Targeting the Hippo Pathway for Breast Cancer Therapy

Breast cancer (BC) is one of the most prominent diseases in the world, and the treatments for BC have many limitations, such as resistance and a lack of reliable biomarkers. Currently the Hippo pathway is emerging as a tumor suppressor pathway with its four core components that regulate downstream transcriptional targets. In this review, we introduce the present targeted therapies of BC, and then discuss the roles of the Hippo pathway in BC. Finally, we summarize the evidence of the small molecule inhibitors that target the Hippo pathway, and then discuss the possibilities and future direction of the Hippo-targeted drugs for BC therapy.

Targeted Treatment of Breast Cancer
BC is the most frequently diagnosed cancer among females, accounting for 25% of all cancer cases worldwide [1]. Over the past decades, despite substantial efforts made to improve the survival and quality of life, BC remains a deadly threat for patients. For most types of BC, treatment involves surgery, radiation therapy, hormone therapy, chemotherapy, and the latest targeted therapy [2]. Up to the present day, multiple targeted drugs have been approved in the treatment of BC by FDA as illustrated in Table 1, including inhibitors of estrogen receptor (ER), aromatase, cyclin-dependent kinase (CDK) 4/6, mTOR (rapamycin), and poly(ADP-ribose) polymerase (PARP), and epithelial growth factor receptor (EGFR) and human epithelial growth factor receptor 2 (HER2)-targeted agents [3]. Additionally, studies about the antitumor effects of many other inhibitors such as inhibitors of vascular endothelial growth factor (VEGF), farnesyl transferase, and fibroblast growth factor receptor (EGFR) also show a promising future and have caught attentions as well [4].
However, current treatment has its own limitations. First of all, BC subtypes show different responses to systemic therapy, suggesting the treatment should be more specific for each patient [5,6]. By detecting the expression levels of ER and progesterone receptor (PgR), as well as the HER2 and Ki-67, BC were identified into categories with similar clinical implications, including Luminal A, Luminal B, HER2-positive, and triple-negative breast cancer (TNBC) subtypes [7]. Among them, TNBC shows a relatively poor prognosis, while the Luminal and HER2-positive subtypes respond sensitively to endocrine therapy and targeted therapy [8]. Secondly, drug resistance, especially to monotherapy, has limited the curative efficacy, resulting in a low response rate. Thirdly, the lack of reliable indicators for most of the targeted agents makes it a challenge to select doses and predict the prognosis of patients [9]. Therefore, identifying novel breast cancer therapeutic targets, revealing the mechanisms underlying drug resistances, discovering new biomarkers and developing rational combinations of targeted therapy remain urgent and important. Competitively inhibits the binding of estradiol to ER, resulting in a reduction in DNA synthesis and cellular response to estrogen [10] Fulvestrant Binds competitively to ER, resulting in ER deformation and decreased estrogen binding [11] Toremifene Chemically related to tamoxifen, binds competitively to ER [12] Aromatase Anastrozole Selectively binds to and reversibly inhibits the enzyme aromatase, which catalyzes the final step in estrogen biosynthesis and may result in growth inhibition of estrogen-dependent breast cancer cells [13] Exemestane Binds irreversibly to and inhibits aromatase [14] Letrozole Selectively and reversibly inhibits aromatase [15] HER2 Trastuzumab Binds to HER2 on the tumor cell surface, induces an antibody-dependent cell-mediated cytotoxicity against tumor cells that overexpress HER2 [16] Pertuzumab Binds to the dimerization domain of the HER2, therefore prevents the activation of HER signaling pathways, resulting in tumor cell apoptosis [9] Ado-trastuzu-mab emtansine The maytansinoid DM conjugated to the HER2-targeting transtuzumab is released and binds to tubulin, thereby inhibiting cell division and the proliferation of cancer cells that overexpress HER2 [17] EGFR, HER2 Lapatinib Selectively inhibits both EGFR and HER2 tyrosine kinases [18] Neratinib maleate Binds to and inhibits both HER2 and EGFR [19] mTOR Everolimus Binds to the immunophilin FKBP-12 to generate an immunosuppressive complex that binds to and inhibits the activation of the mammalian Target of Rapamycin (mTOR) [20] CDK4/6 Palbociclib Selectively inhibits CDK4 and CDK6, thereby inhibiting Rb protein phosphorylation, which suppresses DNA replication and decreases tumor cell proliferation [21] Ribociclib Specifically inhibits CDK4/6 [22] Abemaciclib Specifically inhibits CDK4/6 [23] PARP Olaparib Selectively binds to and inhibits PARP and PARP-mediated repair of single strand DNA breaks [24] ER, Estrogen receptor; HER2, Human epidermal growth factor receptor 2; EGFR, epithelial growth factor receptor; FKBP-12, FK Binding Protein-12; CDK4/6, Cyclin-dependent kinase 4 and 6; Rb, retinoblastoma.

