Deletion of Slc6a14 reduces cancer growth and metastatic spread and improves survival in KPC mouse model of spontaneous pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is lethal. There is a dire need for better therapeutic targets. Cancer cells have increased demand for sugars, amino acids, and lipids and therefore up-regulate various nutrient transporters to meet this demand. In PDAC, SLC6A14 (an amino acid transporter (AAT)) is up-regulated, affecting overall patient survival. Previously we have shown using in vitro cell culture models and in vivo xenograft mouse models that pharmacological inhibition of SLC6A14 with α-methyl-l-tryptophan (α-MLT) attenuates PDAC growth. Mechanistically, blockade of SLC6A14-mediated amino acid transport with α-MLT leads to amino acid deprivation, eventually inhibiting mTORC1 signaling pathway, in tumor cells. Here, we report on the effect of Slc6a14 deletion on various parameters of PDAC in KPC mice, a model for spontaneous PDAC. Pancreatic tumors in KPC mice show evidence of Slc6a14 up-regulation. Deletion of Slc6a14 in this mouse attenuates PDAC growth, decreases the metastatic spread of the tumor, reduces ascites fluid accumulation, and improves overall survival. At the molecular level, we show lower proliferation index and reduced desmoplastic reaction following Slc6a14 deletion. Furthermore, we find that deletion of Slc6a14 does not lead to compensatory up-regulation in any of the other amino transporters. In fact, some of the AATs are actually down-regulated in response to Slc6a14 deletion, most likely related to altered mTORC1 signaling. Taken together, these results underscore the positive role SLC6A14 plays in PDAC growth and metastasis. Therefore, SLC6A14 is a viable drug target for the treatment of PDAC and also for any other cancer that overexpresses this transporter.

Keywords: KPC; SLC6A14; ascites fluid; metastasis; pancreatic cancer; survival.

Figure 1.. KPC mice develop highly invasive and metastatic PDAC with up-regulated Slc6a14.

Gross pathological images of a representative KPC mouse showing abdominal distension due to malignant ascites (A-i), primary tumor in the head of pancreas (red outline) (A-ii), pancreas tumor (red circle) and liver metastasis (red arrows) (A-iii), and metastatic lesions on the diaphragm (red arrows), distended gall bladder (arrow head), and pancreas tumor (red circle) (A-iv). Histopathological images of a representative KPC mouse showing pancreas with tumor on the right side indicated by thick arrows (B-i), small intestine showing tumor invading through the wall into the mucosa of the small intestine (B-ii), tumor invading the diaphragmatic muscle (B-iii), enlarged lymph node near the lung, which at higher power shows metastatic tumor nests beneath its capsule (B-iv), spleen with partly necrotic tumor with central necrosis (B-v), and metastatic extension to the liver (B-vi). RT-PCR showing up-regulation of Slc6a14 mRNA expression in tumor pancreas from KPC mice compared with normal pancreas from wild-type mice. Gapdh was used as an internal control (C). Western blot showing up-regulation of Slc6a14 protein in tumor pancreas from KPC mice compared with normal pancreas from wild-type mice. Hsp60 was used as an endogenous control (D).

Figure 2.. Deletion of Slc6a14 improves several parameters of PDAC in KPC mice.

(A) Kaplan–Meier curve showing survival outcome between KPC and KPCS mice. (B) Bar diagram showing age at endpoint euthanasia in KPC and KPCS mice. (C) Bar diagram showing ascites incidence in KPC and KPCS mice. (D) Box plot showing the difference in percent metastasis per organ per mouse between the KPC vs the KPCS mice. Data are given as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 3.. Deletion of Slc6a14 reduces the proliferation capacity and the desmoplastic reaction in the KPC mice.

Histopathological staining in KPC and KPCS pancreatic tumor sections (A,B); immunohistochemical analysis showing staining of CK19 (C,D), Ki67 (E,F), α-SMA (G,H), and Masson's Trichrome (I,J) in KPC and KPCS pancreatic tumor sections. Scale bar: 100 μm.

Figure 4.. Deletion of Slc6a14 does not lead to a compensatory up-regulation of other amino acid transporters.

Real-time PCR showing relative mRNA expression of 12 amino acid transporters in Slc6a14^+/+ (wild-type) pancreas vs Slc6a14^−/− pancreas. Data are given as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5.. Generation of genetically engineered KPC and KPCS mice.

Breeding strategy for generation of KPC mice using LSL-Kras^G12D/+, LSL-p53^R172H/+and Pdx1-Cre transgenic mice (A). Breeding strategy for generation of KPCS mice using LSL-Kras^G12D/+, LSL-p53^R172H/+and Pdx1-Cre transgenic mice and Slc6a14^−/− mice (B). RT-PCR showing representative KPC and KPCS genotype mice (C,D).