Background
Annexin-1 (ANXA1), also known as lipocortin-1, belongs to a multigene superfamily of calcium-dependent phospholipid binding proteins that regulate inflammatory responses [
1,
2]. ANXA1 is a prime effector of anti-inflammatory effects of glucocorticoids wherein it inhibits phospholipase A2 to suppress eicosanoid production such as prostaglandins, and also leukocyte functions [
3]. Apart from controlling production of proinflammatory mediators, ANXA1 also limits neutrophil recruitment and promotes phagocytosis of apoptotic neutrophils [
4,
5]. While the roles of ANXA1 in innate immune responses are well-established, growing evidence of its functions in adaptive responses that has emerged in recent years, however, has been inconsistent [
6]. While some reports indicate that ANXA1 increases T cell activation and differentiation, others focus on ANXA1-mediated attenuation of T cell responses [
7,
8].
Besides the inflammatory effects, ANXA1 also plays essential roles in cell proliferation, differentiation, apoptosis and cancer [
2]. By modulating the functions of several receptors including epidermal growth factor, ANXA1 influences several downstream signaling cascades [
9,
10]. With its contributions prominent in a wide array of cellular functions, ANXA1 either functions as a pro-cancerous or tumor-suppressive protein depending on the contextual tumor tissue/cell type. In several malignancies associated with liver, pancreas, lung, prostate and brain, high ANXA1 expression has been correlated with tumor progression, aggressiveness and even metastasis [
11‐
15]. Despite ANXA1 being considered a prognostic marker, its status in tumor progression and survival in breast cancer remain contradictory [
16‐
18]. In general, ANXA1-positive cases have been associated with clinically aggressive basal-like breast cancer. In these cells, ANXA1 activates an epithelial-mesenchymal switch (EMT) by enhancing transforming growth factor (TGF)β/Smad signaling thereby exacerbating migration and invasion [
19]. With ANXA1 localizing at F-actin-rich membrane ruffles, a potential role in shaping the cytoskeletal network has also been proposed. Remodeling of the actin cytoskeletal network is instrumental in deciding cell fate towards migration and invasion [
20]. Our previous studies emphasized that ANXA1 modulates cell adhesion and migratory properties during breast cancer initiation and tumorigenesis [
21,
22]. However, the underlying signaling mechanisms for such modulations are not clear on a global scale. The spatial and temporal regulation of many proteins, including their activation status is more often determined by their phosphorylation status. Hence, here we extend our previous work by investigating the phosphorylation landscape in ANXA1
+/- and ANXA1
-/- murine mammary gland cells to understand the ANXA1-modulated signaling network upon breast cancer initiation. Our phosphorylation dataset complements our previous proteome profiling, and provides novel insights into several ANXA1-dependent processes including its role in rewiring adhesion-related machinery.
Discussion
The spectrum of functions attributed to ANXA1 has expanded in recent years from an anti-inflammatory protein to that of a regulator for several cellular processes including proliferation, apoptosis and migration. With several reports suggesting correlation between ANXA1 and breast cancer outcome, we earlier focused on characterizing ANXA1-responsive changes in global proteome during breast cancer initiation and breast cancer tumorigenesis [
21,
22]. The repertoire of proteins that were modulated in normal breast epithelial cells and tumor cells in response to ANXA1 deficiency was distinct. Yet, both the studies emphasized a strong link between ANXA1 and migratory properties of breast cancer cells. Thus following our previous work, in this study we have taken a step further to systematically analyze ANXA1-responsive changes in the phosphorylation profile of mammary gland cells derived from ANXA-1 deficient mice to reveal signaling networks and complexes impacted by ANXA1 and thus understand its role in breast cancer initiation.
Through our combined analysis of the proteome and phosphoproteome datasets we observed functionally distinct fine-tuning of several processes. Collectively, many of the proteins involved in cytoskeletal organization displayed changes either in total abundance, phosphosite abundance or both. However, unlike in the proteome dataset wherein we observed enrichment of the DNA damage response pathway among the downregulated proteins, there was no specific enrichment for this pathway among the regulated phosphoproteins. Similarly, many of the inflammatory-response-associated proteins majorly displayed changes in protein abundance only. This suggested that ANXA1-responsive translational and post-translational mechanisms span discrete functional processes, but for mechanisms involving ECM remodeling and adhesion complexes they work in concert to dictate cell motility fate.