The Hippo Pathway
The Hippo pathway, named after the Drosophila Hpo kinase, is a highly conserved signal transduction pathway that plays important roles in organ size control, tissue regeneration, immune response, stem cell function and tumor suppression [25][26][27]. In mammals, the canonical Hippo pathway consists of four core components that function through phosphorylation: mammalian sterile 20-like kinase (MST; homolog of Drosophila Hpo), large tumor suppressor (LATS) kinases, scaffold proteins Salvador homolog 1 (SAV1) and Mps One Binder kinase activator protein 1 (MOB1) ( Figure 1). In certain conditions such as high cell density, extracellular matrix stiffness and lack of nutrients, the Hippo pathway is activated, with MST and LATS successively phosphorylated with the support of SAV1 and MOB1 [26]. Then, the activated LATS phosphorylates transcriptional co-activator Yes-associated protein (YAP) and its paralog transcriptional coactivator with PDZ-binding motif (TAZ), which prevent TAZ/YAP from entering the nucleus by anchoring them to 14-3-3 protein and/or promoting their degradation in the cytoplasm ( Figure 1) [28,29]. This interrupts their interactions with the transcription factor TEA domain (TEAD) family proteins, which subsequently inhibits cell proliferation and oncogenic transformation and induces apoptosis. Conversely, the dysregulation of the Hippo pathway increases the nuclear functions of TAZ/YAP, leading to active gene expression [30,31], such as several growth-promoting factors, including secretory proteins connective tissue growth factor (CTGF) and CYR61 [32,33], AXL receptor tyrosine kinase [34], c-myc and survivin [35].
promoting their degradation in the cytoplasm ( Figure 1) [28,29]. This interrupts their interactions with the transcription factor TEA domain (TEAD) family proteins, which subsequently inhibits cell proliferation and oncogenic transformation and induces apoptosis. Conversely, the dysregulation of the Hippo pathway increases the nuclear functions of TAZ/YAP, leading to active gene expression [30,31], such as several growth-promoting factors, including secretory proteins connective tissue growth factor (CTGF) and CYR61 [32,33], AXL receptor tyrosine kinase [34], c-myc and survivin [35]. Main components of the Hippo pathway and the current Hippo-targeted inhibitors discussed in this review. In mammals, the canonical Hippo pathway consists of four core components that function through phosphorylation: MST, SAV1, LATS, MOB1. Activated LATS phosphorylates YAP/TAZ, preventing them from entering the nucleus by anchoring them to 14-3-3 protein and/or promoting their degradation in the cytoplasm. This interrupts their interactions with the transcription factor TEAD family proteins, which subsequently inhibits cell proliferation and oncogenic transformation and induces apoptosis. Besides, current Hippo-targeted inhibitors discussed in this review, as well as their targets and major mechanisms, are shown in the figure.
Aside from TAZ/YAP-TEAD interaction, TAZ/YAP can also regulate transcription mediated by RUNX, SMADs, TP73, NKX2.1, OCT4 and PPARγ. When the Hippo pathway engages in crosstalk such as with Wnt, TGFβ, Notch and PI3K, the functions of TAZ/YAP are further stimulated [30,32]. With increasing studies, many regulators of TAZ/YAP have been identified in addition to the core Hippo pathway components. For example, TAZ/YAP activity can be regulated in a LATSindependent way, by binding to Angiomotin (AMOT) family proteins, ZO-1/2, α-catenin, β-catenin, PTPN14 and Scribble [36]; the receptor tyrosine kinase EphA2 could activate TAZ/YAP through Rho-ROCK signaling [37]. In this era of targeted therapy, the Hippo pathway appears to be a promising target for the treatment of breast cancer. Here, we summarize the current evidence to demonstrate the mechanisms beneath and provide an overview of the current development of Hippo-targeted therapy for breast cancer. Main components of the Hippo pathway and the current Hippo-targeted inhibitors discussed in this review. In mammals, the canonical Hippo pathway consists of four core components that function through phosphorylation: MST, SAV1, LATS, MOB1. Activated LATS phosphorylates YAP/TAZ, preventing them from entering the nucleus by anchoring them to 14-3-3 protein and/or promoting their degradation in the cytoplasm. This interrupts their interactions with the transcription factor TEAD family proteins, which subsequently inhibits cell proliferation and oncogenic transformation and induces apoptosis. Besides, current Hippo-targeted inhibitors discussed in this review, as well as their targets and major mechanisms, are shown in the figure.
Aside from TAZ/YAP-TEAD interaction, TAZ/YAP can also regulate transcription mediated by RUNX, SMADs, TP73, NKX2.1, OCT4 and PPARγ. When the Hippo pathway engages in crosstalk such as with Wnt, TGFβ, Notch and PI3K, the functions of TAZ/YAP are further stimulated [30,32]. With increasing studies, many regulators of TAZ/YAP have been identified in addition to the core Hippo pathway components. For example, TAZ/YAP activity can be regulated in a LATS-independent way, by binding to Angiomotin (AMOT) family proteins, ZO-1/2, α-catenin, β-catenin, PTPN14 and Scribble [36]; the receptor tyrosine kinase EphA2 could activate TAZ/YAP through Rho-ROCK signaling [37]. In this era of targeted therapy, the Hippo pathway appears to be a promising target for the treatment of breast cancer. Here, we summarize the current evidence to demonstrate the mechanisms beneath and provide an overview of the current development of Hippo-targeted therapy for breast cancer.

The Roles of the Hippo Pathway in Breast Cancer
In 1999, St John et al. discovered that mice lacking Lats1, a mouse homology of Drosophila lats, display pituitary hyperplasia and develop tumors [38]. Later our studies provided evidence that LATS is a tumor suppressor in human cancer cells [39]. Since then many studies support the role of the Hippo pathway as a tumor suppressor pathway in diverse human cancers, including breast cancer [40,41]. In the following text, we will discuss how each component of the core Hippo pathway is involved in the tumorigenesis and metastasis of breast cancer.

