Mapping the intricacies of GLI1 in hedgehog signaling: A combined bioinformatics and clinical analysis in Head & Neck cancer in Western India.

BACKGROUND: Activation of various cancer stem cell pathways are thought to be responsible for treatment failure and loco-regional recurrence in Head and Neck cancer. Hedgehog signaling, a major cancer stem signaling pathway plays a major role in relapse of disease. GLI1, a transcription activator, plays an important role in canonical/non-canonical activation of Hedgehog signaling. METHODS: Data for H&N cancer patients were collected from The Cancer Genome Atlas- H&N Cancer (TCGA-HNSC). GLI1 co-expressed genes in TCGA-HNSC were then identified using cBioPortal and subjected to KEGG pathway analysis by DAVID tool. Network Analyzer and GeneMania plugins from CytoScape were used to identify hub genes and predict a probable pathway from the identified hub genes respectively. To confirm the hypothesis, real-time gene expression was carried out in 75 patients of head and neck cancer. RESULTS: Significantly higher GLI1 expression was observed in tumor tissues of H&N cancer and it also showed worst overall survival. Using cBioPortal tool, 2345 genes were identified that were significantly co-expressed with GLI1. From which, 15 hub genes were identified through the Network Analyzer plugin in CytoScape. A probable pathway prediction based on hub genes showed the interconnected molecular mechanism and its role in non-canonical activation of Hedgehog pathway by altering the GLI1 activity. The expressions of SHH, GLI1 and AKT1 were significant with each other and were found to be significantly associated with Age, Lymph-Node status and Keratin. CONCLUSION: The study emphasizes the critical role of the Hh pathway's activation modes in H&N cancer, particularly highlighting the non-canonical activation through GLI1 and AKT1. The identification of SHH, GLI1 and AKT1 as potential diagnostic biomarkers and their association with clinic-pathological parameters underscores their relevance in prognostication and treatment planning. Hh pathway activation through GLI1 and its cross-talk with various pathways opens up the possibility of newer treatment strategies and developing a panel of therapeutic targets in H&N cancer patients.

RMRP accelerates ligamentum flavum hypertrophy by regulating GSDMD-mediated pyroptosis through Gli1 SUMOylation.

Hypertrophy of ligamentum flavum (LF) is a significant contributing factor to lumbar spinal canal stenosis (LSCS). lncRNA plays a vital role in organ fibrosis, but its role in LF fibrosis remains unclear. Our previous findings have demonstrated that Hedgehog-Gli1 signaling is a critical driver leading to LF hypertrophy. Through the RIP experiment, our group found lnc-RMRP was physically associated with Gli1 and exhibited enrichment in Gli1-activated LF cells. Histological studies revealed elevated expression of RMRP in hypertrophic LF. In vitro experiments further confirmed that RMRP promoted Gli1 SUMO modification and nucleus transfer. Mechanistically, RMRP induced GSDMD-mediated pyroptosis, proinflammatory activation, and collagen expression through the Hedgehog pathway. Notably, the mechanical stress-induced hypertrophy of LF in rabbit exhibited analogous pathological changes of LF fibrosis occurred in human and showed enhanced levels of collagen and α-SMA. Knockdown of RMRP resulted in the decreased expression of fibrosis and pyroptosis-related proteins, ultimately ameliorating fibrosis. The above data concluded that RMRP exerts a crucial role in regulating GSDMD-mediated pyroptosis of LF cells via Gli1 SUMOylation, thus indicating that targeting RMRP could serve as a potential and effective therapeutic strategy for LF hypertrophy and fibrosis.

Tumor-derived GLI1 promotes remodeling of the immune tumor microenvironment in melanoma.

