Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation

1Solid cancers are heterocellular systems containing both tumor cells and stromal cells. Coercion of stromal cells by tumor cell oncogenes profoundly impacts cancer biology and aberrant tumor-stroma signaling regulates many hallmarks of cancer. While individual oncogene-driven regulators of tumor-stroma signaling have been identified, the propagation of oncogene-dependent signals throughout a heterocellular system is poorly understood. Consequently, our perspective of oncogenic signaling is biased toward how oncogenes regulate tumor cells in isolation.

2In a heterocellular cancer, tumor cell oncogenes drive aberrant signaling both within tumor cells (cell-autonomous signaling) and adjacent stromal cells (non-cell-autonomous signaling). As different cell types process signals via distinct pathways, heterocellular systems (containing different cell types) theoretically provide increased signal processing capacity over homocellular systems (containing a single cell type). By extension, oncogene-dependent signaling can theoretically engage additional signaling pathways in a heterocellular system when compared to a homocellular system. However, to what extent activated stromal cells reciprocally regulate tumor cells beyond cell-autonomous signaling is not well understood.

3We hypothesized that the expanded signaling capacity provided by stromal heterocellularity allows oncogenes to establish a differential reciprocal signaling state in tumor cells. To test this hypothesis, we studied oncogenic KRAS (KRASG12D) signaling in pancreatic ductal adenocarcinoma (PDA). KRAS is one of the most frequently activated oncogenic drivers in cancer and is mutated in >90% of PDA tumor cells. PDA is an extremely heterocellular malignancy—composed of mutated tumor cells, stromal fibroblasts, endothelial cells, and immune cells. Crucially, the gross stromal pancreatic stellate cell (PSC) expansion observed in the PDA microenvironment is non-cell-autonomously controlled by tumor cell KRASG12D in vivo. As a result, understanding the heterocellular signaling consequences of KRASG12D is essential to comprehend PDA tumor biology.

4Comprehensive analysis of tumor-stroma signaling requires concurrent measurement of cell-specific phosphorylation events. Recent advances in proteome labeling now permit cell-specific phosphoproteome analysis in heterocellular systems. Furthermore, advances in proteomic multiplexing enable deep multivariate phospho-signaling analysis.

5Here, we combine cell-specific proteome labeling, multivariate phosphoproteomics, and inducible oncogenic mutations to describe KRASG12D cell-autonomous, non-cell-autonomous, and reciprocal signaling across a heterocellular system. This study reveals KRASG12D uniquely regulates tumor cells via heterotypic stromal cells. By exploiting heterocellularity, reciprocal signaling enables KRASG12D to engage oncogenic signaling pathways beyond those regulated in a cell-autonomous manner. Expansion of KRASG12D signaling via stromal reciprocation suggests oncogenic communication should be viewed as a heterocellular process.

6To investigate how KRASG12D supports heterocellular communication, we first analyzed tumor cell-secreted signals (using PDA tumor cells containing an endogenous doxycycline inducible KRASG12D). Measuring 144 growth factors, cytokines, and receptors across three unique PDA isolations, we observed that KRASG12D increased secretion of GM-CSF, GCSF cytokines, and the growth morphogen sonic hedgehog (SHH) (Figure 1A). As SHH regulates pancreatic myofibroblast expansion, and ablation of SHH signaling reduces PDA tumor stroma in vivo, we focused on understanding the trans-cellular signaling consequences of SHH.

7As previously established, KRASG12D simultaneously induces SHH secretion (Figure 1B) and disrupts primary cilium in PDA cells (Figure 1C). Concordantly, PSCs and KRASWT PDA cells transduce canonical SHH signaling (via SMO-GLI), while KRASG12D cells do not (Figure 1D). This enables KRASG12D PDA cells to non-cell-autonomously signal to PSCs via SHH, while remaining insensitive to autocrine SHH (Figure 1E).

8Quantitative proteomic analysis revealed SHH induces widespread changes across the cytoplasmic, membrane, and secreted PSC proteome (Figures 1F, 1G, and S1A; Data S1). SHH upregulates multiple extracellular matrix components (collagens, MMPs, fibrillin-1, LOX)—suggesting KRASG12D controls PDA desmoplasia via SHH-activated PSCs. Notably, SHH also upregulates IGF1 and GAS6 across multiple PSC isolations but not in PDA cells (Figures 1H, 1I, and S1B). Since IGF1 and GAS6 are growth factors capable of activating the receptor tyrosine kinases (RTKs) IGF1R and AXL, respectively, this suggests that SHH-activation alters the intercellular signaling potential of PSCs.

