Pancreatic Cancer Sensitive to Selective p38 Pathway Inhibition
Triple-immunofluorescence of human pancreatic cancer for p38 pY323 (cyan), TNF-α (green), and IL-17A (red). DAPI (blue) was used to stain the nucleus. Examples of positive cells are denoted with arrows.
Pancreatic ductal adenocarcinoma (PDAC), the most-common cancer of the pancreas, is an aggressive disease that is estimated by the American Cancer Society to be the fourth leading cause of cancer death in men and women in 2015. Like most solid tumors, PDAC is surrounded by an inflammatory microenvironment containing numerous infiltrating immune cells. These cells are unable to eliminate the tumor and instead create a hospitable environment by providing tumor growth-promoting cytokines, the production of which lies downstream of the kinase p38. Unlike most immune cells, which use the classical pathway to activate p38, T cells employ an alternative p38 pathway that involves phosphorylation of tyrosine 323 (pY323) by the T cell receptor. Jonathan Ashwell, M.D., of CCR’s Laboratory of Immune Cell Biology and his colleagues decided to assess the role of the alternative p38 pathway in pancreatic cancer.
The researchers began their studies by evaluating pancreatic tissue from 192 patients with histologically-confirmed PDAC who had not received treatment at the time of surgery. All of the samples stained positive for infiltrating T cells using antibodies to pY323, and these cells also expressed the cytokines TNF-α and IL-17A. The investigators were able to separate patients into two groups depending on the level of pY323 staining: those with less than 10 percent of CD3-positive T cells also positive for pY323 and those with 10 percent or more positive cells. While there was no difference in the amount of T cell infiltration in the two groups, there was a much higher percentage of CD4-positive T cells producing TNF-α and IL-17A and higher levels of pro-angiogenic VEGF, which is downstream of the cytokines, in patients with 10 percent or more pY323-positive cells. Importantly, these patients had a significantly poorer prognosis than patients with fewer than 10 percent of cells expressing pY323. The former group had a median survival of 9.8 months and a five-year survival of 5.3 percent compared to a 20.3-month median survival and 16.1 percent five-year survival for the latter. Thus, the prevalence of p38 pY323-positive T cells is an independent prognostic marker for PDAC.
The scientists then investigated the role of the p38 alternative pathway in tumor progression by using two mouse models of pancreatic cancer. In the first, they injected pancreatic tumor cells under the skin of wild type (WT) mice or mice in which tyrosine 323 in p38α and β was changed to phenylalanine to block the alternative pathway, referred to as double knock-in (DKI) mice. The DKI mice had normal stress-induced p38 activity but reduced signaling downstream of the T cell receptor and, consequently, reduced pro-inflammatory cytokine production. Tumors in the DKI mice were smaller than those in the WT mice, but T cell infiltration and activation were unchanged. Levels of pro-inflammatory cytokines, such as TNF-α, and pro-angiogenic VEGF were also reduced in tumors from DKI mice.
The second model involved mice expressing a mutant K-ras oncogene in the pancreas and lacking one copy of the tumor suppressor p53, known as KPC mice. These mice develop spontaneous pancreatic tumors that mimic human disease. Similar to the patient samples, the researchers detected pY323 p38 and TNF-α in regions with many infiltrating T cells around tumors. To determine whether the alternative p38 pathway was important to oncogene-mediated tumor formation, the investigators irradiated 6-week old KPC mice and reconstituted the bone marrow with normal marrow or marrow from DKI mice. Mice that received DKI marrow survived much longer than mice that received normal marrow. Together, the results from the two models demonstrate that the p38 alternative pathway promotes pancreatic tumor progression.
Because of the importance of the alternative p38 pathway, the scientists developed an inhibitor using a peptide from Gadd45α residues 71-85, which inhibits alternatively-, but not classically-, activated p38. They added 11 arginine residues to the peptide to improve its cellular uptake. The researchers then tested the cell-permeable peptide’s activity as a therapy in their two mouse models. Treatment with the active peptide inhibited the growth of pancreatic tumors injected under the skin, whereas a scrambled control or water had no effect. Tumor growth resumed when the investigators stopped administering the active peptide. Importantly, they observed no toxic side effects from treatment with the peptide. Similarly, in the KPC mouse model treatment with the active peptide before tumor formation reduced the area of inflammation in the pancreas and slowed progression, but not initiation, of cancer. Treating mice after confirmed tumor formation with the active peptide also prolonged survival compared with the scrambled peptide. These results suggest that inhibiting the alternative p38 pathway could be a useful therapy for PDAC.Summary Posted: Thu, 10/01/2015
Alam MS, Gaida MM, Bergmann F, Lasitschka F, Giese T, Giese NA, Hackert T, Hinz U, Hussain SP, Kozlov SV, and Ashwell JD. Selective inhibition of the p38 alternative pathway in infiltrating T cells inhibits pancreatic cancer progression. Nature Medicine. October 19, 2015 PubMed Link