Our understanding of physiology and disease is hampered by the difficulty of measuring the circuitry and plasticity of signaling networks that regulate cell biology, and how these relate to phenotypes. Here, using mass spectrometry-based phosphoproteomics, we systematically characterized the topology of a network comprising the PI3K/Akt/mTOR and MEK/ERK signaling axes and confirmed its biological relevance by assessing its dynamics upon EGF and IGF1 stimulation. Measuring the activity of this network in models of acquired drug resistance revealed that cells chronically treated with PI3K or mTORC1/2 inhibitors differed in the way their networks were remodeled. Unexpectedly, we also observed a degree of heterogeneity in the network state between cells resistant to the same inhibitor, indicating that even identical and carefully controlled experimental conditions can give rise to the evolution of distinct kinase network statuses. These data suggest that the initial conditions of the system do not necessarily determine the mechanism by which cancer cells become resistant to PI3K/mTOR targeted therapies. The patterns of signaling network activity observed in the resistant cells mirrored the patterns of response to several drug combination treatments, suggesting that the activity of the defined signaling network truly reflected the evolved phenotypic diversity.signaling network | phosphoproteomics | systems biology | PI3K | MEK

The tumor microenvironment evolves during malignant progression with major changes in non-malignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein transforming growth factor beta induced (TGFBI -also known as βig-h3). STIC lesions in the fimbria expressed high levels of TGFBI which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFβ and IL4 induced secretion of TGFBI, whereas IFNɣ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvβ3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro. Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated β3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumorpromoting actions of TGFβ and a potential therapeutic target. Statement of significanceAnalysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer.Research.

The Frank–Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16—a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank–Starling response.

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low 2-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs and toxicity of therapy toward healthy hematopoietic cells. We studied the role of cyclin-dependent kinase regulatory subunit 1 (CKS1)–dependent protein degradation in primary human AML and healthy hematopoiesis xenograft models in vivo. Using a small-molecule inhibitor (CKS1i), we demonstrate a dual role for CKS1-dependent protein degradation in reducing patient-derived AML blasts in vivo and, importantly, depleting LSCs, whereas inhibition of CKS1 has the opposite effect on normal hematopoiesis, protecting normal hematopoietic stem cells from chemotherapeutic toxicity. Proteomic analysis of responses to CKS1i in our patient-derived xenograft mouse model demonstrate that inhibition of CKS1 in AML leads to hyperactivation of RAC1 and accumulation of lethal reactive oxygen species, whereas healthy hematopoietic cells enter quiescence in response to CKS1i, protecting hematopoietic stem cells. Together, these findings demonstrate that CKS1-dependent proteostasis is a key vulnerability in malignant stem cell biology.

Class IA phosphoinositide 3-kinases (PI3Ks) are signaling enzymes with key roles in the regulation of essential cellular functions and disease, including cancer. Accordingly, their activity is tightly controlled in cells to maintain homeostasis. The formation of multiprotein complexes is a ubiquitous mechanism to regulate enzyme activity but the contribution of protein-protein interactions to the regulation of PI3K signaling is not fully understood. We designed an affinity purification quantitative mass spectrometry strategy to identify proteins interacting dynamically with PI3K in response to pathway activation, with the view that such binding partners may have a functional role in pathway regulation. Our study reveals that calpain small subunit 1 interacts with PI3K and that the association between these proteins is lower in cells stimulated with serum compared to starved cells. Calpain and PI3K activity assays confirmed these results, thus demonstrating that active calpain heterodimers associate dynamically with PI3K. In addition, calpains were found to cleave PI3K proteins in vitro (resulting in a reduction of PI3K lipid kinase activity) and to regulate endogenous PI3K protein levels in vivo. Further investigations revealed that calpains have a role in the negative regulation of PI3K/Akt pathway activity (as measured by Akt and ribosomal S6 phosphorylation) and that their inhibition promotes cell survival during serum starvation. These results indicate that the interaction between calpain and PI3K is a novel mechanism for the regulation of class IA PI3K stability and activity.lipid signaling | proteomics | quantitative analysis