9These results demonstrate KRASG12D non-cell-autonomously communicates with stromal cells via SHH-SMO-GLI and renders tumor cells insensitive to autocrine SHH. Moreover, KRASG12D achieves a unique signaling output (e.g., production of ECM, IGF1, and GAS6) via stromal cells that is distinct from that produced by tumor cell KRASG12D alone.

10To provide a baseline of cell-autonomous oncogene-regulated signaling from which to compare stromal-dependent reciprocal signaling, we first determined the effect of KRASG12D expression on the PDA phosphoproteome (Figures 2A, 2B, and S2A). Despite being the primary oncogenic driver in PDA, KRASG12D only regulates 7% of the observed tumor cell phosphoproteome (+/−1 log2, p < 0.01) (Figure 2C; Data S1). KRASG12D expression induces canonical activation of ERK1/2 and increases phosphorylation of MAPK/CDK1/CKII-directed kinase motifs. However, while the PI3K-AKT axis is often presumed directly downstream of KRASG12D in PDA—expression of KRASG12D does not activate AKT in a cell-autonomous manner (Figures 2D and S2). This observation is consistent across multiple PDA cell isolations from several independently developed genetic mouse models (Figure S3). To further investigate the dependency of MEK and AKT activity in KRASG12D cell-autonomous signaling, KRASWT and KRASG12D PDA cells were perturbed with MEK (PD-184352) and/or AKT (MK-2206) inhibitors and analyzed by quantitative phosphoproteomics. This analysis confirmed MEK-ERK1/2, not AKT, controls the differential phosphoproteome of KRASG12D (Figure 2E; Data S1).

11Collectively, these observations demonstrate cell-autonomous KRASG12D regulates a distinct section of the tumor cell phosphoproteome. Notably, KRASG12D induces MAPK/CDK/CK kinase motifs via MEK-ERK and does not regulate AKT.

12Given that KRASG12D non-cell-autonomously regulates growth factor production from PSCs (e.g., IGF1 and GAS6), we hypothesized that KRASG12D-activated PSCs initiate a reciprocal signaling axis back in the tumor cells. However, given that tumor cells already undergo phosphoproteomic deregulation by KRASG12D, it was unclear whether additional reciprocal signals from PSCs can further regulate the tumor cell phosphoproteome. To investigate this, the phosphoproteome of KRASWT and KRASG12D PDA cells were directly compared to PDA cells treated with conditioned media from SHH-activated PSCs (Figure 3A).

13Despite the considerable regulation of cell-autonomous signaling by KRASG12D, PDA cells are further modulated by signals from SHH-activated PSCs (Figure 3B). In fact, PSC-signaling regulates (+/−1 log2, p < 0.01) comparable numbers of PDA tumor cell phosphosites (6.7% phosphoproteome) when compared to KRASG12D alone (7.2% phosphoproteome) (Figures 3C, S4A, and S4B; Data S1). This implies stromal cells can substantially alter tumor cell signaling beyond cell-autonomous KRASG12D. Notably, while PDA KRASG12D expression does not activate AKT in a cell-autonomous manner (Figures 2 and S3), tumor cell AKT substrate phosphosites (e.g., AKTS1 [pT247] and GSK3α [pS21]) are exclusively regulated by stromal PSCs (Figures S4C–S4E).

14Targeted temporal analysis revealed SHH-activated PSCs induce rapid phosphorylation of IGF1R (receptor for IGF-1), AXL/TYRO3 (receptor for GAS6), and downstream IRS-1 and AKT (pT308/pS473) in KRASG12D PDA cells (Figure 3D). Tumor cells treated with conditioned media from control or SHH-activated PSCs and perturbed with either MEK and/or AKT inhibitors further confirmed PSCs drive a differential phosphoproteome in PDA cells. However, unlike cell-autonomous KRASG12D, stromal-driven signaling depends on both active MEK and AKT (Figure 3E; Data S1).

15As IGF1 and GAS6 are secreted by activated PSCs, we investigated the dependency of IGF1R and AXL activity on the PSC-induced tumor cell phosphoproteome. Combined IGF1R and AXL inhibitors are required to block the PSC-induced tumor cell phosphoproteome—suggesting a Boolean “OR” axis between PSC IGF1/GAS6 and PDA pAKT (Figures 3F, 3G, and S4F; Data S1).

