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Ⅴ. Discussion
Part 1. COL6A3-derived endotrophin links reciprocal interactions among hepatic cells in the
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Moreover, CCl4 driven hepatic cell apoptosis, inflammation, and fibrogenesis were attenuated by using endotrophin neutralizing antibody. Paracrine interactions of hepatocytes with NPCs were also efficiently suppressed by endotrophin inhibition in our ex vivo settings, suggesting that endotrophin inhibition is a promising therapeutic strategy for CLD.
Part 2. Notch and HER3 signaling crosstalk via Nrg1 promotes breast cancer progression in patients with diabetes
Glucose is the primary energy source for multiple cells, and cells regulate glucose homeostasis through multiple glucose-sensing mechanisms [50]. One way of sensing glucose availability is via O- GlcNAc modification, wherein high glucose levels enhance HBP flow and subsequent O- GlcNAcylation of target proteins [27]. This modification regulates protein activities, allowing cells to adapt to environmental conditions [51]. In chronic hyperglycemia, several target proteins undergo aberrant O-glycosylation, including Sp1 and FoxO1 [51]. O-GlcNAc of NOTCH1 was promoted by HG, which facilitated Notch activation and subsequent Nrg1 overexpression. However, it remained unclear whether glucose triggered PTM in other proteins, such as P300 and Setd1A, to facilitate the Nrg1 enhancer recruitment. Although several studies reported that promoter binding of P300 is increased by high glucose stimulus, the detailed mechanism that links glucose and P300 recruitment is still poorly understood [52, 53]. Beyond the PTM of NOTCH or other targets, tumorigenic signals could be attributed to direct involvement of excess glucose through activation of the Nrg1 enhancer. As the epigenetic modifications rely on the metabolic status of cells using metabolites as cofactors or substrates [54], hyperglycemia could contribute the hyperacetylation of the Nrg1 enhancer. Acetyl-CoA abundance is regulated by glucose accessibility, and its production facilitates histone acetylation [55, 56]. Although we did not perform an investigation of metabolomics in this study, we propose that hyperglycemia- induced reprogramming of glucose/acetyl-CoA metabolism could sustain tumor development [55].
RBPJ is a statically DNA-bound protein independent of Notch activation; however, recent studies show that RBPJ can be dynamically recruited to target elements in a Notch-inducible manner [57, 58]. A genome-wide ChIP-seq study identified a subset of dynamic Notch targets characterized by sensitive responses to Notch on/off status and lack of Notch-binding elements in promoter regions;
instead, their transcriptional regulation was largely dictated by a distal enhancer enriched with H3K27ac [59]. In this study, Notch activation promoted the binding of NICD-RBPJ complex to the distal enhancer region of Nrg1 in an HG-inducible manner, suggesting that Nrg1 is a dynamic Notch target under the control of the enhancer. HG reduced the binding occupancy of HDAC1, a component of RBPJ co- repressor complex, as it was dissociated from the Nrg1 enhancer. Inhibiting HDAC1 with TSA was
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sufficient to switch on Nrg1 expression even under LG conditions. Thus, HDAC1 suppresses Nrg1 expression under LG conditions, and its dissociation by HG-linked Notch activation seems to be crucial in shaping HG-inducible enhanceosome assembly.
The therapeutic potential of the metabolic status of breast cancer patients has been largely ignored, as therapeutic outcomes have been unsatisfactory. Approximately 35% of HER2-positive breast cancer patients initially responded to trastuzumab, of which only 30% exhibited no progression within a year [60]. One possible reason for the resistance to HER2-targeted therapy is hyperglycemia, which may arise during anticancer treatment or before cancer develops. The detrimental impact of diabetes on clinical outcomes of HER2-positive breast cancer treatment using trastuzumab was reported [61, 62].
