Furthermore, GEM treatment stimulates IL-35 expression through activation of the NF-κB pathway, resulting in acquired chemoresistance. In the mouse model, a neutralizing antibody against IL-35 enhances the tumor suppressive effect of GEM. Collectively, our data suggests that IL-35 is critical in mediating GEM resistance in pancreatic cancer, and therefore could be a valuable therapeutic target in overcoming PDAC chemoresistance.

Systematic exploration of the underlying mechanism of gemcitabine resistance in pancreatic adenocarcinoma.

Resistance to gemcitabine is the main challenge of chemotherapy for pancreatic ductal adenocarcinoma (PDAC). Hence, the development of a response signature to gemcitabine is essential for precision therapy of PDAC. However, existing quantitative signatures of gemcitabine are susceptible to batch effects and variations in sequencing platforms. Therefore, based on within‐sample relative expression ordering of pairwise genes, we developed a transcriptome‐based gemcitabine signature consisting of 28 gene pairs (28‐GPS) that could predict response to gemcitabine for PDAC at the individual level. The 28‐GPS was superior to previous quantitative signatures in terms of classification accuracy and prognostic performance. Resistant samples classified by 28‐GPS showed poorer overall survival, higher genomic instability, lower immune infiltration, higher metabolic level and higher‐fidelity DNA damage repair compared with sensitive samples. In addition, we found that gemcitabine combined with phosphoinositide 3‐kinase (PI3K) inhibitor may be an alternative treatment strategy for PDAC. Single‐cell analysis revealed that cancer cells in the same PDAC sample showed both the characteristics of sensitivity and resistance to gemcitabine, and the activation of the TGFβ signalling pathway could promote progression of PDAC. In brief, 28‐GPS could robustly determine whether PDAC is resistant or sensitive to gemcitabine, and may be an auxiliary tool for clinical treatment.

Role of hepatocyte nuclear factor 4 alpha in cell proliferation and gemcitabine resistance in pancreatic adenocarcinoma

BackgroundHepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor that regulates the expression of numerous genes in hepatocytes and pancreatic β cells. HNF4α has been reported to affect cell proliferation and chemoresistance in several cancers. However, the role of HNF4α in pancreatic adenocarcinoma (PDAC) has not been studied extensively and remains unclear.MethodsBy utilizing immunohistochemical (IHC) staining, we measured the expression of HNF4α in PDAC tissues. By silencing HNF4α in PDAC cell lines, we assessed the impact of HNF4α on pancreatic cancer cell proliferation and gemcitabine sensitivity. We used CCK8 and colony formation assays to examine the effect of HNF4α on cell proliferation. A flow cytometry assay was used to assess cell apoptosis. The expression of gemcitabine-related genes was detected by quantitative real‑time PCR (qRT-PCR) and Western blotting. IHC was utilized to assess the correlation between HNF4α and human equilibrative nucleoside transporter 1 (hENT1) expression in PDAC patients. Chromatin immunoprecipitation (ChIP) and dual‑luciferase reporter assays were used to confirm that hENT1 is a target gene of HNF4α.ResultsIncreased HNF4α expression was detected in PDAC tissues; patients with higher HNF4α expression displayed worse prognosis. To elucidate the function of HNF4α, we examined its role in pancreatic cancer cell proliferation, apoptosis and gemcitabine resistance. In HNF4α-silenced Capan-1 and MiaPaCa-2 cells, we observed decreased cell proliferation and increased sensitivity to gemcitabine compared to those of controls. The mechanism of HNF4α in gemcitabine-related chemosensitivity was then explored. In response to HNF4α silencing, the expression levels of gemcitabine-related proteins, hENT1 and deoxycytidine kinase (dCK) were significantly increased. Additionally, hENT1 was negatively correlated with HNF4α in PDAC tissue samples. Moreover, we identified hENT1 as a downstream target of HNF4α.ConclusionHNF4α is a prognostic marker for overall survival, is required for pancreatic cancer cell proliferation and promotes resistance to gemcitabine by downregulating hENT1. Therefore, targeting HNF4α might reverse gemcitabine resistance and provide novel treatment strategies for PDAC.

Pancreatic cancer: Intra-tumour bacteria promote gemcitabine resistance in pancreatic adenocarcinoma.