YAP and TAZ
High YAP are more common in BC lacking functional adherens junctions [40]. The expression status of YAP is also associated with the molecular subtypes, tumoral and cellular components of BC, and could be a prognostic marker for patents, pointing to an oncogenic role [42]. YAP overexpression enhances multiple processes for tumorigenesis and metastasis in BC cells, including cellular proliferation, transformation, migration, and invasion [43]. Previous studies reported that overexpression of YAP in human non-transformed mammary epithelial cells induces epithelial-to-mesenchymal transition (EMT), suppression of apoptosis, growth factor-independent proliferation, and anchorage-independent growth on soft agar [44].
Besides, the divergent tumor-suppressive roles of YAP have also been recognized [41,[45][46][47][48][49][50][51], which could be explained by many reasons. YAP exists in two major isoforms (YAP1 and YAP2) that may have their own transcriptional targets. Therefore, the relative levels of those two isoforms might decide whether YAP is acting as a tumor suppressor or otherwise in BC cells [52,53]. In addition, YAP1 can translocate to the nucleus and associate with tumor suppressor p73, resulting in apoptosis through transcriptional activation of the proapoptotic gene puma [46]. Another explanation for the tumor-suppressive role of YAP is that deregulated TAZ/YAP activity in BC cells induces an anti-tumorigenic immune surveillance response, ultimately leading to the eradication of tumor cells so BC cells have to restrain YAP activity consequently [54,55]. Moreover, studies reported that YAP can bind and signal through anti-apoptotic protein (delta)∆Np63 isoform to protect cancer cells from DNA damage. Therefore, it is possible that only in certain conditions like DNA damage, YAP can selectively induce p73-mediated apoptosis [56,57]. Additionally, more investigations considering different intrinsic subtypes of BC and stem cells should be done to explain the dramatic effects of YAP [41].
TAZ has also been identified as an oncogene that plays a critical role in the migration, invasion, and tumorigenesis of BC cells [58,59]. It is conspicuously overexpressed in human breast cancer tissues from patients in which its expression levels generally correlate with the TNBC diagnosis [60] and patient prognosis [41]. Overexpression of TAZ in low-expressing MCF10A non-tumorigenic mammary cells leads to the acquisition of a spindle-shaped morphology and increases migratory and invasiveness [58], while the depletion of TAZ inhibits cell growth in 184A1 and HCC1937 breast cancer cells [61] and retards the anchorage-independent growth on soft agar and tumorigenesis in nude mice in MCF7 cells [58]. Additionally, TAZ has been implicated in BC-associated metastatic bone disease, partly through its interaction with hypoxia inducible factor-1α [62]. Recent studies show that TAZ is required for sustaining self-renewal, tumor-initiation capacities [63], and metastatic activity [59] in BC stem cells (BCSCs). The connection between TAZ and BCSCs has been correlated with its interaction with established inducers of the cancer stem cell phenotype such as hypoxia-inducible factor 1 (HIF1) and extracellular cues [64][65][66].
YAP/TAZ act as central players of multiple cancer-associated features such as proliferation and cell survival, migration and metastasis [41], and the tumor-initiating functions. All of these functions rely on their interaction with TEAD transcriptional factors. TAZ/YAP-TEAD complexes directly promote the expression of many oncogenic factors that contribute to BC progression [43], such as cysteine-rich angiogenic inducer 61 (CYR61) and connective tissue growth factor (CTGF) (also known as CCN1 and CCN2, respectively) [32,67,68]. TAZ-TEAD can activate BMP4, which will enhance signaling downstream of TAZ, and then promote Smad1/5 intracellular signaling and cell migration [69]. YAP-TEAD also could control receptor for hyaluronan-mediated motility (RHAMM) transcription leading to ERK activation and cancer metastasis by binding to RHAMM promoter at specific sites [70]. Glutamine metabolism is critical to many tumor cells including BC cells, and it could be regulated by TAZ/YAP. Through the increased expression of downstream genes SLC1A5 and GLS, TAZ/YAP could promote glutamine uptake and therefore upregulate the amount of intracellular glutamine [37]. Besides TEADs, TAZ/YAP can bind to other transcriptional factors, such as the krueppel-like factor 5 (KLF5) and transforming growth factor β (TGFβ)-activated SMAD2/3. The overexpression of YAP could upregulate KLF5 protein levels and mRNA expression levels of its downstream target genes including FGFBP1 and ITGB2 that promote BC cell proliferation and survival [71]; the interaction between TAZ/YAP and SMAD2/3 regulates novel targets such as NEGR1 and UCA1 that are necessary for tumorigenic activity in metastatic BC cells [68]. YAP function is also required for cancer-associated fibroblasts (CAFs) to promote matrix stiffening, cancer cell invasion and angiogenesis [72].

Other Components
The upstream components of the Hippo pathway ( Figure 1) were found to be tumor suppressors in human breast cancer, and their functions are not limited through the inactivation of TAZ/YAP. In human BC, the downregulation of MST has been identified to be a predictable biomarker for prognosis [73]. MST regulates a diverse array of substrates in addition to the core Hippo pathway components such as LATS, SAV1 and MOB1. Studies have shown that MST also target histone H2B, FOXO, GA-binding protein (GABP) and LATS-related kinases Ndr1/Ndr2 [74][75][76][77], pointing to a tumor-suppressive role.
Clinical evidence has shown reduced expression of LATS in human BC, and functional studies show that overexpression of LATS1 can modulate CDC2 kinase activity and induce pro-apoptotic Bax expression, which causes G2/M cell cycle arrest and induction of apoptosis [78]. Additionally, LATS1 can interact with actin, and Zyxin and LIMK1 [79,80], two regulators of actin filament assembly, regulating actin polymerization [81]. Besides, LATS could phosphorylate angiomotin (AMOT) and thus inhibit cell migration in vitro and angiogenesis [82]. More recently, a direct interaction between LATS and ERα signaling was identified, suggesting that in the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation, which is a novel non-canonical effect of LATS in the regulation of human breast cell fate [83].
Together, these studies showed that the Hippo pathway is involved in the development of human BC through diverse mechanisms, thus could be a therapeutic target of BC. The therapeutic strategy could be divided into two directions: to inhibit the TAZ/YAP-TEAD interaction, or to up-regulate the upstream components and regulators (Figure 1), since TAZ/YAP activity is mainly governed by LATS kinases [84].

Resistance to Chemotherapy
Our laboratory for the first time identified TAZ as a novel gene target responsible for drug resistance in BC. Enhanced levels of TAZ render resistance of mammary epithelial cells to chemotherapeutic drug Taxol (paclitaxel) through the downstream activation of CYR61/CTGF promoters, while TAZ knockdown in TAZ-high/drug-resistant MDA-MB231 BC cells turned them sensitive to Taxol [33]. Later we found that TAZ phosphorylation by CDK1 sensitizes BC cells to antitubulin drugs, suggesting a possible novel target for the treatment of antitubulin-resistant cancers [85]. Moreover, TAZ-expressing BC cells and stem cells were reported to be more resistant than control groups to two widely used chemotherapeutic drugs: doxorubicin and paclitaxel [63,86].
Previous study revealed that the expression of YAP could protect BC cells from chemotherapeutic agents Taxol and cisplatin [44]. Recent evidence suggested that YAP resistance to antitubulin drugs is modulated by a Hippo-independent pathway, since antitubulin drugs activate CDK1 and then YAP is phosphorylated on five sites independent of the Hippo pathway [87]. This result suggests YAP and its phosphorylation status to be novel prognostic predictor for antitubulin treatment for BC patients. Besides, low levels of LATS2 mRNA could be a predictor for favorable response to epirubicin plus cyclophosphamide in breast cancers [88]. This role of LATS2 may be explained by the disruption of the checkpoint function at the G1/S phase induced by down-regulation of LATS2.

Resistance to Targeted Therapy
Several targeted drugs have been approved for the treatment of BC, but the effectiveness varies greatly because of innate and acquired resistance. Current study revealed that the expression levels of TAZ could predict the response to trastuzumab and chemotherapy in Luminal B and HER2-positive BC patients [89]. Besides, in HER2-positive BC cells, TAZ/YAP have been discovered to play a role in the resistance to EGFR/HER2 inhibitor lapatinib by improving the matrix rigidity via the mechanotransduction arm of the Hippo pathway. In HER2-positive BC tumor xenograft mouse model, YAP inhibition increases the sensitivity to lapatinib, suggesting targeting matrix stiffness could be an adjuvant strategy for treating drug-resistant patients [90,91].