Additionally, Notch signaling has recently been identified as critically important for trastuzumab resistance. Thus, a combination of a Notch antagonist and HER2-targeted drugs (trastuzumab, lapatinib) has been investigated as a therapeutic strategy to prevent drug resistance or cancer recurrence [39, 63]. The role of Notch pathway in tumor progression has been largely explored [64], and numerous studies have focused on the potential of Notch inhibitors as cancer therapeutic drugs [65]. In clinical trials, several Notch inhibitors have been tested for their safety and therapeutic efficacy, including γ-secretase inhibitors (GSI), antibody-based drugs, and small molecules targeting the Notch- Rbpj complex [65, 66]. Notch activation is also implicated in diabetes and its complications, and Notch- targeted therapy is proposed as a promising strategy for diabetic intervention [67, 68]. Considering the applications of the Notch inhibitors in cancer and diabetes, Notch-targeting drugs are expected to be beneficial, especially in diabetic cancer patients. However, these drugs still lack detailed mechanisms and require further elucidation. Because Notch activity is elevated in HG-treated cancer cells and tissues from HER2-positive breast cancer patients with hyperglycemia, we postulated that hyperglycemia- driven NOTCH activation in breast cancer cells aggravated resistance to HER2-targeted therapy.
Lapatinib resistance in hyperglycemic mice was reversed by combining lapatinib with a NOTCH antagonist. As the NRG1-HER3 axis contributes to resistance to HER2-targeted therapy [69], blocking the Notch pathway and the subsequent NRG1 downregulation might account for the beneficial effect of lapatinib-DAPT combination treatment on hyperglycemia-driven drug resistance. These findings suggest that NRG1 mediates the crosstalk between HER2 and Notch pathway under hyperglycemic conditions, possibly by acting as a NOTCH target and a HER3 ligand.
There were no matching blood glucose or HbA1c data associated with the gene expression data in TCGA; hence, we postulated that elevated NOTCH activity might represent the patients’
hyperglycemic status. Although the diabetic status of breast cancer patients was unknown, we
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demonstrated that patients with high NOTCH activity had upregulated NRG1 expression, regardless of breast cancer subtype, and had more accessible chromatin structure within the NRG1 enhancer region.
Additionally, NRG1 overexpression predicted poor outcome in hyperglycemic HER2-positive cancer patients but not in HER2-negative patients. Due to a complexity of the diabetic pathophysiology, it is challenging to select proper hyperglycemic mouse model; Obesity-linked type 2 diabetes is considered to be relevant model that recapitulates most of hyperglycemic patients, however, other confounding factors could influence tumor progression, including hyperinsulinemia, dyslipidemia, and inflammation, and thus make it difficult to distinguish the effect of hyperglycemia from that of other factors. It remains to be further investigated the impact of hyperglycemia on tumor progression, in the context of obesity associated type 2 diabetes.
Part 3. Natural product derived Verminoside confers cisplatin sensitivity in metastatic breast cancer
Chemotherapeutic drug treatment often fails, and causes cancer recurrence when used in single therapy. Thus, it is generally admitted to apply combination of multiple cancer therapeutic drugs to achieve improved clinical outcomes, which is called multidrug treatment. From the several few decades, numerous efforts were attempting to investigate pharmacological or molecular factors involved in drug combinations, including signaling pathway, side effects, interaction of drugs, administration ratio, and as a result, gave us a better insight of multidrug treatment [70]. Due to the progression, we developed therapeutic combination regimens that profoundly improved the outcomes in cancer survival. One example in successful multidrug treatment is an administration of cisplatin combined with gemcitabine or docetaxel, which is current first-line chemo protocols for breast cancer patients with metastasis [71, 72]. On the other hand, chemoadjuvant therapy is a combinatorial form that adopts different type of interventions, like radiation therapy with chemo drugs, instead of applying multiple cancer therapeutic drug [73, 74]. Guideline from National Cancer Institute, adjuvant therapy is described as an additional interventions for cancer patients who received primary therapeutic treatment to minimize the risk of cancer recurrence. Due to the recent improvements in adjuvant therapy investigations, newer agents are applied with conventional chemotherapeutic drugs. Among the diverse regimens, natural compounds are documented to have beneficial effect when combined with radiotherapy or chemotherapy [75]. The chemoadjuvant therapy based on natural products could enhance cytotoxic effect to cancer cells, and moreover, they could elicit activation of immune system in tumor stroma [76].
