SummaryOncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRASG12D) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer.Video Abstract

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We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC M.tub-KDEL) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr M37-KDEL ) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC M.tub-KDEL and Lyr Despite advances in the understanding of intracellular signaling networks in monoculture, our capacity to biochemically investigate communication between cells in direct co-culture remains limited. This is primarily due to the technical difficulty of discerning cell-specific protein signaling events from a co-culture. Stable isotope labeling with amino acids in cell culture (SILAC) 1 (2) can be used to identify the discrete proteomes of different cell populations in co-culture (3). However, as L-lysine and L-arginine are equally metabolized by all cell types in a co-culture, SILAC technology is not suitable for investigating long-term cell-specific proteomic changes in proliferating co-cultures.To address this limitation, Gauthier et al. recently reported an alternative cell-specific isotopic labeling technology (4). This approach, entitled cell-type-specific labeling with amino acid precursors (CTAP), relies on the ectopic expression of non-mammalian amino acid processing enzymes to convert lysine precursors into essential L-lysine. Two distinct enzymes specifically convert their respective lysine precursor substrates into L-lysine: diaminopimelate decarboxylase (DDC) converts meso-2,6-diaminopimelate (DAP) into L-lysine (5), and lysine racemase (Lyr) converts D-lysine into L-lysine (6). When these enzymes are combined with isotopically labeled D-lysine, their cell-specific expression confers perpetual and cell-specific labeling of two cell populations in coculture (supplemental Figs. S1A and S1B).As this technique uses amino acid processing enzymes to confer isotopic labeling, the ectopic performance of DDC and Lyr is directly responsible for cell-specific labeling fidelity. Thus, for biologically relevant CTAP experiments, optimal DDC and Lyr enzymes are essential. Optimal cell-specific labeling enzymes should (i) confer excellent precursor-specific proliferation, (ii) be strictly intracellular, (iii) be applicable From the ‡Division

Ageing and cancer is often associated with altered T cell distributions and this phenomenon has been suggested to be the main driver in the development of immunosenescence. Memory phenotype PD-1+ CD4+ T cells accumulate with age and during leukemic development, and they might account for the attenuated T cell response in elderly or diseased individuals. The transcription factor C/EBPα has been suggested to be responsible for the accumulation as well as for the senescent features of these cells including impaired TCR signaling and decreased proliferation. Thus modulating the activity of C/EBPα could potentially target PD-1+ CD4+ T cells and consequently, impede the development of immunosenescence. To exploit this possibility we tested the importance of C/EBPα for the development of age-dependent PD-1+ CD4+ T cells as well as its role in the accumulation of PD-1+ CD4+ T cells during leukemic progression. In contrast to earlier suggestions, we find that loss of C/EBPα expression in the lymphoid compartment led to an increase of PD-1+ CD4+ T cells specifically in old mice, suggesting that C/EBPα repress the accumulation of these cells in elderly by inhibiting their proliferation. Furthermore, C/EBPα-deficiency in the lymphoid compartment had no effect on leukemic development and did not affect the accumulation of PD-1+ CD4+ T cells. Thus, in addition to contradict earlier suggestions of a role for C/EBPα in immunosenescence, these findings efficiently discard the potential of using C/EBPα as a target for the alleviation of ageing/cancer-associated immunosenescence.

Pancreatic Ductal Adenocarcinoma is characterized by a reactive stroma, which modifies tumor progression and response to therapy. Oncogenic mutations regulate signaling both within tumor cells and adjacent stromal cells. However, defining whether oncogenes can regulate tumor cell signaling and phenotypic behavior via stromal cells is of importance to understand mechanisms of disease progression and response to therapy. Here we show that oncogenic KRAS (KRASG12D) regulates pancreatic cancer cell signaling via stromal stellate cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in Pancreatic Ductal Adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic stellate cells, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation, apoptosis, and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. Citation Format: Christopher Tape, Stephanie Ling, Maria Dimitriadi, Kelly McMahon, Jonathan Worboys, Hui S. Leong, Ida Norrie, Crispin Miller, George Poulogiannis, Douglas Lauffenburger, Claus Jorgensen.{Authors}. Oncogenic KRAS regulates pancreatic cancer cell signaling via stromal reciprocation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A34.