BACKGROUND: Melanoma progression is based on a close interaction between cancer cells and immune cells in the tumor microenvironment (TME). Thus, a better understanding of the mechanisms controlling TME dynamics and composition will help improve the management of this dismal disease. Work from our and other groups has reported the requirement of an active Hedgehog-GLI (HH-GLI) signaling for melanoma growth and stemness. However, the role of the downstream GLI1 transcription factor in melanoma TME remains largely unexplored. METHODS: The immune-modulatory activity of GLI1 was evaluated in a syngeneic B16F10 melanoma mouse model assessing immune populations by flow cytometry. Murine polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were differentiated from bone marrow cells and their immunosuppressive ability was assessed by inhibition of T cells. Conditioned media (CM) from GLI1-overexpressing mouse melanoma cells was used to culture PMN-MDSCs, and the effects of CM were evaluated by Transwell invasion assay and T cell inhibition. Cytokine array analysis, qPCR and chromatin immunoprecipitation were performed to explore the regulation of CX3CL1 expression by GLI1. Human monocyte-derived dendritic cells (moDCs) were cultured in CM from GLI1-silenced patient-derived melanoma cells to assess their activation and recruitment. Blocking antibodies anti-CX3CL1, anti-CCL7 and anti-CXCL8 were used for in vitro functional assays. RESULTS: Melanoma cell-intrinsic activation of GLI1 promotes changes in the infiltration of immune cells, leading to accumulation of immunosuppressive PMN-MDSCs and regulatory T cells, and to decreased infiltration of dendric cells (DCs), CD8 + and CD4 + T cells in the TME. In addition, we show that ectopic expression of GLI1 in melanoma cells enables PMN-MDSC expansion and recruitment, and increases their ability to inhibit T cells. The chemokine CX3CL1, a direct transcriptional target of GLI1, contributes to PMN-MDSC expansion and recruitment. Finally, silencing of GLI1 in patient-derived melanoma cells promotes the activation of human monocyte-derived dendritic cells (moDCs), increasing cytoskeleton remodeling and invasion ability. This phenotype is partially prevented by blocking the chemokine CCL7, but not CXCL8. CONCLUSION: Our findings highlight the relevance of tumor-derived GLI1 in promoting an immune-suppressive TME, which allows melanoma cells to evade the immune system, and pave the way for the design of new combination treatments targeting GLI1.

Combined targeting of Hedgehog/GLI1 and Wnt/ß-catenin pathways in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a rare and aggressive form of non-Hodgkin lymphoma. Challenges in its treatment include relapse, drug resistance, and a short survival period. The Hedgehog/GLI1 (Hh/GLI1) and Wnt/ß-catenin pathways are crucial in cancer cell proliferation, survival, and drug resistance, making them significant targets for anticancer research. This study aimed to assess the effectiveness of combining inhibitors for both pathways against MCL and investigate the underlying molecular mechanisms. The co-expression of key proteins from the Hh/GLI1 and Wnt/ß-catenin pathways was observed in MCL. Targeting the Hh/GLI1 pathway with the GLI1 inhibitor GANT61 and the Wnt/ß-catenin pathway with the CBP/ß-catenin transcription inhibitor ICG-001, dual-target therapy was demonstrated to synergistically suppressed the activity of MCL cells. This approach promoted MCL cell apoptosis, induced G0/G1 phase blockade, decreased the percentage of S-phase cells, and enhanced the sensitivity of MCL cells to the drugs adriamycin and ibrutinib. Both GANT61 and ICG-001 downregulated GLI1 and ß-catenin while upregulating GSK-3ß expression. The interaction between Hh/GLI1 and Wnt/ß-catenin pathways was mediated by GANT61-dependent Hh/GLI1 inhibition. Moreover, GLI1 knockdown combined with ICG-001 synergistically induced apoptosis and increased drug sensitivity of MCL cells to doxorubicin and ibrutinib. GANT61 attenuated the overexpression of ß-catenin and decreased the inhibition of GSK-3ß in MCL cells. Overall, the combined targeting of both the Hh/GLI1 and Wnt/ß-catenin pathways was more effective in suppressing proliferation, inducing G0/G1 cycle retardation, promoting apoptosis, and increasing drug sensitivity of MCL cells than mono treatments. These findings emphasize the potential of combinatorial therapy for treating MCL patients.

Bone or Tooth dentin: The TGF-ß signaling is the key.