16Collectively, these results reveal activated stromal cells can return a differential signal to tumor cells via an IGF1R/AXL-AKT axis. The stromal-driven tumor cell phosphoproteome is distinct from the KRASG12D regulated cell-autonomous phosphoproteome and responds differently to pharmacological perturbation.

17Our data suggests that oncogenic KRAS in tumor cells establishes a reciprocal signaling axis between stromal cells and tumor cells. Herein, we define an oncogenic reciprocal signaling axis as an oncogenic cue that signals via an adjacent heterotypic cell to produce a distinct response in the oncogene-expressing cell. For this heterocellular variation on the “cue-signal-response” systems biology paradigm to be valid, we hypothesized that oncogenic reciprocal signaling requires three essential features: (1) an oncogenic cue (e.g., KRASG12D), (2) a cue-driven non-cell-autonomous signal (e.g., KRASG12D-induced SHH), and (3) a heterotypic cell capable of transducing the signal response back to the instigating oncogenic cell (e.g., PSC). To test this multi-node reciprocal signaling hypothesis, we systematically perturbed each reciprocal feature in a native heterocellular tumor-stroma context.

18To measure multivariate signaling in a heterocellular system, concurrent cell-specific and variable-specific phosphoproteomic data are required. We have previously shown that stable isotopic proteome labeling can resolve between discrete cell types in direct culture of heterotypic cells and recently introduced cell type-specific labeling with amino acid precursors (CTAP) LyrM37-KDEL and DDCM.tub-KDEL enzymes for cell-specific isotopic labeling. To this end, we combined CTAP labeling (spatial resolution) with isobaric tandem mass tag (TMT) phosphoproteomics (variable resolution) to enable heterocellular multivariate phosphoproteomic analysis of each reciprocal signaling component (Figure 4A). This technique allows simultaneous observation of cell-autonomous, non-cell-autonomous, and reciprocal oncogenic phosphoproteomes at cell-specific resolution.

19Cell-specific phosphoproteomes were interrogated in PDA cells expressing either KRASWT or KRASG12D, either in homo- or heteroculture with isotopically “heavy”-labeled PSCs, and treated with either SHH inhibitor or vehicle. We monitored 3,695 lysine-containing (8,566 total) phosphopeptides across eight conditions, two heterotypic cell types, and three biological replicates with cell-specific resolution (Figures 4B and S5; Data S1). As expected, expression of KRASG12D in tumor cells alone regulates (+/−1 log2) 7.2% of the identified cell-autonomous phosphoproteome. In parallel, tumor cell KRASG12D non-cell-autonomously regulates 4.7% of the PSC phosphoproteome. Moreover, when KRASG12D is allowed to communicate with PSCs via SHH, a reciprocal axis is completed and the differentially regulated tumor cell phosphoproteome almost doubles to 13.8%. Importantly, perturbation by a SHH blocking antibody decreases the phosphoproteomic regulation on PSCs back down to 1.2% and PDA phosphoproteome to 8.1% (close to cell-autonomous at 7.2%).

20Heterocellular multivariate phosphoproteomics demonstrates how tumor cell oncogenes exploit the differential signaling capacity of stromal cells to achieve a unique signaling state in the inceptive tumor cell. KRASG12D reciprocal signaling engages additional phospho-nodes to cell-autonomous KRASG12D alone, allowing KRASG12D to extend the oncogenic signaling capacity in the inceptive tumor cells. Crucially, these observations are the product of native tumor-stroma signaling and are independent of exogenous stimulation.

21Comprehensive phosphoproteomic quantification of reciprocally engaged PDA cells (Figures 5A–5C and S6A; Data S1) revealed upregulation of several AKT substrates (e.g., BAD [pS136], PDCD4 [pS457], CHSP1 [pS53], AKTS1 [T247], and GSK-3α [pS21]). Interestingly, cell-autonomous targets of KRASG12D (e.g., RAF1 [pS621] and ERK1/2 [pT183/pY185; pT203/pY205]) were not regulated by reciprocal signaling—further implying reciprocal KRASG12D supplements cell-autonomous KRASG12D by engaging additional tumor cell kinases (Figure S6B).

