Wnt signaling has been linked to various types of cancers including colon, cutaneous melanoma, hepatocellular carcinoma (HCC) and breast cancer. It is also involved in metastasis as Wnt regulates cell morphology and motility. Increased Wnt ligand 5a (Wnt5a) in melanoma was correlated with increased invasiveness, cell motility and changes in morphology through changes in calcium signaling. Wnt5a has been extensively associated with proto-oncogenic cellular phenotypes. Wnt5a has been shown to act as a proto-oncogene in melanoma, breast cancer, prostate and pancreatic cancer, and a tumor suppressor in breast cancer, colon, thyroid and esophageal squamous cell carcinoma, acute lymphoblastic lymphoma, acute myeloid lymphoma, and neuroblastoma (Taciak et al., 2018).

In canonical Wnt signaling, binding of nuclear β-catenin to TCF/LEF transcription factors stimulate expression of cyclin D1 and c-MYC which alters cell cycle progression and promotes tumorigenesis in cutaneous melanoma (Taciak et al., 2018). β-catenin/TCF2 is a negative regulator of IFIT1 and IFIT2, host antiviral defense mediators through apoptosis. In colorectal cancer, IFIT2 expression is decreased which creates a pro-survival environment for cancer cells through inhibition of apoptosis. The β-catenin/TCF2 complex and down regulation of IFIT1/2 is commonly seen in colorectal cancer compared to normal tissue ( Table 1 ) (Taciak et al., 2018).

The non-canonical Wnt/PCP pathway plays an important role in tumor development through its influence on cancer metastasis. Downstream signaling of the PCP pathway induces cytoskeletal rearrangement which facilitates cellular motility (Humphries and Mlodzik, 2018; Di Bartolomeo et al., 2023). In breast cancer, fibroblast-derived exosomes promote autocrine Wnt11/PCP signaling to enhance invasiveness. The invasive breast cancer cells displayed asymmetric localization of core PCP complexes like that found in development (Humphries and Mlodzik, 2018). In addition, there is a correlation between non-canonical Wnt, proinflammatory cytokines, and epithelial-mesenchymal transition (EMT). EMT induces metastasis in various cancer types and non-canonical Wnt signaling is commonly associated with EMT due to its role in cellular differentiation (Taciak et al., 2018). Likewise, proinflammatory cytokine interleukin (IL)-8 was found to induce EMT through Wnt signaling. Macrophages can limit cancer cell division through inhibition of canonical Wnt, but this increases non-canonical pathways. In cancer cells, increased non-canonical Wnt promotes differentiation, polarization, and separation from the tumor by EMT resulting in metastasis.

E. chaffeensis repurposes Wnt signaling to evade host immune responses and promote survival. Silencing of Wnt pathway components significantly reduces E. chaffeensis bacterial load, indicating the importance of Wnt signaling during infection. TRP120 contains a repetitive Wnt SLiM mimic (QDVASH) within the tandem repeat domain (TRD) (Rogan et al., 2021; Byerly et al., 2023). E. chaffeensis TRP120 Wnt SLiM mimic binds Fzd5 and induces nuclear translocation of β-catenin to modulate transcription of downstream Wnt target genes ( Figure 2 ; Table 1 ) (Luo et al., 2016; Rogan et al., 2021). In this context, Wnt pathway activation results in cytoskeletal rearrangement and the induction of phagocytosis which contributes to ehrlichial host cell entry (Luo et al., 2016). Moreover, TRP120 has been shown to exploit the Wnt pathway to prevent autolysosome formation and allow E. chaffeensis to evade oxidative killing (Lina et al., 2017). Specifically, TRP120 binds to Wnt receptor and activates Dvl which subsequently activates the PI3K/AKT pathway and inhibits GSK3. PI3K/AKT activation inhibits negative regulator TSC2, which activates mTORC1. Activated mTOR phosphorylates TFEB, preventing nuclear translocation and subsequent upregulation of lysosomal target genes which prevents autolysosome formation (Lina et al., 2017). Additionally, canonical Wnt/β-catenin activation represses p62, an autophagy protein, as a mechanism for intracellular survival (Petherick et al., 2013). Interestingly, E. chaffeensis also activates the transcription factor nuclear factor of activated T-cells (NFAT) and initiates nuclear translocation of NFAT through the non-canonical Wnt/Ca2+ pathway. While knockdown of NFAT has been shown to significantly reduce E. chaffeensis bacterial load, the function of NFAT during infection has not been elucidated ( Table 1 ) (Luo et al., 2016; Rogan et al., 2019).

