PTEN Is a Major Tumor Suppressor in Pancreatic Ductal Adenocarcinoma and Regulates an NF-κB–Cytokine Network

Pten deficiency induces strong stromal reaction with immune cell infiltration and promotes the development of invasive and metastatic pancreatic tumors in cooperation with Kras^G12D. A, six-week-old pancreata from Kras^G12D (i–v) and Kras^G12D Pten^L/+ (vi–x) stained with H&E (i, vi), or with antibodies to SMA (ii, vii), S100A4 (iii, viii), Gr-1 (iv, ix), and FoxP3 (v, x). B, left, analysis of myeloid cells. Bar graph showing percentage of CD45⁺CD11b⁺Gr-1^low (white) and Gr-1^high (black) cells in total live cells of wild-type (n = 3), Kras^G12D (n = 3), and Kras^G12D Pten^L/+ (n = 3) pancreata. Right, Treg analysis. Percentage of CD25⁺FoxP3⁺ cells within CD4⁺ T cells in pancreas of wild-type (n = 2), Kras^G12D (n = 2) and Kras^G12D Pten^L/+ (n = 2) mice. *, P < 0.05; **, P < 0.01. C, tumor invasion and metastases. Gross picture shows liver metastases (i). H&E staining shows local invasion into duodenum wall (ii), lymph node metastasis (iii), liver metastasis (iv), and microscopic lung metastasis (vi). Liver metastasis is stained with cytokeratin-19 antibody (v). Scale bars, 100 μm.

Molecular analysis of Kras^G12D Pten^L/+ PDACs. A, PCR analysis of the Pten locus in genomic DNA from primary murine PDAC cell lines (lanes 2, 4, 6, 8, and 10) and control tail DNA from the same animal (lanes 1, 3, 5, 7, and 9). Although all control tail DNA showed both wild-type (Pten⁺) and Pten lox (Pten^L) alleles (lanes 1, 3, 5, 7, and 9), and deleted Pten (Pten^D) allele was present in all tumor DNA (lanes 2, 4, 6, 8, and 10), 3 tumor lines maintain the wild-type Pten allele (lanes 4, 6, and 8). B, Pten IHC staining in tumors with Pten LOH (top) or wild-type Pten allele (bottom) Scale bar, 100 μm. C, Western blot analysis for p16^Ink4a, p19^Arf, and Smad4 expression in early-passage Kras^G12D (lanes 1–3) or Kras^G12D Pten^L/+ (lanes 4–7) PDEC lines and Kras^G12D Pten^L/+ PDAC lines (lanes 8–12). PDAC line from Kras^G12D p53^L/+ was used as a control (lane 13). D, Kras^G12D Pten^L/+ PDAC cell lines were treated with doxorubicin 0.4 μg/mL for 16 hours and collected for Western blot analysis for p53 and p21. Kras^G12D p53^L/+ tumor line was used as a negative control (lanes 11 and 12).

Pten deficiency activates the NF-κB pathway. A, cytoplasmic (C) or nuclear (N) extract was prepared from Kras^G12D (lanes 1–4) or Kras^G12D Pten^L/+ (lanes 5–12) PDECs and blotted for NF-κB p65. Lamin A/C and caspase 3 were used as markers for nuclear and cytoplasmic fraction, respectively. Kras^G12D Pten^L/+ PDECs were also treated with either vehicle (Ctr) or LY, 20 μM, for 24 hours before collection. B, Kras^G12D or Kras^G12D Pten^L/+ PDECs were transfected with NF-κB–luciferase reporter, and Kras^G12D Pten^L/+ PDECs were treated with either vehicle (Ctr) or LY, 20 μM, for 24 hours before being collected for luciferase activity assay. *, P < 0.05; **, P < 0.01. C, six-week-old pancreata from Kras^G12D (top) and Kras^G12D Pten^L/+ (bottom) mice were stained with phospho–NF-κB p65 (pRelA) Scale bars, 100 μm. D. Kras^G12D PDECs were infected with pSuper (Ctr) or pSuper-shPten (shPten) and blotted for Pten, pAkt, and Akt (lanes 1–4). Cytoplasmic (C) or nuclear (N) extract was blotted for NF-κB p65 (lanes 5–12).

Suppression of NF-κB inhibits tumorigenic activity. A, PDAC from Kras^G12D Pten^L/+ animals was stained for phospho–NF-κB p65 (pRelA) Scale bar, 100 μm. B, colony formation assay for Kras^G12D Pten^L/+ PDAC cell lines infected with pBabe (Vec) or pBabe-IκBαM. Quantification of colony numbers was shown on the right. C, Kras^G12D Pten^L/+ PDAC cell lines were infected with pBabe (Vec) or pBabe-IκBαM and injected s.c. into nude mice with Vec in the left flank and IκBαM in the right flank, respectively. Tumor volumes were measured in D. *, P < 0.05; **, P < 0.01.