To investigate the cell linkage between tooth dentin and bones, we studied TGF-ß roles during postnatal dentin development using TGF-ß receptor 2 (Tgfßr2) cKO models and cell lineage tracing approaches. Micro-CT showed that the early Tgfßr2 cKO exhibit short roots and thin root dentin (n = 4; p<0.01), a switch from multilayer pre-odontoblasts/odontoblasts to a single-layer of bone-like cells with a significant loss of ~85% of dentinal tubules (n = 4; p<0.01), and a matrix shift from dentin to bone. Mechanistic studies revealed a statistically significant decrease in odontogenic markers, and a sharp increase in bone markers. The late Tgfßr2 cKO teeth displayed losses of odontoblast polarity, a significant reduction in crown dentin volume, and the onset of massive bone-like structures in the crown pulp with high expression levels of bone markers and low levels of dentin markers. We thus concluded that bones and tooth dentin are in the same evolutionary linkage in which TGF-ß signaling defines the odontogenic fate of dental mesenchymal cells and odontoblasts. This finding also raises the possibility of switching the pulp odontogenic to the osteogenic feature of pulp cells via a local manipulation of gene programs in future treatment of tooth fractures.

Distraction force promotes the osteogenic differentiation of Gli1+ cells in facial sutures via primary cilia-mediated Hedgehog signaling pathway.

BACKGROUND: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown. METHODS: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling. RESULTS: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force. CONCLUSIONS: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.

The Combined Immunohistochemical Expression of GLI1 and BCOR in Synovial Sarcomas for the Identification of Three Risk Groups and Their Prognostic Outcomes: A Study of 52 Patients.

Synovial sarcoma (SS) is a rare soft-tissue tumor characterized by a monomorphic blue spindle cell histology and variable epithelial differentiation. Morphologically, SSs may be confused with other sarcomas. Systemic treatment is more effective for patients with high-risk SSs, patients with advanced disease, and younger patients. However, further studies are required to find new prognostic biomarkers. Herein, we describe the morphological, molecular, and clinical findings, using a wide immunohistochemical panel, of a series of SS cases. We studied 52 cases confirmed as SSs by morphological diagnosis and/or molecular studies. Clinical data (gender, age, tumor size, tumor location, resection margins, adjuvant treatment, recurrences, metastasis, and survival) were also retrieved for each patient. All the available H&E slides were examined by four pathologists. Three tissue microarrays (TMAs) were constructed for each of the tumors, and a wide immunohistochemical panel was performed. For time-to-event variables, survival analysis was performed using Kaplan-Meier curves and log-rank testing, or Cox regression. Statistical significance was considered at p < 0.05. The mean age of our patients was 40.33, and the median was 40.5 years. We found a predominance of males versus females (1.7:1). The most frequent morphological subtype was monophasic. TRPS1, SS18-SSX, and SSX-C-terminus were positive in 96% of cases. GLI1 expression was strong in six and focal (cytoplasmic) in twenty patients. Moreover, BCOR was expressed in more than half of SSs. Positive expression of both proteins, BCOR and GLI1, was correlated with a worse prognosis. Multivariate analysis was also performed, but only BCOR expression appeared to be significant. The combination of GLI1 and BCOR antibodies can be used to group SSs into three risk groups (low, intermediate, and high risk). We hypothesize that these findings could identify which patients would benefit from receiving adjuvant treatment and which would not. Moreover, these markers could represent therapeutic targets in advanced stages. However, further, larger series of SSs and molecular studies are necessary to corroborate our present findings.

Identification of Novel GANT61 Analogs with Activity in Hedgehog Functional Assays and GLI1-Dependent Cancer Cells.

Aberrant activation of hedgehog (Hh) signaling has been implicated in various cancers. Current FDA-approved inhibitors target the seven-transmembrane receptor Smoothened, but resistance to these drugs has been observed. It has been proposed that a more promising strategy to target this pathway is at the GLI1 transcription factor level. GANT61 was the first small molecule identified to directly suppress GLI-mediated activity; however, its development as a potential anti-cancer agent has been hindered by its modest activity and aqueous chemical instability. Our study aimed to identify novel GLI1 inhibitors. JChem searches identified fifty-two compounds similar to GANT61 and its active metabolite, GANT61-D. We combined high-throughput cell-based assays and molecular docking to evaluate these analogs. Five of the fifty-two GANT61 analogs inhibited activity in Hh-responsive C3H10T1/2 and Gli-reporter NIH3T3 cellular assays without cytotoxicity. Two of the GANT61 analogs, BAS 07019774 and Z27610715, reduced Gli1 mRNA expression in C3H10T1/2 cells. Treatment with BAS 07019774 significantly reduced cell viability in Hh-dependent glioblastoma and lung cancer cell lines. Molecular docking indicated that BAS 07019774 is predicted to bind to the ZF4 region of GLI1, potentially interfering with its ability to bind DNA. Our findings show promise in developing more effective and potent GLI inhibitors.