22Reciprocal signaling also activates several translational mediators (e.g., RPS6 [pS235/pS236], PDCD4 [pS457], and EIF4B [pS422]). Concordantly, RNA sequencing (RNA-seq) analysis of PDA cells revealed reciprocal signaling upregulates RNA associated with translational control (Figures S6C–S6F), further suggesting a de novo control of PDA protein abundance. To validate whether the SHH-driven reciprocal signaling axis regulates de novo tumor cell protein turnover, PSC+DDCM.tub-KDEL and PDA+LyrM37-KDEL CTAP cells were differentially isotopically labeled, treated with a SHH inhibitor or vehicle, and cell-specific proteomes were quantified in heteroculture (Figure 5D). This experimental format permitted cell-specific quantification of changes to the KRASG12D tumor cell proteome following inhibition of the PSC targeting signal (SHH). Parallel perturbations with AKT and IGF1R/AXL inhibitors provided additional insight into the role of each reciprocal node.

23Cell-specific proteomics confirmed KRASG12D reciprocally regulates the PDA proteome and is dependent on active SHH, IGF1R/AXL, and AKT signaling (Figure 5E; Data S1). As with the PDA phosphoproteome, reciprocal signaling regulates the PDA proteome differently to cell-autonomous KRASG12D. For example, while cell-autonomous KRASG12D rapidly depletes distinct mitochondrial components from PDA cells (Data S1), reciprocally engaged KRASG12D restores mitochondrial proteins in an SHH-, IGF1R/AXL-, and AKT-dependent manner. Moreover, PDA proteins involved with DNA replication are also upregulated under reciprocal conditions. These results demonstrate reciprocal signaling uniquely regulates both the tumor cell phosphoproteome and global proteome when compared to cell-autonomous signaling. Reciprocal signaling states are unique to a heterocellular environment and are not observed in tumor cells alone.

24Reciprocal signaling regulates proteins and phospho-sites known to control several important biological processes. For example, while cell-autonomous and reciprocal KRASG12D signaling both regulate mitochondrial proteins, many of these are asymmetrically regulated. As a result, we hypothesized PDA mitochondrial activity would be differentially regulated by cell-autonomous and reciprocal KRASG12D. Concordantly, cell-autonomous KRASG12D decreases PDA mitochondria polarization (Δψm) and mitochondrial superoxide production, whereas reciprocal signaling increases these processes (via SHH, IGF1R/AXL, and AKT) (Figures 6A and S7). Furthermore, reciprocal signaling increases spare mitochondrial respiratory capacity in tumor cells (Figure 6B). These results demonstrate KRASG12D can differentially regulate mitochondrial performance via heterocellular communication.

25Reciprocal signaling also regulates proteins known to control cell proliferation and survival. In agreement, cell-specific analysis of PDA proliferation in homo and heterocellular cultures revealed increased tumor cell proliferation under heterocellular conditions (via SHH, IGF1R/AXL, and AKT activity) (Figure 6C). Upregulation of AKT substrates (e.g., inhibition of BAD [pS136]) also suggested reciprocal signaling might protect tumor cells from apoptosis. Concordantly, TUNEL and caspase 3/7 profiling revealed activated PSCs protect tumor cells from apoptosis and sensitize tumor cells to reciprocal node inhibitors (IGF1R/AXL and AKT) (Figures 6D–6E).

26Increased mitochondrial performance, proliferative capacity, and resistance to apoptosis collectively implied reciprocal signaling supports tumor cell phenotypes beyond cell-autonomous KRASG12D. In accordance, reciprocal signaling increases semi-solid colony growth relative to cell-autonomous KRASG12D alone (Figure 6F). Reciprocal colony growth is dependent on SHH activation of PSCs and IGF1R/AXL-AKT activity in tumor cells. Collectively, these results demonstrate the unique signals produced by reciprocal KRASG12D control distinct metabolic, proliferative, anti-apoptotic, and anchorage-independent growth phenotypes in tumor cells.

27Whether oncogenes regulate tumor cell signaling via stromal cells is a fundamental question in tumor biology. Using heterocellular multivariate phosphoproteomics, we demonstrate how oncogenic KRAS signals through local non-tumor cells to achieve a differential reciprocal signaling state in the inceptive tumor cells. In PDA, this reciprocal axis supplements oncogenic cell-autonomous signaling to control protein abundance, transcription, mitochondrial activity, proliferation, apoptosis, and colony formation. Reciprocal signaling is the exclusive product of heterocellularity and cannot be achieved by tumor cells alone. These observations imply oncogenes expand their capacity to deregulate cellular signaling via stromal heterocellularity (Figure 7).