Resistance to Endocrine Therapy
Over two-thirds of breast cancer patients express ERα and respond to ERα antagonists (e.g., tamoxifen and fulvestrant), or drugs that reduce ER ligand estrogen (e.g., letrozole). However, resistance to endocrine therapy occurs frequently and the prognosis of patients does not meet the expectations [92]. Phosphorylation of ERα is one of the mechanisms associated with resistance to endocrine therapy, and LATS2 has been demonstrated to activate ERα transcription. LATS2 co-localizes with ERα in the nucleus, and thus contributes to the resistance to tamoxifen and other ER antagonists in ER+ breast cancer [93].

MST and LATS Activation
As mentioned before, MST and LATS are crucial kinases in the Hippo pathway, and they are frequently found to be hypermethylated in BC. Although up till the present moment no direct agents to activate MST and LATS has been discovered, but those indirect activators of MST and LATS still have the potentialities to be targeted drugs for breast cancer cells.
ISIS 5132 is an antisense oligonucleotide designed to hybridize to c-Raf mRNA ( Table 2) [94], and Raf-1 was recently found to be an upstream regulator of MST2 [95] (Figure 1). By sequestering MST2 into an inactive complex, Raf-1 could inhibit the apoptosis of BC cells. Preclinical data for ISIS 5132 showed anti-tumor effects in breast cancer and other solid xenograft mouse models, but the agent was withdrawn because of the failure in Phase II clinical trials in patients with colorectal, ovarian or prostate cancer [96]. However, the possibility of ISIS 5132 for the treatment of BC shall need further investigation.
Likewise, since F-actin is the inhibitor of MST/LATS, then the negative regulators of F-actin can indirectly activate MST/LATS activity [97]. The marine-derived macrolides latrunculin are known to reversibly bind actin monomers, disrupting their polymerization. Studies have demonstrated the anti-proliferative and anti-invasive effects of latrunculin in BC cell lines (Table 2) [98][99][100]. Besides, Y27632 could indirectly activate MST/LATS through the inhibition of Rho-associated, coiled-coil containing protein kinase (ROCK) (Figure 1) [101], making itself another targeted agent for BC. Furthermore, anti-Rho siRNAs also could inhibit the proliferation and invasiveness of BC cells in vitro and in vivo [102].
Additionally, statins can also activate LATS through Rho inhibition by suppressing HMG-CoA reductase activity (Figure 1; Table 2) [103]. A case control study suggests that the use of statins is associated with a 51% risk reduction of BC after controlling for age, smoking, alcohol use and diabetes [104]. In a new research, phosphatidic acid (PA) was identified to be a key player in the Hippo pathway, mainly by binding to LATS and NF2 (Figure 1). Therefore, inhibitors of PA and its regulator phospholipase D (PLD) could suppress the oncogenic function of YAP. In MDA-MB-231 cells, PLD inhibitor CAY10594 (Table 2) could suppress cell viability and cell migration in vitro, while another PLD inhibitor FIPI could suppress BC xenograft tumor growth [105]. QLT0267, an integrin-linked kinase (ILK) inhibitor, reduces BC cell growth by activating MST [106]. Moreover, QLT0267 can combine with docetaxel to enhance cytotoxicity, reduce phosphorylated AKT (pAKT) levels, alter F-actin architecture and improve treatment outcomes in an orthotopic BC tumor xenograft mouse model [107].

Targeting TAZ/YAP Regulators
The SRC kinase inhibitor dasatinib is able to inhibits YAP1 nuclear localization and stabilization by reducing Yes-mediated YAP1 phosphorylation or by increasing YAP1 phosphorylation mediated by SRC-PI3K-LATS [108]. Since it suggests promising sensitivity in TNBC cell lines, a Phase II trial examined the efficacy and safety of single-agent dasatinib in unselected patients with advanced TNBC [109]. However, the result showed the effect of single-agent dasatinib is quite limited, so future studies shall investigate other therapeutic settings, such as chemotherapy combinations.
Auranofin was originally used to cure rheumatoid arthritis but recent studies have revealed its antitumor effects [110]. In lung and ovarian cancer, a PKC-AMOT-YAP axis was revealed and auranofin thus could inhibit YAP through AMOT by inhibiting PKC (Figure 1; Table 2) [111]. In breast cancer cells, auranofin induces apoptosis. However, the effect of auranofin could also be explained by prolonged elevation of calcium so further experiments are needed [112].
Additionally, studies showed that taxol also can inactivate TAZ/YAP by activating CDK1 [85,87]. In addition, Bromodomain-containing protein 4 (BRD4), a chromatin-binding protein [113], has been reported to be able to regulate YAP/TAZ transcriptional activity. A potent BRD4 inhibitor named BAY1238097 was tested in human TNBC cell lines and showed satisfying antitumor effect, which might offer new perspectives on the treatment of TNBC patients through modulation of YAP/TAZ ( Figure 1; Table 2) [114]. Moreover, energy stress induced by metformin can activate AMP kinase, which directly phosphorylates AMOTL1 and consequently promotes YAP activity in a LATS-independent way ( Figure 1; Table 2) [115,116]. A short-term clinical trial in patients with breast cancer showed that, the tumor associated antigen CA15-3 significantly decreased after metformin treatment. However, since metformin may affect breast cancer in other ways as systemic changes in insulin metabolism, more trials should be performed in the future [117].

