Modulation of the leptin receptor mediates tumor growth and migration of pancreatic cancer cells

Abstract

Obesity has been implicated as a significant risk factor for development of pancreatic cancer. In the setting of obesity, a systemic chronic inflammatory response is characterized by alterations in the production and secretion of a wide variety of growth factors. Leptin is a hormone whose level increases drastically in the serum of obese patients. High fat diet induced obesity in mice leads to an overall increased body weight, pancreatic weight, serum leptin, and pancreatic tissue leptin levels. Here we report the contribution of obesity and leptin to pancreatic cancer growth utilizing an in vivo orthotopic murine pancreatic cancer model, which resulted in increased tumor proliferation with concomitant increased tumor burden in the diet induced obese mice compared to lean mice. Human and murine pancreatic cancer cell lines were found to express the short as well as the long form of the leptin receptor and functionally responded to leptin induced activation through an increased phosphorylation of AKT473. In vitro, leptin stimulation increased cellular migration which was blocked by addition of a PI3K inhibitor. In vivo, depletion of the leptin receptor through shRNA knockdown partially abrogated increased orthotopic tumor growth in obese mice. These findings suggest that leptin contributes to pancreatic tumor growth through activation of the PI3K/AKT pathway, which promotes pancreatic tumor cell migration.

Fig 1. High fat diet induced obesity contributes to pancreatic adiposity in mice.

Mice were maintained on a 42% high fat diet for 3 months to induce obesity. DIO mice gained significantly more total body weight (A) as well as pancreatic weight (B). Histological analysis of total pancreata from Lean (C) compared to DIO mice (D) showed accumulation of interpancreatic (white arrow) and intrapancreatic (black arrowheads) adipose in DIO pancreas. The total number of intrapancreatic adipocytes (E) as well as the size of peripancreatic adipocytes (F) was greater in the DIO pancreas. Leptin levels were increased in both plasma samples and pancreatic tissue of obese mice (G). Scale bars are 100μm.

Fig 2. Diet induced obesity increases orthotopic Panc02 pancreatic tumor growth.

Bioluminescence revealed increased growth over time in DIO mice compared to lean mice (A). Total flux from luciferase imaging was statistically different by Day09 after orthotopic tumor cell injection (B). Endpoint tumor weight (C) as well as tumor area (D) were significantly increased in the DIO mice. Proliferation assessed by Ki67 staining was increased in DIO tumors compared to lean tumors (E). Radiance heat map scale is x10⁵ p/sec/cm²/sr. Statistical analysis by Mann-Whitney of p<0.0079 (*).

Fig 3. Pancreatic cancer cell lines express functional leptin receptors.

Western and real-time qPCR analysis verified leptin receptor expression for the long and short forms of the leptin receptor in murine and human pancreatic cell lines (A,B,C). Western anlayis demonstrated stimulation of pancreatic cancer cell lines with leptin lead to the phosphorylation of AKT in the Panc02 and Panc1 cell lines, and to the phosphorylation of STAT3 (D). Addition of the PI3K/AKT inhibitor LY294002 was able to block leptin induced phosphorylation of pAKT but did not affect leptin induced pSTAT3 activation in the Panc1 cell line. Densitometric analysis was used to quantify the amount of pSTAT3 and pAKT relative to total levels for each protein and then normalized to zero timepoint.

Fig 4. Leptin induced proliferation and migration of pancreatic cancer cells is cell line dependent.

Leptin stimulation at 5, 50, and 250ng/ml caused an increase in proliferation assessed through EdU incorporation in murine Panc02 and human Panc1 cell lines but did not alter proliferation in human MiaPaca cells (A). Leptin stimulation caused an increase in migration recorded as distance migrated for Panc02 and Panc1 cells assessed through scratch assay which was blocked by PI3K inhibitor LY294002 (B). Statistical analysis by *ANOVA of p<0.0001, and **T-test of p<0.05.

Fig 5. Knockdown of leptin receptor in pancreatic cancer cells leads to functional impairment of leptin signaling.

Long and short leptin receptor RNA levels measured through realtime PCR analysis were both significantly decreased using two different shRNAmir lentiviral titers in the Panc02 cell line (A). Western and densitometric analysis confirmed leptin receptor knockdown as well as reduced activation of pAKT (B). Basal proliferation was significantly decreased in both the LRKD1 and LRKD2 knockdown cell lines when grown in serum free conditions (C). Stimulation with leptin at 50ng/mL induced proliferation in parental and control cells but failed to induce proliferation (percent change compared to untreated) in either of the knockdown cell lines compared to parental or control shRNA (D). Statistical analysis by ANOVA *represents p<0.014 short, p<0.0023 long, p<0.0003 common (A); ANOVA p<0.0008 (C).; ANOVA p<0.0001 (D). *p<0.05 in C,D.

Fig 6. Leptin receptor knockdown in Panc02 cells antagonizes obesity stimulated orthotopic tumor growth in mice.

Panc02 cells with leptin receptor knockdown variants LRKD1 and LRKD2 were orthotopically injected into lean and DIO mice. (A) Differences between parental (P), control shRNA (C), and knockdown variants were not statistically different when comparisons were made between control and knockdown tumors in lean mice. Leptin receptor knockdown variant LRKD2 showed a significantly decreased tumor weight in DIO mice when compared to the parental and control tumors in DIO mice. (ANOVA p<0.0001, *p<0.05). (B) Assessment of tumor cell proliferation through Ki67 staining was not different between control DIO tumors compared to LRKD2 DIO tumors, but was significant between lean and DIO tumors (ANOVA p<0.0004, *p<0.05).