Inhibition of phospholipase D1 reduces pancreatic carcinogenesis in mice partly through a FAK-dependent mechanism

Abstract

Phospholipase D (PLD) plays a critical role in cancer progression. However, its role in pancreatic cancer remains unclear. Thus, we evaluated the role of PLD1, one of two classical isoforms of PLD, in pancreatic carcinogenesis in vivo. The role of PLD1 in tumor growth was evaluated by subcutaneously transplanting human MIA PaCa-2 cells expressing endogenous PLD1 levels (Ctr KD cells) or cells in which PLD1 was knocked down (Pld1 KD cells) into immunodeficient mice. Twenty days post-implantation, tumors that arose from Pld1-KD cells were significantly smaller, compared to controls (Ctr KD). Then, we assessed the role of PLD1 in the tumor microenvironment, by subcutaneously implanting mouse LSL-KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) cells into wild-type or PLD1 knockout (Pld1-/-) mice. Compared to wild type, tumor growth was attenuated in Pld1-/- mice by 39%, whereas treatment of Pld1-/- mice with gemcitabine reduced tumor growth by 79%. When PLD1 was ablated in LSL-KrasG12D;Ptf1Cre/+ (KC) mice, no reduction in acinar cell loss was observed, compared to KC mice. Finally, treatment of KC mice with a small molecule inhibitor of PLD1 and PLD2 (FIPI) significantly reduced acinar cell loss and cell proliferation, compared to vehicle-treated mice. Mechanistically, the effect of PLD on tumor growth is mediated, partly, by the focal adhesion kinase pathway. In conclusion, while PLD1 is a critical regulator of pancreatic xenograft and allograft growth, playing an important role at the tumor and at the microenvironment levels, the inhibition of PLD1 and PLD2 is necessary to reduce pancreatic carcinogenesis in KC mice and might represent a novel therapeutic target.

Keywords: FAK; PLD1; lipids; pancreatic cancer; pancreatic carcinogenesis; phospholipase D.

Figure 1:. PLD1 downregulation in pancreatic cancer cells reduces tumor growth in vivo.

(A) Immunoblot of PLD1 in MIA PaCa-2 cells expressing endogenous PLD1 expression (Ctr KD) or PLD1 knock-down (Pld1 KD) MIA PaCa-2 cells. β-Tubulin is shown as a loading control. (B) Tumor volume progression over time for Ctr KD and Pld1 KD subcutaneous xenografts in female BALB/c nude mice. (C) Body weight at sacrifice, n= 6-10 per group. (D and E) Immunoblots of Bcl-xL, Bcl-2, p-ERK, ERK, p-AKT, AKT, p-STAT3, STAT3 and p-4E-BP1, and 4E-BP1 in total tumor homogenates collected from transplanted tumor xenografts, *p<0.05. n= 6-9 per group. Values are presented as mean ± SD.

Figure 2:. Pancreatic tumor growth is impaired in mice lacking PLD1

(A) Tumor volume progression over time for C57BL/6 (WT) and Pld1^−/− mice subcutaneously transplanted with mouse KPC cells. (B) Tumor weight at sacrifice, *p<0.05. n= 6-10 per group. (C and D) Immunoblots for Bcl-xL, Cyclin D1, Osteopontin, p-ERK, ERK, p-STAT5, STAT5, p-p38, p38, p-4E-BP1, 4E-BP1, p-FAK, FAK, p-STAT3 and STAT3 in total tumor homogenates collected from transplanted WT and Pld1^−/− mice, *p<0.01. n= 6-9 per group. (E) Kaplan-Meier survival curve of WT (black) and Pld1^−/− mice (grey) injected with 30,000 KPC cells, n= 5-6 per group. (F) Kaplan-Meier survival curve of WT (black) and Pld1^−/− mice (grey) injected with 100,000 KPC cells, n= 9 per group. Values are presented as mean ± SD.

Figure 3:. Pancreatic tumor growth inhibition of gemcitabine is comparable to that of mice lacking PLD1

(A) Tumor volume progression over time of subcutaneous xenograft of mouse KPC cells in C57BL/6 (WT), Pld1^−/− mice, and C57BL/6 treated with 100mg/kg gemcitabine 2x/week (GEM). (B) Body weight at five and twelve days of treatment. (C) Immunoblots for p-ERK, ERK, p-STAT3, STAT3, p-AKT, AKT, p-4E-BP1, 4E-BP1, and Bcl-xL, *p<0.05, ***p<0.0005. n= 8 per group. Values are presented as mean ± SD. (D) Tumor volume progression over time of subcutaneous xenograft of mouse KPC cells in C57BL/6 (WT), Pld1^−/− mice, and Pld1^−/− mice treated with 100mg/kg GEM 2x/week. *p<0.05 vs Pld1^−/− mice.

Figure 4:. PLD1 ablation does not impair pancreatic carcinogenesis in mice with active Kras

(A) PCR analysis of wild-type (+/+) and Pld1 (−/−) genomic data (left) and immunoblot of PLD1 in LSL-Kras^G12D;Ptf1^Cre/+ (KC) and LSL-Kras^G12D;Ptf1^Cre/+;Pld1^−/− (KC;Pld1^−/−) mice (right). (B) Pancreas weight of male and female KC and KC;Pld1^−/− mice at sacrifice, *p<0.05, **p<0.005. n=7-10 per sex per group (C) H&E and immunostaining of PCNA, p-ERK, and Bcl-xL were performed on pancreas sections. Representative images (20x magnification) are shown. Scale bar: 100 μm. Acinar cell loss and mononuclear inflammatory cell infiltration results are expressed as percent area as described in Materials and Methods. Immunostaining results are expressed as percent of PCNA(+), p-ERK(+), or Bcl-xL(+) cells ± SD, *p<0.05, **p<0.001. n=6-10 per sex/group (D) Immunoblots of p-FAK, FAK, p-STAT3, STAT3, p-ERK, ERK, p-AKT, AKT, p-4E-BP1, and 4E-BP1, ***p<0.0005. n= 4-7 per sex per group. Values are presented as mean ± SD.

Figure 5:. Pharmacological inhibition of PLD reduces pancreatic carcinogenesis in KC mice

(A) Pancreas weight of vehicle control and FIPI treated mice at sacrifice. (B) H&E and immunostaining of PCNA and p-ERK were performed on pancreas sections. Representative images (20x magnification) are shown. Scale bar: 100 μm. Acinar cell loss and mononuclear inflammatory cell infiltration results are expressed as percent area as described in Materials and Methods. Yellow lines delineate areas of acinar cell loss. Immunostaining results are expressed as percent of PCNA(+) or p-ERK(+) cells ± SD. (C) Immunoblots of p-FAK, FAK, p-STAT3, STAT3, p-ERK, ERK, p-AKT, AKT, p-4E-BP1, and 4E-BP1, *p<0.05, **p<0.005. n=8-13 per group. Values are presented as mean ± SD.