As a key regulator of the immune response and cell fate, Notch associated genes are commonly mutated in several cancers. In breast cancer, genetic mutations upregulate Notch through gain-of-function mutations in Notch1 (5-10%) and loss-of-function mutations in Numb (50%), a negative regulator of Notch (Stylianou et al., 2006; Aster et al., 2017). In T-cell acute lymphoblastic leukemia (T-ALL), loss-of-function mutations in F-Box and WD domain repeating containing 7 (FBW7) (20%) and gain-of-function mutation in Notch1 (50-60%) also result in upregulated Notch signaling (Aster et al., 2017; Sanchez-Martin and Ferrando, 2017).

In both T-ALL and breast cancer, aberrant expression of Notch promotes cell proliferation and inhibits apoptosis (Stylianou et al., 2006; Palomero et al., 2007; Aster et al., 2017; Sanchez-Martin and Ferrando, 2017; Baker et al., 2018). Downstream effects include the inhibition of the JNK and p53 pathways resulting in decreased levels of pro-apoptotic factors, Puma and Noxa (Stylianou et al., 2006). Furthermore, Notch modulates the PI3K/AKT pathway through transcriptional downregulation of PTEN, a negative regulator of the PI3K/AKT pathway, in both T-ALL and breast cancer (Palomero et al., 2007; Baker et al., 2018).

Intriguingly, downregulation of PTEN and inhibition of FBW7 have both been associated with chemotherapeutic resistance. Both PTEN and FBW7 inhibition in cancer are associated with resistance to γ-secretase inhibitors (GSI) which are used to treat breast cancer and are known to downregulate Notch (Palomero et al., 2007; Thompson et al., 2007; Baker et al., 2018; Fan et al., 2022; Chen et al., 2023). In these cancers, upregulation of Notch promotes a tumorigenic environment through inhibition of apoptosis and regulation of cell growth and proliferation which encourages further resistance to chemotherapeutics.

During E. chaffeensis infection the Notch pathway is activated by TRP120 to inhibit apoptosis and promote infection. TRP120 promotes Notch activation through three mechanisms: direct activation of Notch, degradation of negative regulators, and transcriptional upregulation of Notch genes (Lina et al., 2016; Wang et al., 2020; Patterson et al., 2022, 2023). TRP120 contains an 11 amino acid SLiM (EDEIVSQPSSE) that mimics Notch ligands thereby activating Notch signaling during infection (Patterson et al., 2022). Moreover, TRP120 contains a HECT E3 ubiquitin ligase which ubiquitinates host FBW7, a negative regulator of NICD to maintain Notch activation ( Figure 2 ) (Wang et al., 2020). FBW7 negatively regulates several oncoproteins (NICD, MCL-1, c-Jun, and c-MYC) through ubiquitination and subsequent proteasomal degradation (Wang et al., 2020). To further promote Notch activation, TRP120 binds the promoter regions of Notch1 and ADAM17 to promote transcription during infection (Lina et al., 2016). Upregulation of Notch1 promotes generation of the Notch-1 receptor while ADAM17 increases NICD S2 cleavage of the Notch receptors (Lina et al., 2016). E. chaffeensis activation of Notch inhibits PU.1, Toll-like receptor 2 and 4 (TLR2/4) expression through manipulation of ERK1/2 and p38 pathways (Lina et al., 2016). Further, X-linked inhibitor of apoptosis protein (XIAP) is sequestered and stabilized during infection due to increased cytoplasmic NICD (Patterson et al., 2023). Equally important, Notch activation leads to transcription of Notch target genes that modulate cell fate and proliferation to promote infection ( Table 1 ) (Patterson et al., 2022).

Hh signaling is involved in numerous developmental processes, and thus is implicated in various genetic diseases, including cancer. The Hh signaling pathway is known to promote tumor formation via ligand-independent or ligand-dependent mechanisms. Hh ligand-independent cancers include basal cell carcinoma, medulloblastoma (MB) and pediatric brain tumors. Mechanisms required for ligand-independent cancers involve mutations in Hh pathway components that lead to constitutive activation of smoothened (SMO) and GLI and repression of PTCH and the suppressor of fused (SUFU) (Sari et al., 2018). Hh ligand-dependent cancers such as colorectal, ovarian, breast, prostate, pancreatic and liver cancers utilize either autocrine or paracrine signaling to promote tumorigenesis whereby endogenous ligands are copiously secreted, facilitating feed-forward pathway activation. Paracrine ligand-dependent Hh signaling requires endogenous ligands to bind stromal cell PTCH receptors thereby initiating the release of growth signals such as interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), bone morphogenetic protein (BMP), and insulin-like growth factor (IGF) to promote tumor progression (Sari et al., 2018).