Inhibition of YAP/TAZ-TEAD Interaction
Through a screen using a luciferase reporter for TEAD response element, verteporfin (VP) was identified to be able to disrupt the interaction between YAP and TEADs ( Figure 1; Table 2) in vitro and in vivo [57]. Later, similar study confirmed that VP acts as a potential inhibitor of TAZ/YAP-driven signaling and tumorigenicity in BC [118]. A recent research explored the possibility to combine VP and paclitaxel treatment for patients with TNBC, and the result suggested that both agents are capable of eliminating BC cells and do not interfere with each other [119]. There is an ongoing Phase II clinical trial of continuous low-irradiance photodynamic therapy (CLIPT) using VP for cutaneous BC patients and so far, the null hypothesis of RR ≤ 5% has been rejected [120]. However, VP itself may not be a very promising Hippo-targeted drug for BC treatment due to difficulty for large-scale synthesis, low solubility and stability, and Hippo-independent effects [121,122].
Besides VP, the other members of the porphyrin family, such as hematoporphyrin and protoporphyrin IX are both currently identified as disruptors of YAP-TEAD interaction in xenograft mouse models [123], which could be the next candidates for BC treatment. In addition, current in vitro studies found that tankyrase inhibitor XAV939 could suppress YAP-TEAD transcriptional activities by maintaining the stabilization of AMOT (Figure 1; Table 2) [124,125], while another study revealed XAV939 could reduce tumorsphere formation in TNBC model by suppressing Wnt pathway [126]. Thus, the mechanism of how XAV939 affects BC cells still need more investigations.
Previous studies also show that in BC cells thiazovivin, dasatinib, lovastatin, cucurbitacin I, and pazopanib inhibited YAP-TEAD interaction by changing the nuclear localization of YAP (Figure 1). Among these drugs, dasatinib, statins, and pazopanib are approved as clinically used drugs (Table 2). Therefore, more researches shall be done focusing on those agents. It is notable that pazopanib can also induce proteasomal degradation of TAZ/YAP by the ubiquitin-proteasome system [127]. Additionally, cyclic YAP-like peptides have been designed to occupy the interface 3 on TEAD, which disrupts YAP-TEAD interaction and proves its therapeutic potency [128]. These and the previously mentioned ones are listed in Table 2.

Ongoing Challenges
There is no doubt that the Hippo pathway plays a critical role in cancer development and therefore presents a promising target for the treatment of BC. However, some ongoing challenges remain urgent and unresolved. First, the exact effects of pathway crosstalk and signaling circuitry on therapeutic outcomes are unknown. For example, a relationship between the Hippo pathway and the Wnt pathway has been discussed for a long time, as the Hippo pathway can restrict or activate the Wnt pathway under certain conditions, while CD44, a target of the Wnt pathway, could interact with the Hippo upstream regulator NF2 and therefore activate the Hippo pathway [129,130]. The Hippo pathway is known to engage other pathways such as TGF-β [131], Ras [132], Hedgehog [133], Notch [134] signaling, so the underlying mechanisms require more investigations. Second, it is clear that the Hippo pathway can serve as a therapeutic target for BC patients, but more experiments and clinical trials shall be conducted to get the data about the sensitivity and the response rate of the Hippo-targeted agents, and to identify reliable biomarkers to predict drug responses. Our lab established the first LATS biosensor that could monitor LATS activity in real-time with high sensitivity non-invasively [135]. Since the system could work in vitro and in vivo, it could be used to examine the effects of the potential targeted agents for BC in mice. Third, we shall discover more novel targets and corresponding inhibitors. For example, PP1A phosphatase was identified to be cable to antagonize the function of LATS, and thus regulate the reversible activation of TAZ, suggesting that PP1A phosphatase inhibitor may acts as an antitumor agent [136]. Additionally, no Hippo-targeted agent has been approved to clinical use for breast cancer yet, so to find out their doses and combination strategies, we still have a long way to go.

Conclusions and Future Directions
The past decade has witnessed the raising time of the Hippo pathway and the era of targeted therapy. From all the studies mentioned, the Hippo pathway represents both opportunities and challenges for the treatment of breast cancer. In the future, it might be urgent and meaningful to develop drugs that directly target Hippo components (e.g., LATS and YAP/TAZ) or dissociate the TAZ/YAP-TEAD interaction. These drugs can be used alone or combined with other therapeutic drugs (e.g., chemotherapy, target therapy, and immunotherapy) for more effective treatment of drug-resistant or/and metastatic BC in the future.
Author Contributions: L.W. and X.Y. wrote and revised the review.


extfieldsofstudy: Chemistry Medicine

text:
Tumor-derived mesenchymal-stem-cell-secreted IL-6 enhances resistance to cisplatin via the STAT3 pathway in breast cancer

Cisplatin is used for the treatment of a range of solid malignant tumors; however, with prolonged treatment durations, the sensitivity of tumor cells to the drug decreases owing to an unclear mechanism of drug resistance. The present study aimed to investigate whether breast-cancer-tissue-derived mesenchymal stem cells (BC-MSCs) are involved in mediating the effects of cisplatin on breast cancer cells, and which component of the BC-MSC conditioned medium (BC-MSC-CM) exhibited an anti-apoptotic effect. Cytokines/chemokines in BC-MSC-CM were quantified using a Luminex immunoassay, and reverse transcription-quantitative polymerase chain reaction analysis detected interleukin-6 (IL-6) levels in MCF-7 cells following different treatments. MTT and flow cytometry analysis measured cell vitality and apoptosis, respectively, and activation of signal transduced and activator of transcription 3 (STAT3) was evaluated by western blotting. BC-MSCs reversed the pro-apoptotic effect of cisplatin and enhanced the proliferation of MCF-7 cells more potently than bone-marrow-derived MSCs. Further study revealed that BC-MSCs secreted IL-6 to protect MCF-7 cells from apoptosis and promote their survival. Neutralizing IL-6 with a specific antibody partially inhibited the IL-6/STAT3 signaling pathway and reversed the promoter role of BC-MSCs in MCF-7 cells. Taken together, the findings of the present study indicated that BC-MSCs decreased the level of cisplatin-induced apoptosis in MCF-7 cells by activating the IL-6/STAT3 pathway in cancer cells. BC-MSCs, as important cells in the tumor microenvironment, have a key role in the treatment of breast cancer.