28Despite the well-established heterocellularity of cancer, our understanding of oncogenic signaling within tumor cells has largely excluded non-tumor cells. We observe that stromal cells approximately double the number of tumor cell signaling nodes regulated by oncogenic KRAS, suggesting both cell-autonomous (internal) and reciprocal (external) stimuli should be considered when defining aberrant oncogenic signaling states. For example, although KRAS is thought to cell-autonomously regulate AKT in PDA, we show that KRASG12D activates AKT, not cell-autonomously, but reciprocally. As PI3K signaling is essential for PDA formation in vivo reciprocal signaling may control oncogene-dependent tumorigenesis. Our findings suggest future genetic studies should consider the heterocellular signaling consequences of oncogene/tumor-suppressor deregulation.

29The observation that many oncogene-dependent tumor cell signaling nodes require reciprocal activation has important implications for identifying pharmacological inhibitors of oncogene signaling. For example, if PDA tumor cells were screened alone, one would expect MEK, MAPK, and CDK inhibitors to perturb KRASG12D signaling. However, when screened in conjunction with heterotypic stromal cells, our study additionally identified SHH, AKT, and IGF1R/AXL inhibitors as KRASG12D-dependent targets in tumor cells. Inhibitors of signaling specific to reciprocally engaged tumor cells, such as or AKT or IGF1R/AXL, block heterocellular phenotypes (e.g., protein expression, proliferation, mitochondrial performance, and anti-apoptosis), but have little effect on KRASG12D tumor cells alone. An appreciation of reciprocal nodes increases our molecular understanding of drug targets downstream of oncogenic drivers and highlights focal points where reciprocal signals converge (e.g., AKT). These trans-cellular observations reinforce the importance of understanding cancer as a heterocellular disease.

30Previous work in PDA tumor cells under homocellular conditions demonstrated cell-autonomous KRASG12D shifts metabolism toward the non-oxidative pentose phosphate pathway, whereas KRASG12D-ablated cells depend on mitochondrial activity. Here, we show that heterocellular reciprocal signaling can restore the expression of mitochondrial proteins and subsequently re-establish both mitochondrial polarity and superoxide levels. This suggests KRASG12D regulates non-oxidative flux through cell-autonomous signaling and mitochondrial oxidative phosphorylation through reciprocal signaling. These results provide a unique example of context-dependent metabolic control by oncogenes and reinforce the emerging role of tumor-stroma communication in regulating cancer metabolism.

31In PDA, the stroma has dichotomous pro-tumor and anti-tumor properties. It is becoming increasingly evident that non-cell-autonomously activated stromal cells vary within a tumor and can influence tumors in a non-obvious manner. For example, while vitamin D receptor normalization of stromal fibroblasts improves PDA therapeutic response, total stromal ablation increases malignant behavior. Thus, while stromal purging is unlikely to provide therapeutic benefit in PDA, “stromal reprogramming” toward an anti-tumor stroma is now desirable. Although we describe a largely pro-tumor reciprocal axis, both pro- and anti-tumor stromal phenotypes likely transduce across reciprocal signaling networks. Our work suggests future efforts to therapeutically reprogram the PDA stroma toward anti-tumor phenotypes will require an understanding of reciprocal signaling. In describing the first oncogenic reciprocal axis, this study provides a foundation to measure the cell-cell communication required for anti-tumor stromal reprogramming.

32We demonstrate heterocellular multivariate phosphoproteomics can be used to observe reciprocal signaling in vitro. Unfortunately, cell-specific isotopic phosphoproteomics is not currently possible in vivo. To delineate reciprocal signaling in vivo, experimental systems must support manipulation of multiple cell-specific variables and provide cell-specific signaling readouts. Simple pharmacological perturbation of reciprocal nodes (e.g., IGF1R, AXL, AKT, etc.) in existing PDA GEMMs will in principle affect all cell types (e.g., tumor cells, PSCs, immune cells) and cannot provide axis-specific information in vivo. Future in vivo studies of reciprocal signaling will require parallel inducible genetic manipulation (e.g., oncogene activation in cancer cell and/or inhibition of reciprocal node in stromal cell), combined with cell-specific signaling data (e.g., using epithelial tissue mass-cytometry).