E. chaffeensis TRP120 engages the PTCH2 receptor through a repeated SLiM ligand mimic (NPEVLIKD) to activate Hh signaling. This activation results in nuclear translocation of GLI-1 in THP-1 cells and primary human monocytes (PHM) ( Figure 2 ) (Byerly et al., 2022). Informational spectrum method (ISM) predicted the TRP120 Hh SLiM shares sequence and functional similarity with endogenous Hh ligands. This prediction was supported by protein interaction assays which demonstrated the tandem repeat domain of TRP120 co-localizes and directly interacts with the PTCH2 receptor. Furthermore, TRP120-mediated GLI-1 nuclear translocation resulted in upregulation of key target genes that were consistent with classical Hh ligands ( Table 1 ) (Byerly et al., 2022).

During E. chaffeensis infection, Hh activation has been shown to significantly increase the expression of anti-apoptotic protein, BCL-2, thus preventing Bax-mediated cytochrome c release to maintain mitochondrial membrane integrity ( Table 1 ) (Byerly et al., 2022). This ehrlichial survival strategy blocks intrinsic cell death signals and appropriates host cell nutrients for survival and dissemination. Further, knockdown of pathway components including GLI-1, PTCH2 and SMO decreases E. chaffeensis infection. In addition, THP-1 cell treatment with an antibody against the TRP120 Hh SLiM or treatment with a TRP120 Hh SLiM mutant prevented GLI-1 nuclear translocation and subsequent pathway activation. Moreover, E. chaffeensis-infected THP-1 cells showed decreased GLI-1 nuclear translocation and increased cell death after treatment with a Hh pathway inhibitor (Vismodegib/GDC-0449), suggesting that Hh signaling plays a significant role in E. chaffeensis infection by inhibiting apoptosis (Byerly et al., 2022). This study was the first to show E. chaffeensis TRP120 SLiM-mediated Hh activation, highlighting the necessity to understand the nuances of Hh signaling which will be fundamental in defining distinct mechanisms of pathway regulation in various diseases.

Although the role of Hippo signaling in cancer is context-dependent, the pathway is typically considered tumor suppressing. Thus, inactivation of Hippo signaling and downstream activation of Yes-associated protein 1 (YAP) and WW-domain-containing transcription regulator 1 (TAZ) is common in a variety of malignancies such as breast, gastric, renal, hepatic, and hematologic cancers (Xu et al., 2019; Kyriazoglou et al., 2021; Ma et al., 2021; Noorbakhsh et al., 2021; Song et al., 2022; Wu et al., 2022; Liu et al., 2023a; Messina et al., 2023; Li et al., 2024; Yang et al., 2024). Elevated activation of YAP/TAZ is implicated in tumor initiation, metastasis, and drug resistance through mechanisms including inhibition of apoptosis and reprogramming of metabolic pathways in tumor cells (Liu et al., 2021; Fu et al., 2022). Additionally, Hippo signaling engages in crosstalk with other pathways, such as Wnt signaling (discussed above), further mediating cancer cell survival and tumor progression (Jiang et al., 2020; Noorbakhsh et al., 2021; Li et al., 2019b).

When bound to transcriptional enhanced associated domain family of proteins (TEAD 1-4), YAP/TAZ may upregulate the expression of anti-apoptotic factors such as members of the BCL-2 and inhibitor of apoptosis protein (IAP) families (Cheng et al., 2022; Zhang et al., 2022). These proteins directly inhibit apoptosis by preventing apoptotic signals such as Bax/Bak-mediated mitochondrial outer membrane permeabilization or caspase cleavage, respectively (Cetraro et al., 2022; Czabotar and Garcia-Saez, 2023). Such mechanisms have been observed in malignancies including colorectal cancer and adenocarcinoma, amongst others (Zhang et al., 2015, 2022; Jin et al., 2021). YAP/TAZ activation can also contribute to metabolic reprograming in cancer cells, specifically in the upregulation of proteins that facilitate increased glucose uptake and glycolytic flux such as GLUT1-3, phosphofructokinase and others (Liu et al., 2021). These proteins not only support cancer cell growth by elevating energy acquisition but may also contribute to maintaining an anti-apoptotic profile by positive regulation of BCL-2 family proteins (Coloff et al., 2011; Liu et al., 2014; Fang et al., 2015; Lin and Xu, 2017; Li et al., 2019a). For example, GLUT1 upregulation is a consequence of YAP activation in both breast and gastric cancer and has been correlated with BCL-xL expression in colorectal and gastric cancer (Wincewicz et al., 2007; Lin and Xu, 2017; Li et al., 2019a). Interestingly, Hippo signaling also interacts with other signaling pathways and modulates cell survival through crosstalk (Jiang et al., 2020).