Introduction
Breast cancer is the leading cause of cancer-associated mortality among women worldwide (1). Although there are a range of therapeutic methods available to treat breast cancer, including surgery, chemotherapy, endocrine therapy, radiation and targeted therapy, there remains a large proportion therapeutic failures, subsequently resulting in cancer recurrence and metastasis (2).
Cisplatin, a platinum-containing antineoplastic drug, is a chemotherapeutic agent used to treat a range of solid malignant tumors, including ovarian cancer, breast cancer, gastric carcinoma and rectal carcinoma (3,4). Previous studies have revealed that cisplatin exerts its anticancer effects by activating the apoptotic pathway and inducing cell death (5)(6)(7). Apoptosis is the main response induced in tumor cells upon treatment chemotherapeutic agents (8). Cisplatin is an antitumor agent with a high rate of success in treating tumors; however, resistance to this therapy can develop. Evidence indicates the involvement of the tumor microenvironment (TME) in acquisition of chemoresistance (9,10). The TME influences the growth of tumors and affects the outcome of chemotherapy (11,12). Whether the TME has a role in cisplatin-triggered chemoresistance in breast cancer is unclear.
The TME is a dynamic system that consists of complex non-malignant cells, extracellular matrix (ECM) and signaling molecules that communicate with cancer cells. The non-malignant cells include fibroblasts, endothelial cells and immune cells that, together with the surrounding ECM, affect tumor activity. Mesenchymal stem cells (MSCs), one of the pivotal components of the TME, are a focus of research in the progression of tumors (13,14). Notably, there is evidence to suggest that MSCs release cytokines and growth factors to influence the behavior of tumor in a paracrine manner (15)(16)(17). In particular, owing to the influence on the tumor itself and the inflammatory TME, tumor-tissue-derived MSCs are widely studied (14,18). Previous studies have demonstrated that tumor progression, enhancement of metastatic potential, and resistance to chemo-and radiotherapy may all be attributed to MSCs (19)(20)(21). Whether breast-cancer-tissue-derived mesenchymal stem cells (BC-MSCs) are able to promote chemoresistance of cisplatin requires further investigation.
In the present study, it was revealed that BC-MSCs enhanced the proliferation of breast cancer cells and decreased Tumor-derived mesenchymal-stem-cell-secreted IL-6 enhances resistance to cisplatin via the STAT3 pathway in breast cancer cisplatin triggered apoptosis in MCF-7 cell. Interleukin-6 (IL-6) was released by BC-MSCs, which mediated a reduction in apoptosis induced by cisplatin by activating the signal transducer and activator of transcription 3 (STAT3) signaling pathway. This study revealed a novel mechanism of drug resistance of cisplatin.

Materials and methods
Cells and reagents. The human breast cancer MCF-7 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO 2 atmosphere in a humidified incubator. Breast cancer tissues were obtained from female patients (n=12) aged 40-60 years (mean 52 years), who underwent surgical operation in the First People's Hospital of Lianyungang from April 2015 to July 2015 (Jiangsu, China), all patients provided written informed consent to participate in the study and all experiment protocols were approved by the Medical Ethics Committee of First People's Hospital of Lianyungang. The cell isolation procedure was typically undertaken within 4 h of tumor removal. In brief, fresh tissue specimens were collected, washed with PBS, cut into 1 mm 3 -sized pieces and put into culture dishes for 30 min at 37˚C. Then, tissue samples were maintained in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin at 37˚C with 5% CO 2 . Medium was replaced every three days after the initial plating. When adherent fibroblast-like cells confluence reached ~80%, the cells were passaged into new flasks for further expansion. Cells at passage 3-4 were used for the evaluation of the experiments. Bone marrow-mesenchymal stem cells (BM-MSCs) from patients (n=12) aged 3-60 years (mean, 22 years) with suspected blood system diseases who were diagnosed with no hematological disease or other cancers at the First People's Hospital of Lianyungang from April 2015 to July 2015 (Jiangsu, China) were chosen as the MSC controls.  Multidifferentiation capacity. To determine the ability of BC-MSCs to undergo osteogenic and adipogenic differentiation, BC-MSCs were seeded in 35-mm plates at 3x10 4 cells/cm 2 and cultured in L-DMEM containing 15% FBS. The next day, the medium was changed to osteogenic medium or adipogenic medium (both from Cyagen Biosciences, Sunnyvale, CA, USA) in accordance with the culture protocol. After 2 weeks, the osteogenic differentiation of BC-MSCs was examined by alkaline phosphatase staining at room temperature for 15 min. At 3 weeks later, adipocytes were stained with Oil-Red-O at room temperature for 15 min to confirm the ability of adipogenic differentiation of BC-MSCs. A fluorescence-inverted microscope (Olympus Corporation, Tokyo, Japan) was used to observe the staining results at x100 and x400 magnification.
Preparation of MSC-conditioned medium. A total of (1.0x10 6 ) BC-MSC cells were seeded in 35-mm plates and cultured in L-DMEM with 15% FBS. The following day, the media was removed and the cells were washed with PBS, re-incubated with fresh medium for 48 h. Next, BC-MSC-conditioned medium (BC-MSC-CM) and BM-MSC-conditioned medium (BM-MSC-CM) were collected, and centrifuged at room temperature to remove possible cell debris (1,000 x g for 10 min).
MTT assay. To investigate the effect of cisplatin, BC-MSC-CM combination cisplatin on the viability of MCF-7 cells, an MTT assay was performed. Briefly, MCF-7 cells were plated at a density of 1x10 4 cells/well in a 96-well rounded bottom plate. After incubation for 12 h, cells were incubated with cisplatin for another 48 h at 37˚C by a continuous induction from 2.5 to 80 µM (2.5, 5, 10, 20, 40 and 80 µM) in a stepwise increasing concentration manner in the presence or absence of BC-MSC-CM. Control medium was used as a control. All the cells were cultured for a further 48 h. Subsequently, MTT reagent was added into each well and the cells were incubated for an additional 4 h at 37˚C. Following incubation and removal of the supernatant, 150 µl DMSO was added to dissolve the dye and the absorbance was measured at 490 nm with a microplate reader. IC 50 was defined as the drug concentration causing a 50% apoptosis relative to the negative control. In this experiment, the IC 50 was 20 µM. The experiment was repeated three times; six parallel samples were detected each time. Antibody blocking assay. To evaluate the effect of IL-6 on cisplatin-induced apoptosis in MCF-7 cells, 10 µg/ml IL-6 neutralization antibody (R&D Systems, Inc., Minneapolis, MN, USA, MAB206.) was added to a total of 5.0x10 4 MCF-7 cells cultured with BC-MSC-CM. Following incubation at 37˚C for 48 h, cells were collected for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses.
Statistical analysis. All values were expressed as the mean ± standard deviation. Statistically significant differences between groups were assessed by two-way analysis of variance or Student's t-test using GraphPad Prism software (version 6.0; GraphPad, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results
Characterization and identification of BC-MSCs. After 10-14 days of primary culture of tissue samples, a small population of fibroblastic cells was observed. When the confluence of adherent fibroblast-like cells reached ~80%, the cells were passaged into new flasks, and spindle-shaped morphologies were observed on BC-MSCs and BM-MSCs (Fig. 1A). Flow cytometric analysis demonstrated that BC-MSCs and BM-MSCs expressed high levels of CD29, CD44, CD90, and CD105, but were negative for CD14, CD34 and CD45 ( Fig. 1B and C). In addition, in order to evaluate the pluripotent differentiation potential of BC-MSCs, cells were cultured in osteoblastic-induction medium for two weeks and numerous Alkaline phosphatase staining-positive cells were observed. In addition, a number of BC-MSCs were positive for Oil-Red-O intracellular staining following three weeks of incubation indicating adipogenic induction. Control cells cultured in the complete medium were negative for Oil-Red-O staining (Fig. 1D).