Pancreatic cancer: Intra-tumour bacteria promote gemcitabine resistance in pancreatic adenocarcinoma

Abstract 3273: The efficiency of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma

Abstract Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have revealed the anti-cancer effects of metformin (MET), including against pancreatic ductal carcinoma (PDC). Gemcitabine (GEM) has become the standard chemotherapy for PDC but tolerance to GEM becomes an important issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDC in a xenograft mouse model. For this in vivo study, wild-type BxPC-3 was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (without any treatment); (ii) GEM-treated group (100 mg/kg); (iii) MET-treated group (600 mg/kg); and (iv) combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally every day. GEM was given by intraperitoneal injection every week. In the final tumor volumes, the two groups treated with GEM had significantly reduced tumors compared with control, but there were no differences between control and MET groups. The anti-tumor effect of MET for BxG30 (the cell line for GEM-resistant PDC) was evaluated using the method described above. Tumor volumes were decreased significantly in GEM+MET groups compared with control. The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; GEM+MET, 47.2%. The anti-tumor effect of GEM for BxG30 was limited. The MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the GEM+MET group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDC. Western blot analysis was performed to confirm mammalian target of rapamycin (mTOR) expression level was surely inhibited. The results revealed that the expression level of mTOR treated with MET was significantly decreased, but no in GEM group. The expression level of hypoxia-inducible factor 1 (HIF-1) was evaluated by western blot analysis also. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM, again. Then the expression of VEGF mRNA, downstream of HIF-1 signaling pathway, was evaluated by qRT-PCR. The result showed inhibition of VEGF mRNA expression by MET treatment. Our results showed that MET has a partial anti-tumor effect for PDC. Combination therapy has an excellent effect not only for wild-type PDC but also for GEM-resistant PDC. The mechanism of anti-tumor effect of MET seems to be, at least partly, the inhibition of HIF-1/mTOR signaling pathway. These data suggest that MET could be used to treat PDC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yoshiyuki Ishii, Masayoshi Osaku. The efficiency of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3273.

Abstract 2570: The mechanism of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma

Abstract Several epidemiological studies have revealed the anti-cancer effects of metformin (MET), first-line treatment for type-2 diabetes mellitus, including against pancreatic ductal carcinoma (PDC). Gemcitabine (GEM) has become the standard chemotherapy for PDC but tolerance to GEM becomes an important issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDC in a xenograft mouse model. For this in vivo study, wild-type BxPC-3 was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (without any treatment); (ii) GEM-treated group (100 mg/kg); (iii) MET-treated group (600 mg/kg); and (iv) combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally every day. GEM was given by intraperitoneal injection every week. In the final tumor volumes, the two groups treated with GEM had significantly reduced tumors compared with control, but there were no differences between control and MET groups. The anti-tumor effect of MET for BxG30 (the cell line for GEM-resistant PDC) was evaluated using the method described above. Tumor volumes were decreased significantly in GEM+MET groups compared with control. The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; GEM+MET, 47.2%. The anti-tumor effect of GEM for BxG30 was limited. The MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the GEM+MET group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDC. Western blot analysis was performed to confirm mammalian target of rapamycin (mTOR) expression level was surely inhibited. The results revealed that the expression level of mTOR treated with MET was significantly decreased, but no in GEM group. Then the expression level of hypoxia-inducible factor 1 (HIF-1) was evaluated by western blot analysis also. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM, again. Our results showed that MET has a partial anti-tumor effect for PDC. Combination therapy has an excellent effect not only for wild-type PDC but also for GEM-resistant PDC. mTOR is well known as an upstream activator of HIF-1 function in cancer cells. That is, the mechanism of anti-tumor effect of MET seems to be, at least partly, the inhibition of HIF-1 through the mTOR regulation. These data suggest that MET could be used to treat PDC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yoshiyuki Ishii, Masayoshi Osaku. The mechanism of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2570. doi:10.1158/1538-7445.AM2015-2570

Abstract 3122: Combination effect of metformin with gemcitabine for gemcitabine-resistant pancreatic adenocarcinoma

Abstract Metformin (MET) is the first-line treatment for type-2 diabetes mellitus. Several epidemiological studies have revealed the anti-cancer effects of metformin, including against pancreatic ductal carcinoma (PDC). Gemcitabine (GEM) has become the standard chemotherapy for PDC but tolerance to GEM is an important issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDC in a xenograft mouse model. For this in vivo study, wild-type BxPC-3 was implanted into both flanks of 7-week-old female BALB/c nude mice. Mice were divided into four groups: (i) control (without any treatment); (ii) GEM-treated group; (iii) MET-treated group; and (iv) combined treatment group (GEM+MET). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET (600 mg/kg) was administrated orally every day. GEM (100 mg/kg) was given by intraperitoneal injection every week. The final tumor volumes (in g) were: control, 0.59±0.05; GEM, 0.32±0.07; MET, 0.42±0.08; GEM+MET, 0.23±0.06. The two groups treated with GEM had significantly reduced tumors compared with control, but there were no differences between control and MET groups. The anti-tumor effect of MET for BxG30 (the cell line for GEM-resistant PDC) was evaluated using the method described above. The final tumor volumes were: control, 0.26±0.05; GEM, 0.21±0.05; MET, 0.15±0.06; GEM+MET, 0.11±0.03. Tumor volumes were decreased significantly in GEM+MET groups compared with control. The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; GEM+MET, 47.2%. The anti-tumor effect of GEM for BxG30 was limited. The MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the GEM+MET group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDC. The IC50 was 20.25 mM for GEM and 0.004 µg/mL for MET upon single-drug use. Our results showed that MET has a partial anti-tumor effect for PDC. Combination therapy has an excellent effect not only for wild-type PDC but also for GEM-resistant PDC. These data suggest that MET could be used to treat PDC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Masayoshi Osaku, Yoshinori Yamada. Combination effect of metformin with gemcitabine for gemcitabine-resistant pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3122. doi:10.1158/1538-7445.AM2014-3122