A key example of Hippo pathway crosstalk is that with the Wnt pathway. Activation of Hippo signaling impedes Wnt signaling, as phosphorylated, cytoplasmic YAP/TAZ sequesters β-catenin outside the nucleus, preventing its translocation and subsequent upregulation of Wnt target genes (Imajo et al., 2012). Therefore, it is unsurprising that some Wnt ligands, notably Wnt3a and Wnt5a, can inactivate Hippo signaling through Fzd5 to ensure successful β-catenin nuclear translocation (Park et al., 2015). The crosstalk between these pathways is particularly relevant in cancer, as Hippo inactivation and Wnt activation are common mechanisms of cancer cell survival (Jiang et al., 2020; Noorbakhsh et al., 2021; Li et al., 2019b; Andl and Zhang, 2017). YAP activation is essential for β-catenin function in a variety of cancers including melanoma, hepatoblastoma, colon cancer, and breast cancer, and a screen of 85 cancer cell lines determined that those driven by β-catenin were dependent on YAP (Rosenbluh et al., 2012; Tao et al., 2014; Liu et al., 2019; Quinn et al., 2021). Additionally, the latter study also concluded that β-catenin and YAP act as transcriptional coregulators, forming a complex that upregulates gene expression of anti-apoptotic proteins, survivin and BCL2L1 (Rosenbluh et al., 2012). Hippo and Wnt signaling are therefore inextricably linked and possess a great deal of influence over cell proliferation and survival. Intriguingly, co-option of the crosstalk between these two pathways has been observed during infection of host cells by E. chaffeensis, one of the few bacterial pathogens associated with Hippo signaling (Byerly et al., 2023).

The role of Hippo signaling in bacterial infection is critically understudied for such a ubiquitous and influential signaling pathway. Interestingly, one of the best examples of Hippo pathway involvement in bacterial infection is E. chaffeensis, which is extensively demonstrated to inactivate Hippo through SLiM-icry. In fact, E. chaffeensis uses the same SLiM to both activate Wnt signaling and inactivate Hippo signaling, taking advantage of the crosstalk between these pathways. By inactivating Hippo signaling, E. chaffeensis ensures the efficacy of Wnt signaling activation while promoting YAP-mediated anti-apoptotic gene expression in host cells ( Figure 2 ) (Byerly et al., 2023).

Motivated by the role of Wnt in E. chaffeensis pathology, and the existence of Wnt/Hippo crosstalk, YAP activation was investigated in a cell culture model of E. chaffeensis infection. In E. chaffeensis-infected cells, YAP is activated and translocates to the nucleus, where it upregulates a diverse panel of target genes. YAP activation during E. chaffeensis infection was attributed to the TRP120 Wnt SLiM, suggesting this sequence is responsible for Hippo inactivation in addition to activation of Wnt signaling. Furthermore, in Fzd5 knockout cells, YAP is not activated by E. chaffeensis or the TRP120 Wnt SLiM (Byerly et al., 2023). Finally, TRP120 ubiquitinates adenomatous polyposis coli (APC), a negative regulator of YAP and β-catenin, targeting it for degradation ( Figure 2 ; Table 1 ) (Byerly et al., 2024). Taken together, these findings demonstrate that E. chaffeensis inactivates Hippo signaling through the same mechanism as Wnt signaling activation (Byerly et al., 2023).

As described above, inactivation of Hippo signaling may contribute to excessive cellular survival and metabolic reprogramming through YAP-mediated genetic regulation. Notably, this phenomenon is also observed in E. chaffeensis-infected cells. Hippo inactivation by E. chaffeensis is critical for pathogen survival as knockdown of YAP and TEAD family transcription factors significantly decreases infection (Byerly et al., 2023). Both E. chaffeensis and TRP120 Wnt SLiM significantly increase expression of GLUT1, while GLUT1 knockdown significantly decreases infection, suggesting this metabolic protein is crucial for maintaining infection ( Table 1 ). Further investigation revealed that BCL-xL levels increase while Bax levels decrease in response to infection and the TRP120 Wnt SLiM, and this result is abrogated by treatment with Verteporfin, a YAP inhibitor (Liu-Chittenden et al., 2012; Byerly et al., 2023). Verteporfin also significantly decreases bacterial load and cell viability in infected cells, and significantly increases caspase activation, indicating an increase in apoptosis. Collectively, these results illustrate that E. chaffeensis inactivates Hippo signaling to engage the YAP-GLUT1-BCL-xL axis and establishes an anti-apoptotic profile in host cells, a mechanism like that observed in multiple cancers (Byerly et al., 2023).