Odontogenesis-Empowered Extracellular Vesicles Safeguard Donor-Recipient Stem Cell Interplay to Support Tooth Regeneration.

Harnessing the developmental events of mesenchymal condensation to direct postnatal dental stem cell aggregation represents a cutting-edge and promising approach to tooth regeneration. Tooth avulsion is among the most prevalent and serious dental injuries, and odontogenic aggregates assembled by stem cells from human exfoliated deciduous teeth (SHED) have proven effective in revitalizing avulsed teeth after replantation in the clinical trial. However, whether and how SHED aggregates (SA) communicate with recipient components and promote synergistic tissue regeneration to support replanted teeth remains elusive. Here, it is shown that SA-mediated avulsed tooth regeneration involves periodontal restoration and recovery of recipient Gli1+ stem cells, which are mobilized and necessarily contribute to the reestablishment of the tooth-periodontal ligament-bone interface. Mechanistically, the release of extracellular vesicles (EVs) is revealed indispensable for the implanted SA to mobilize recipient Gli1+ cells and regenerate avulsed teeth. Furthermore, SHED aggregates-released EVs (SA-EVs) are featured with odontogenic properties linked to tissue regeneration, which enhance migration, proliferation, and differentiation of Gli1+ cells. Importantly, local application of SA-EVs per se empowers recipient Gli1+ cells and safeguards regeneration of avulsed teeth. Collectively, the findings establish a paradigm in which odontogenesis-featured EVs govern donor-recipient stem cell interplay to achieve tooth regeneration, inspiring cell-free translational regenerative strategies.

Effects of mesenchymal stem cells versus curcumin on sonic hedgehog signaling in experimental model of Hepatocellular Carcinoma.

BACKGROUND: Sonic Hedgehog (SHH) is a fundamental signaling pathway that controls tissue reconstruction, stem cell biology, and differentiation and has a role in gut tissue homeostasis and development. Dysregulation of SHH leads to the development of HCC. METHODS, AND RESULTS: The present study was conducted to compare the effects of mesenchymal stem cells (MSCs) and curcumin on SHH molecular targets in an experimental model of HCC in rats. One hundred rats were divided equally into the following groups: control group, HCC group, HCC group received MSCs, HCC group received curcumin, and HCC group received MSCs and curcumin. Histopathological examinations were performed, and gene expression of SHH signaling target genes (SHH, PTCH1, SMOH, and GLI1) was assessed by real-time PCR in rat liver tissue. Results showed that SHH target genes were significantly upregulated in HCC-untreated rat groups and in MSC-treated groups, with no significant difference between them. Administration of curcumin with or without combined administration of MSCs led to a significant down-regulation of SHH target genes, with no significant differences between both groups. As regards the histopathological examination of liver tissues, both curcumin and MSCs, either through separate use or their combined use, led to a significant restoration of normal liver pathology. CONCLUSIONS: In conclusion, SHH signaling is upregulated in the HCC experimental model. MSCs do not inhibit the upregulated SHH target genes in HCC. Curcumin use with or without MSCs administration led to a significant down-regulation of SHH signaling in HCC and a significant restoration of normal liver pathology.

GLI1 and PTTG1 expression in colorectal carcinoma patients undergoing radical surgery and their correlation with lymph node metastasis.