Here, we sought to develop the natural products as chemoadjuvants for conventional chemotherapy. We firstly provided evidence that natural compound NC13 and its bioactive constituent,
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Vms had a therapeutic potential as chemoadjuvants in cisplatin combined regimens. The target compounds were screened on the basis of cytotoxic effects to the breast cancer cells, where the synergistic (or additive) cytotoxic effect is exerted only when used in combined with cisplatin. Using the criteria, NC13 was selected and their major component were also tested including Vms. In our experimental settings, NC13 and Vms exhibited beneficial effects with cisplatin treatment to inhibit lung metastasis in vivo. The combination treatment displayed a limited effect on tumor growth, however, pulmonary metastases were efficiently suppressed by the combination treatment in MMTV-PyMT and 4T1 tumor bearing mice model.
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ⅤI. Conclusion
Currently, the endotrophin signaling pathway is not fully elucidated; endotrophin receptors and effector molecules have not yet been identified. In this study, we proposed the molecular and cellular mechanisms how endotrophin could affect the pathological process of CLD, using various in vivo and ex vivo models. The JNK signaling acts as a crucial pathway involved in conversion of external cues into a broad spectrum of cell responses, such as cellular proliferation, differentiations, and apoptosis [77, 78], and especially tumorigenesis [79]. Several studies reported that HCC development is attenuated in xenograft and DEN-induced cancer models by pharmacological inhibition or genetic depletion of JNK1 [20, 22]. Here, we demonstrated that endotrophin overexpression promotes JNK1- dependent hepatic apoptosis under the additional liver stress conditions, such as CCl4, where the endotrophin-driven JNK1 activation contributes to establish a pathological niche in CLD. Those results suggest that endotrophin could augment the liver insults at the early step of CLD through JNK1 pathway.
Thus, inhibition of endotrophin-JNK1 axis would be profitable strategy for liver cancer patients, especially for high expression of endotrophin.
Here, we showed that the Nrg1 enhancer is activated by a hyperglycemic cue mediated by Notch-driven active histone modification following Nrg1 enhanceosome assembly. Previous reports reported that Notch activation could promote NRG1 expression through direct binding of NICD to the Nrg1 promoter in melanoma [80] or indirectly through the involvement of other mediators in cardiac tissues [81]. Beyond these regulatory mechanisms of Nrg1, our study showed that the NICD-RBPJ complex is assembled on the Nrg1 enhancer and drives Nrg1 overexpression upon HG stimulus. NRG1 overexpression leads to activation of NRG1-HER3 signaling, and subsequent malignant cancer growth.
We further showed that the oncogenic effect of NRG1 could be blocked by inhibiting both Notch and HER2. In conclusion, although the metabolic state (i.e. obesity) of patients is often overlooked, it should be taken into consideration to improve predicted responses and identify patients for whom drug combination therapy would be suitable. We propose that the diabetic status and NOTCH-NRG1-HER3 axis can be used as prognostic markers to predict responses to HER2-targeted therapy, offering a unique therapeutic strategy for treating breast cancer patients with diabetes.
Over 90% of cancer mortality is attriwbutable for tumor metastasis in solid cancers, and thus, it is urgent to develop cancer therapeutic drugs targeting cancer invasion or metastatic growth [82].
Numerous studies reported the promising effects of anti-cancer drugs for metastasis in the breast cancer models [83-85], and furthermore, several drugs for metastatic breast cancer patients are under the phase III clinical trials, including sacituzumab and govitecan-hziy [86]. In this study, we proposed that NC13- derived Vms exerted a beneficial effect on cisplatin therapy, mediated through suppression of EMT and
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metastatic capacity of the cancer. Diverse applications of combination therapy have been investigated to overcome the limits of conventional chemotherapy, and our results proposed that Vms could be developed as chemoadjuvant for metastatic breast cancer patients.
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