BC-MSCs decrease MCF-7 cell apoptosis and promote MCF-7 cell survival from cisplatin.
To evaluate the effects of BC-MSCs in the cisplatin-induced MCF-7 cell response, cells were seeded in 96-well plates or 6-well plates for different experiments. As presented in Fig. 2A, the results of an MTT assay revealed that MCF-7 cells exposed to cisplatin in the presence of BC-MSC-CM exhibited significantly higher growth rates compared with those treated with cisplatin alone (P<0.01) and cisplatin plus BM-MSC-CM-treated cells (P<0.05). FACS analysis revealed that the proportion of annexin-V-bound MCF-7 cells cultured with control medium increased significantly in response to cisplatin treatment compared with those cultured with BC-MSC-CM (P<0.01; Fig. 2B). The anti-apoptosis effect of BC-MSC-CM was significantly increased compared with that in the BM-MSC-CM group (P <0.05; Fig. 2B). Co-treatment with BC-MSC-CM and cisplatin revealed that the vitality of MCF-7 cells increased significantly when compared with cells treated with cisplatin alone (Fig. 2C; P<0.01). Furthermore, BC-MSCs had a significantly higher potential to decrease MCF-7 cell apoptosis and promote cell proliferation compared with BM-MSCs (P<0.05; Fig. 2). These results suggested that BC-MSCs weakened the antitumor effect of cisplatin more effectively than BM-MSCs.   Furthermore, MCF-7 cell viability evidently decreased following IL-6 neutralization antibody treatment (Fig. 4B). To assess whether the change in response of MCF-7 cells to cisplatin treatment caused by BC-MSCs was mediated through activation of the IL-6/STAT3 signaling pathway, western blot analysis was performed (Fig. 4C). Data indicated that in the presence of BC-MSC-CM, levels of p-STAT3 were raised markedly in MCF-7 cells, which could be reversed by incubation with IL-6-neutralizing antibody. The expression of the apoptosis-associated protein Bax was reduced in MCF-7 cells on incubation with BC-MSC-CM compared with cisplatin alone treated cells. The aforementioned data indicated that IL-6 is crucial in the BC-MSC-mediated reduction in apoptosis induced by cisplatin in breast cancer cells.

Discussion
The interactions between the stromal microenvironment and tumor cells have a central role in patient survival, and the response to chemotherapy. MSCs are commonly used as stromal cells for in vitro studies on multiple tumor types, including gastric, breast and ovarian cancer (24)(25)(26). Tumor-tissue-derived MSCs have been widely studied owing to their proximity to tumors and the influence they exert on tumors (16,(27)(28)(29)(30). MSCs act as regulators of apoptosis, proliferation, angiogenesis and immune regulation, and, when in contact with tumor cells, produce a variety of cytokines that affect proliferation, survival and the acquisition of chemoresistance (31,32). The present study focused on the paracrine effects of BC-MSCs on the behavior of MCF-7 cells during cisplatin treatment. MSCs were isolated from human breast cancer tissues and revealed to exhibit a heterogeneous immunophenotype with fibroblastic morphology and the potential to differentiated into multiple cell types. First of all, BC-MSC-CM was prepared for the subsequent experiments. BC-MSC-CM significantly decreased the inhibitory effect of cisplatin treatment on MCF-7 cell growth and promoted MCF-7 cell survival. The results of flow cytometric analysis revealed that in the presence of BC-MSC-CM, the degree of cisplatin-triggered apoptosis was evidently decreased and the proportion of apoptotic cells was more evidently reduced in the presence of BC-MSC-CM compared with in the control medium. A prior study revealed that MSCs protect tumor cells exposed to chemotherapeutic drugs from apoptosis more potently than BM-MSC-CM (33).
To investigate the underlying mechanism by which BC-MSC-CM enhances the survival of the MCF-7 cells and protects them from drug-induced apoptosis, cytokine levels in BC-MSC-CM were examined by a Luminex immunoassay. The present study demonstrated that the level of IL-6 was markedly higher in the BC-MSC-CM compared with in BM-MSC-CM, suggesting that IL-6 may act as a key mediator of the tumor-promoting activity of BC-MSCs. IL-6, as a key mediator of the inflammatory response, has a pathological role in the development of several neoplasms, including malignant mesothelioma, breast tumor, endometrial cancer and lung cancer (16,34,35). It has been reported that bone-marrow-and glioma-derived MSCs enhance cancer cell proliferation via the IL-6/STAT3 signaling pathway (15,36). It is well established that the IL-6/glycoprotein 130/STAT3 signaling pathway further enhances the growth of cancer cells and reduces the sensitivity of cancer cells to antitumor drugs (37,38). The present study was designed to determine the role of BC-MSCs on cisplatin treatment of tumor cells via the IL-6/STAT3 signaling pathway. The current study demonstrated that the degree of proliferation, viability and apoptosis of MCF-7 cells in response to cisplatin treatment regulated by BC-MSCs may be attenuated by incubation with an IL-6 neutralizing antibody. Western blot analysis revealed that incubation of MCF-7 cells with BC-MSC-CM activated the IL-6/STAT3 signaling pathway in MCF-7 cells, markedly decreasing Bax expression. In addition, this effect was partially abolished in the presence of IL-6 neutralizing antibody. The data of the present study indicated that BC-MSC-secreted IL-6 attenuated the function of cisplatin on MCF-7 cells, preventing apoptosis and thus promoting breast cancer growth and survival. A more marked promotion was observed in breast cancer growth and survival when MCF-7 cells were incubated with BC-MSC-CM compared with BM-MSC-CM, suggesting that BC-MSCs have a greater potential to promote breast cancer growth and decrease apoptosis upon exposure to cisplatin than BM-MSCs.
In summary, BC-MSCs significantly enhanced the survival of MCF-7 cells that were exposed to cisplatin, one of the reasons behind the development of drug resistance. Furthermore, IL-6 was demonstrated to contribute to the BC-MSC-induced protection of MCF-7 cells from apoptosis. Therefore, BC-MSC-secreted IL-6 should be considered as a novel therapeutic target to aid the improvement of patient responses to cisplatin.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Tumour-associated and non-tumour-associated microbiota: Addendum