33We describe KRASG12D reciprocal signaling between PDA tumor cells and PSCs. However, it is likely oncogenic reciprocal signaling occurs across multiple different cell types in the tumor microenvironment. For example, in PDA, FAP+ stromal fibroblasts secrete SDF1 that binds tumor cells to suppress T cells. Our model predicts oncogene signaling expands across several cell types in the tumor microenvironment—including immune cells. Moreover, as oncogenes non-cell-autonomously regulate the stroma in many other tumor types, our model predicts oncogenic reciprocal signaling to be a broad phenomenon across all heterocellular cancers. The presented heterocellular multivariate phosphoproteomic workflow now enables future characterization of oncogenic reciprocal signaling in alternative cancer types.

34As differentiated cells process signals in unique ways, heterocellularity provides increased signal processing space over homocellularity. We provide evidence that KRASG12D exploits heterocellularity via reciprocal signaling to expand tumor cell signaling space beyond cell-autonomous pathways. Given the frequent heterocellularity of solid tumors, we suspect reciprocal signaling to be a common—albeit under-studied—axis in oncogene-dependent signal transduction.

35KRASWT PDA cells (1 × 106) were plated in a 6-well dish and cultured in DMEM + 0.5% FBS ± 1 μg/ml doxycycline for 72 hr. Conditioned media was analyzed for relative changes in KRASG12D-driven cytokines and growth factors using the RayBio Mouse Cytokine Antibody Array G2000 (RayBiotech AAM-CYT-G2000-8) (144 proteins quantified in duplicate per sample). SHH-N expression after 24 hr was further validated by sandwich ELISA (R&D Systems DY461).

36For comprehensive phosphoproteomic quantification of KRASG12D-dependent cell-autonomous signaling, 1 × 106 KRASWT PDA cells were plated in a 6-well dish (DMEM + 0.5% FBS) and cultured ± 1 μg/ml doxycycline for 24 hr (biological replicates n = 5). Cells were lysed in 6 M urea, 10 mM NaPPi, 20 mM HEPES, pH 8.0, sonicated, centrifuged to clear cell debris, and protein concentration was determined by BCA (Pierce 23225). One hundred micrograms of each condition were individually digested by FASP, amine-TMT-10-plex-labeled (Pierce 90111) on membrane (iFASP), eluted, pooled, lyophilized, and subjected to automated phosphopeptide enrichment (APE). Phosphopeptides were desalted using OLIGO R3 resin (Life Technologies 1-1339-03) and lyophilized prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (see the Supplemental Experimental Procedures).

37For TMT-labeled samples, phosphopeptides were enriched from each fraction using the automated phosphopeptide enrichment (APE) method described by. Phosphopeptide fractions were individually desalted using OLIGO R3 resin (Life Technologies 1-1339-03) and resuspended in 0.1% formic acid prior to Q-Exactive Plus HCD FT/FT LC-MS/MS (see the Supplemental Experimental Procedures). For reciprocal phosphoproteomics PSC-PDA co-cultures, 15 mg protein was digested with 150 μg Lys-C (Wako 125-05061) (24 hr) and 150 μg Trypsin (Worthington) (24 hr) using 2 ml FASP. Lyophilized tryptic peptides were re-suspended in 60% MeCN and resolved using a Ultimate 3000 (Dionex) high-performance liquid chromatography fitted with a 10 μm particle size, 7.8 mm ID, and 30 cm TSKgel Amide-80 hydrophilic interaction liquid chromatography (HILIC) column (Tosoh 14459) into 24 fractions. Phosphopeptides were enriched from each fraction by APE. Phosphopeptide fractions (n = 192) were individually desalted using OLIGO R3 resin (Life Technologies 1-1339-03) and re-suspended in 0.1% formic acid prior to LTQ Velos HCD FT/FT LC-MS/MS (see the Supplemental Experimental Procedures).

38For concurrent PDA cell-autonomous and reciprocal phosphoproteomics, 1 × 106 PSCs were plated in a 6-well dish, stimulated with 5 nM SHH-N (C25II) (R&D Systems 464-SH-025/CF) in DMEM + 0.5% FBS, and conditioned media was collected after 48 hr. PDA cells (1 × 106) were cultured without doxycycline (KRASWT), with 1 μg/ml doxycycline (KRASG12D), and with 1 μg/ml doxycycline (KRASG12D) + PSC+SHH conditioned media (biological n = 3) (all in +0.5% dialyzed FBS). One hundred micrograms of each condition was then processed for TMT and APE analysis as described above.