High Expression of Survivin Is Prognostic of Shorter Survival but Not Predictive of Adjuvant Gemcitabine Benefit in Patients with Resected Pancreatic Adenocarcinoma

Overexpression of survivin has been associated with gemcitabine resistance in pancreatic adenocarcinoma, but previous studies have shown conflicting results. This study aims to determine its prognostic value in resected pancreatic adenocarcinoma with or without adjuvant therapy and its predictive value in adjuvant gemcitabine benefit in patients with resected pancreatic adenocarcinoma. This study included 118 patients who underwent pancreaticoduodectomy from 1999 to 2007, with no neoadjuvant chemoradiation. Forty-five patients received adjuvant gemcitabine. Survivin expression was assessed immunohistochemically and was graded as low (≤10% positive cells) and high (>10% positive cells) by recursive partitioning analysis. Prognostic factors, including tumor size, number of positive lymph nodes, perineural invasion, and stage, were identified for overall survival (OS) and progression-free survival (PFS) using Cox proportional hazards models. Multivariable analysis of the entire cohort revealed that both high survivin expression and perineural invasion predict significantly shorter OS (hazard ratio [HR] 2.0, p=0.01; HR 1.9, p=0.01, respectively) and shorter PFS (HR 1.9, p=0.04; HR 3.1, p=0.0006, respectively). Expression of survivin predicts neither OS nor PFS in patients treated with adjuvant gemcitabine. In summary, high expression of survivin is associated with shorter OS and PFS in patients with resected pancreatic adenocarcinoma after adjusting for other histopathological factors. However, survivin has no predictive value of adjuvant gemcitabine benefit.

Armed and targeted measles virus for chemovirotherapy of pancreatic cancer

No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high- and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.

Inhibition of SRC tyrosine kinase impairs inherent and acquired gemcitabine resistance in human pancreatic adenocarcinoma cells.

We tested the hypotheses that Src tyrosine kinase overactivity represents a chemoresistance mechanism and that Src inhibition may enhance gemcitabine cytotoxicity in pancreatic adenocarcinoma cells. Pancreatic adenocarcinoma cells PANC1, MiaPaCa2, Capan2, BxPC3, and PANC1(GemRes), a stably gemcitabine-resistant subline of PANC1, were exposed to combinations of gemcitabine and Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Src expression, phosphorylation (Tyr-416), and activity were analyzed by immunoblotting and in vitro kinase assay. Expression of the M2 subunit of ribonucleotide reductase (RRM2), a putative chemoresistance enzyme, was quantified by Northern and Western blot. Cellular proliferation was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was characterized by YO-PRO-1/propidium iodide staining, fluorometric caspase profiling, and caspase inhibition (Z-Val-Ala-Asp-fluoromethyl ketone). The effects of constitutively active and dominant negative Src were determined. The therapeutic efficacy of PP2 in combination with gemcitabine was tested in nude mice orthotopically xenografted with PANC1(GemRes). Greater gemcitabine resistance was associated with higher Src phosphorylation and activity, both of which were higher in PANC1(GemRes), relative to PANC1; total Src levels were alike. PANC1(GemRes) overexpressed RRM2. PP2 enhanced inherent gemcitabine chemosensitivity and attenuated gemcitabine resistance in PANC1(GemRes). Constitutively active Src increased gemcitabine chemoresistance; dominant negative Src impaired gemcitabine chemoresistance. PP2 augmented gemcitabine-induced caspase-mediated apoptosis, suppressed RRM2 expression, and decreased activity of the RRM2-regulating transcription factor E2F1 in PANC1(GemRes). PP2 and gemcitabine in combination substantially decreased tumor growth and inhibited metastasis in vivo. Increased Src tyrosine kinase activity represents a potential chemoresistance mechanism and a promising therapeutic target warranting further investigation in gemcitabine-resistant pancreatic adenocarcinoma.