ABSTRACT In a recent study1 we reported that tissue-associated microbial Co-abundance Groups (CAGs) were differentially associated with colorectal cancer (CRC). Two of the CAGs, which we named Pathogen CAG and Prevotella CAG, were correlated with a gene expression signature indicative of a TH17 response. A TH17 response has been associated with decreased survival in patients with CRC2, and members of the Pathogen CAG such as Fusobacterium nucleatum, Escherichia coli and Bacteroides fragilis have been repeatedly reported to be associated with CRC-development. Thus we hypothesized that the abundance of these CAGs may be associated with poor survival. In this Addendum we extend our analysis of the at-surgery microbiota to microbiota profiles obtained after surgery for CRC which we analyzed in the context of survival data for patients with CRC. Surprisingly we found that high tissue-associated abundance of the previously defined Prevotella- and Pathogen-CAGs at surgery was associated with longer survival. Furthermore, we detected an association of the Bacteroidetes CAG in pre-surgery faecal microbiota with stability of the microbiota after surgery.

Results
Survival after surgery may be associated with the microbiota profile at surgery In the current analysis we tested whether tissueassociated microbiota profiles at surgery were predictive of survival. An overview of the studied individuals follows. The median follow-up time was 1371 days post diagnosis, the minimum follow-up time was 630 days. During the follow-up period 16 individuals died. We obtained microbiota data from at-surgery tumour tissue for 47 individuals. For more patient-related details please see Supplementary Table 1.
We then stratified individuals by their American Joint Committee on Cancer (AJCC) tumour stage and we repeated CAG-clustering of the microbiota for this cohort. As expected, using a Cox proportional hazards model we found that AJCC stage had a strong effect on survival (P D 0.015, Likelihood ratio test of the overall model), with stage IV being particularly discriminative (P D 0.026, HR D 12.44, CI [1.36, 113.57]). Similarly, Kaplan-Meier curves and a log-rank test demonstrated a strong association between AJCC stage and survival ( Fig. 1). We then extended our model by including the relative abundance of five tissue-associated bacterial CAGs (see Supplementary Fig. 1 for CAG profiles of the cohort) while adjusting for tumour stage, age, gender, treatment with chemo-and/or radiotherapy and site of the cancer. Surprisingly, Pathogen-and Prevotella-CAG-type microbiotas, which were again detected in this cohort, were associated with longer survival (P D 0.12, HR which we also found to be more abundant in individuals with CRC in our previous study. 1 Overall microbiota profiles are similar before and after surgery If the pre-surgery microbiota is a risk factor or biomarker for CRC, then the post-surgery microbiota might be relevant for relapse or survival. We performed principal coordinate analysis on the faecal microbiota profiles from 28 individuals, measured before and after surgery. The overall microbiota composition did not change to a statistically significant degree after surgery (Fig. 3). Similarly, abundance of only one OTU was statistically significantly different between before and after surgery samples (ANCOM, FDR < .1). Despite these global similarities we observed that the faecal microbiota of several individuals differed strongly after surgery, which was correlated with the overall microbiota profile before surgery (Fig. 4). We then grouped faecal OTUs into CAGs as previously and  again found grouping of bacteria into Firmicutes, Bacteroidetes and Prevotella CAGs ( Supplementary  Fig. 2), thus resembling enterotypes. 3 Strikingly, the abundance of the luminal Bacteroidetes CAG was significantly negatively correlated with microbiota profile alterations after surgery (Fig. 5, middle  panel). Conversely, the collective abundance of bacteria colonizing less than 37% of individuals (which we named Sporadic Colonizers), was positively correlated with change (Fig. 5, left panel). On the level of the bacterial genus, Lachnospira abundance before surgery was associated with microbiota stability (Fig. 5, right panel).

Conclusions
It is striking that the mucosal Bacteroidetes CAG was associated with improved survival and the luminal Bacteroidetes CAG was associated with greater stability of the microbiota. These two CAGs both comprised major microbiota components viz. the genera Alistipes and Bacteroides (Supplementary Fig. 1 and Supplementary Fig. 2). Thus it is tempting to speculate that the stability of the microbiota after surgery is also associated with improved survival. Unfortunately it was not possible for us to directly test this hypothesis because re-sampling of the microbiota after surgery obviously depends on patient survival. Future strategic sampling of faecal samples at pre-defined time-points after surgery will enable testing of this hypothesis.
Our current finding that microbiotas of the Prevotellaand Pathogen-CAGs were associated with improved survival was not expected. We previously found correlations of these microbiotas with the expression of host genes associated with an inflammatory response indicative of a TH17 response. 1 Tosolini et al. 2 reported reduced survival for individuals with CRC that had an increased  . The stability of the faecal microbiota after surgery is positively associated with the pre-surgery abundance of sporadic colonizers and negatively associated with the Bacteroidetes CAG and genus Lachnospira abundance. Graphs are the scatter-plots of the Bray-Curtis distance of samples after surgery compared to their pre-surgery sample (x-axis) and the combined centered log-ratio, pre-surgery abundance (y-axis) of bacteria found in less than 37% of individuals (i.e. bacteria which only sporadically colonize the human gut; panel to the left), of the Bacteroidetes CAG as defined in Supplementary Figure 2 (middle panel) and the genus Lachnospira (panel to the right). OTUs of this genus were mainly found in the Bacteroidetes CAG and had the highest negative correlation of all single genera with microbiota change after surgery. Linear regression trendlines are displayed. Pearson's correlation coefficient r and P-values are given for the correlation between distance of samples after surgery from their presurgery counterpart and location of the pre-surgery sample on the PCoA. The colour of the arrows and the circles is associated with the Bray-Curtis distance of the sample after surgery from the sample before surgery (log-scale), as per the colour scale to the right. TH17 response. Furthermore, F. nucleatum, one of the members of the Pathogen CAG, has been linked with shorter survival 4 and recurrence after chemotherapy. 5 More comprehensive testing of the inflammatory response in our sample set may yet reveal a rather general activation of the immune system associated with elevated levels of the Pathogen-and Prevotella-CAGs which in turn may positively affect cancer-related survival.
We concede that our current findings are preliminary because we could not adjust for factors such as specific tumour genotypes (e.g. microsattelite instability which has been linked with improved disease outcome), and the fact that many of the studied individuals have been followed up for less than two years. Larger and more long-term studies are currently under way at the APC Microbiome Institute.