39Mycobacterium tuberculosis (DDCM.tub-KDEL) (P0A5M4) diaminopimelate decarboxylase (DDC) and Proteus mirabilis lysine racemase (LyrM37-KDEL) (M4GGR9) were synthesized by GeneArt. Full details can be found in. DDC cells were grown in DMEM (-K/-R) (Caisson DMP49) supplemented with 10% (v/v) dialyzed FBS (GIBCO), 0.3 mM L-arginine (Sigma A8094) and 5 mM meso-2,6-diaminopimelate (DAP) (Sigma 07036). Lyr cells were grown in DMEM (-K/-R) supplemented with 10% (v/v) dialyzed FBS, 0.3 mM L-arginine and either 2.5 mM “Medium” D-lysine-4,4,5,5-d4 HCl (C/D/N D-7334) (Delta mass: 4.025107, Delta average mass: 4.0246) or 2.5 mM “Heavy” D-lysine-3,3,4,4,5,5,6,6-d8 2HCl (C/D/N D-6367) (Delta mass: 8.0502136, Delta average mass: 8.04928).

40PDA cells were transfected with DDCM.tub-KDEL and grown on 5 mM DAP (“Light”). PSCs were transfected with LyrM37-KDEL and grown on 2.5 mM D-lysine-3,3,4,4,5,5,6,6-d8 2HCl (“Heavy”). PDA+DDC cells (3 × 106) were cultured in a 10 cm dish ± 1 μg/ml doxycycline, ±10 μg/ml SHH neutralizing monoclonal antibody (mAb) (R&D Systems MAB4641) and ±3 × 106 “Heavy” PSC+Lyr cells (biological triplicates). All cells were grown in DMEM (-K/-R) supplemented with 0.5% (v/v) dialyzed FBS, 0.3 mM L-arginine, 5 mM DAP, and 2.5 mM “Heavy” D-lysine. After 5 days, each condition was lysed in 6 M urea, sonicated, centrifuged to clear cell debris, and protein concentration was determined by BCA. One hundred micrograms of each variable was then processed for TMT and APE analysis as described above.

41To investigate reciprocal regulation of PDA protein abundance, “Heavy” PDA+LyrM37-KDEL cells were co-cultured with “Light” PSC+DDCM.tub-KDEL in the presence of 2.5 mM “Heavy” D-lysine-3,3,4,4,5,5,6,6-d8 and 5 mM “Light” DAP (biological n = 3). For each experiment, a control co-culture of “Medium” PDA+LyrM37-KDEL cells and “Light” PSC+DDCM.tub-KDEL was performed in the presence of either PDA pre-treatment with IGF1R inhibitor (250 nM picropodophyllin [PPP]), AXL inhibitor (500 nM R428), or 20 μg/ml SHH-neutralizing antibody (R&D Systems MAB4641). All co-cultures were performed in +0.5% dialyzed FBS for 72 hr. Co-cultures were lysed in 100 mM Na2CO3 (pH 11.0), pooled, snap-frozen in liquid nitrogen, treated with Benzonase (Novagen 70746), centrifuged at 40,000 rpm (to resolve membrane-bound proteins from cytosolic proteins), and denatured in 6 M urea 2 M thiourea. Differential changes in cytoplasmic and membrane protein levels were determined using “In-gel digestion” (see the Supplemental Experimental Procedures). To investigate the comparative KRASG12D cell-autonomous proteome, KRASWT “Medium” and “Heavy” PDA+LyrM37-KDEL cells were seeded into 10-cm dishes (biological n = 3) (5 × 106 PDA cells/plate). Doxycycline (1 μg/ml) was then added to the “Heavy” PDA cells (i.e., KRASG12D) and the “Medium” cells were left untreated (i.e., KRASWT) (in +0.5% dialyzed FBS). After 72 hr, cells were lysed as above.

42C.J.T. conceived the project, performed all proteomic-signaling experiments, and wrote the paper. S.L. performed PSC secretomics. M.D. and G.P. performed PDA mitochondrial flux analysis. K.M.M. and J.D.W. provided LC-MS/MS support. I.C.N., H.S.L., and C.J.M. performed FACS RNA-seq. D.A.L. oversaw the project. C.J. conceived the project, oversaw the project, and wrote the paper.