We observed several cell adhesion and migration-associated proteins among the ANXA1-responsive phosphoproteins (Additional file
14: Table S9). Of note, our previous study established that ANXA1-deficient mammary epithelial cells are less migratory as compared with ANXA1
+/− cells [
21]. Accordingly, we found several proteins that can function as a part of the adhesome complex to have increased phosphorylation upon ANXA1-deficiency. For instance, several proteins including PDLIM5, vinexin, ponsin, palladin and supervillain, which constitute core cell adhesion machinery, and hence part of the consensus adhesome [
55], displayed changes in phosphorylation in response to ANXA1. Some of the phosphorylation we observed on these proteins including those on adaptor protein vinexin (Ser412 mouse/Ser348 human) have been previously reported as adhesion-complex-specific phosphorylation sites [
56]. While the upregulation of several of these regulated phosphoproteins are known to induce formation of focal adhesion and stress fiber, the precise roles of phosphorylation on these proteins still remain unclear. Also, we observed several GAPs and GEFs with modulated phosphorylation. With predicted activity changes for PAK and ROCK1 kinases that occur downstream of Rac1 and RhoA, respectively, we posit ANXA1-mediated crosstalk between Rho and Rac GTPases that possibly drives cellular decisions on adhesion and migration. Though tyrosine phosphorylation events on adhesion proteins such as focal adhesion kinase (FAK), Src, paxillin etc. are characteristic of adhesion and integrin signaling, we did not detect these tyrosine sites in our data. This may be partly because we used whole cell lysate and not specifically isolated adhesion complexes to perform our phosphoproteome profiling, and also that we did not perform any phosphotyrosine enrichment in this study. However, it is worthy to stress that the importance of several serine/threonine phosphorylation events by far remains underappreciated in the context of adhesion signaling.
Microtubule dynamics are a key factor in controlling cell cycle and other processes such as intracellular trafficking, signaling and events leading to cell migration [
57,
58]. Through coordinated polymerization and depolymerization, the spatial and temporal organization of the microtubule network largely governs directionality and also restrains cell movement. We uncovered several phosphorylation changes on microtubule associated proteins and those that regulate its dynamics in ANXA1-deficient cells. Such a role for ANXA1 in regulating microtubule dynamics has not been established earlier to our knowledge. One of the major regulators of microtubule dynamics, stathmin, was found with elevated phosphorylation on Ser16 in ANXA1-deficient cells. The cell cycle-regulated stathmin promotes disassembly or destabilization of microtubules, and hence is turned off at the onset of mitosis to allow assembly of mitotic spindle [
59]. The “on” and “off” status of stathmin is mainly regulated by phosphorylation at Ser16 with increase in phosphorylation inhibiting its destabilizing activity [
42,
60]. Thus, in ANXA1-deficient mammary cells, we predict a decrease in microtubule depolymerization. Indeed, this process also affects disassembly of focal adhesion, and we attribute the observed increase in adhesion to such defects as well. Of note, low levels of Ser16-phosphorylated stathmin correlate with metastatic states of breast cancer [
61], and an increase in Ser16 has been shown to reduce migration in esophageal cancer cells [
62], thus underscoring its influence in limiting migration and possibly increasing adhesion. Interestingly, our kinase analysis also predicted higher activity for CAMK2, PAK and RSK2 kinases that are responsible for its phosphorylation.
Our phosphoproteome data also highlighted altered phosphorylation on proteins involved in signaling pathways including the WNT, Hippo and AMPK cascades. AMPK activation was also predicted by upstream kinase analysis, and this negative correlation was consistent with previous reports wherein ANXA1 knockdown in breast cancer cells activated AMPK [
63]. We particularly observed modulation of Hippo signaling and reduced transcriptional output via TEAD transcription factors. Loss of adherens junction deregulates Hippo signaling and hyperactivates its downstream effectors YAP/TAZ [
64]. We speculate on adhesion-induced modulation of Hippo signaling pathway in ANXA1-deficient mammary cells, wherein YAP protein levels and activity are suppressed (YAP1 levels are indeed reduced upon ANXA1 knockout [
21]) leading to reduced activation of TEAD transcription factors. Notably, the phosphorylation of a basolateral component SCRIB, the loss of which activates YAP/TAZ [
64], also displayed elevated phosphorylation in ANXA1-deficient cells. Thus we posit there is correlation between ANXA1 and Hippo signaling outcome and hypothesize that high levels of ANXA1 could hyperactivate YAP/TAZ leading to increased TEAD transcriptional output.