BACKGROUND: Few studies have investigated the expression of GLI1 and PTTG1 in patients undergoing radical surgery for colorectal carcinoma (CRC) and their association with lymph node metastasis (LNM). Therefore, more relevant studies and analyses need to be conducted. AIM: To explore GLI1 and PTTG1 expression in patients undergoing radical surgery for CRC and their correlation with LNM. METHODS: This study selected 103 patients with CRC admitted to our hospital between April 2020 and April 2023. Sample specimens of CRC and adjacent tissues were collected to determine the positive rates and expression levels of GLI1 and PTTG1. The correlation of the two genes with patients' clinicopathological data (e.g., LNM) was explored, and differences in GLI1 and PTTG1 expression between patients with LNM and those without were analyzed. Receiver operating characteristic (ROC) curves were plotted to evaluate the predictive potential of the two genes for LNM in patients with CRC. RESULTS: Significantly higher positive rates and expression levels of GLI1 and PTTG1 were observed in CRC tissue samples compared with adjacent tissues. GLI1 and PTTG1 were strongly linked to LNM in patients undergoing radical surgery for CRC, with higher GLI1 and PTTG1 levels found in patients with LNM than in those without. The areas under the ROC curve of GLI1 and PTTG1 in assessing LNM in patients with CRC were 0.824 and 0.811, respectively. CONCLUSION: GLI1 and PTTG1 expression was upregulated in patients undergoing radical surgery for CRC and are significantly related to LNM in these patients. Moreover, high GLI1 and PTTG1 expression can indicate LNM in patients with CRC undergoing radical surgery. The expression of both genes has certain diagnostic and therapeutic significance.

Impaired Sonic Hedgehog Responsiveness of Induced Pluripotent Stem Cell-Derived Floor Plate Cells Carrying the LRRK2-I1371V Mutation Contributes to the Ontogenic Origin of Lower Dopaminergic Neuron Yield.

Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

Daunorubicin induces GLI1­dependent apoptosis in colorectal cancer cell lines.

Daunorubicin, also known as daunomycin, is a DNA­targeting anticancer drug that is used as chemotherapy, mainly for patients with leukemia. It has also been shown to have anticancer effects in monotherapy or combination therapy in solid tumors, but at present it has not been adequately studied in colorectal cancer (CRC). In the present study, from a screening using an FDA­approved drug library, it was found that daunorubicin suppresses GLI­dependent luciferase reporter activity. Daunorubicin also increased p53 levels, which contributed to both GLI1 suppression and apoptosis. The current detailed investigation showed that daunorubicin promoted the ß­TrCP­mediated ubiquitination and proteasomal degradation of GLI1. Moreover, a competition experiment using BODIPY­cyclopamine, a well­known Smo inhibitor, suggested that daunorubicin does not bind to Smo in HCT116 cells. Administration of daunorubicin (2 mg/kg, ip, qod, 15 days) into HCT116 xenograft mice profoundly suppressed tumor progress and the GLI1 level in tumor tissues. Taken together, the present results revealed that daunorubicin suppresses canonical Hedgehog pathways in CRC. Ultimately, the present study discloses a new mechanism of daunorubicin's anticancer effect and might provide a rationale for expanding the clinical application of daunorubicin.

Gastroblastoma: a case report and literature review.

Objective: Gastroblastoma is an extremely rare gastric tumor. Its pathogenesis remains unclear and there is a lack of specific clinical symptoms. The aim of this paper is to report a case of gastroblastoma and provide references for the diagnosis, treatment, and prognosis of this disease. Methods: The diagnosis and treatment of a 51-year-old female patient with gastroblastoma were retrospectively reported. Analyzing this case by combining the clinical data such as imaging and pathological results of patients with the relevant literature. Results: The patient's chief complaint was the presence of melena persisted for over two weeks. Abdominal contrast-enhanced CT showed gastric antral nodules, and micro-probe endoscopic ultrasonography was considered as "gastric antral protruding lesions". The initial diagnosis of "gastric stromal tumor" was made after admission, and surgical treatment was performed on September 23, 2021. Postoperative pathology showed that gastric mixed epithelial and stromal tumor, combined with immunohistochemical staining, was suggestive of gastroblastoma. No signs of tumor recurrence or metastasis were observed during the 2-year follow-up. Conclusion: Combined with the existing literature reports, the incidence of gastroblastoma is mainly higher in young men, and the predilection site is gastric antrum. The biological behavior of the tumor tends to be indolent, and the prognosis of most cases is favorable. However, due to the extremely small number of cases, this conclusion still needs a large number of cases and follow-up data to support. Postoperative pathological and immunohistochemical examination results are the only methods for definite diagnosis at present, and surgery is the first choice for treatment.

siRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression.

BACKGROUND: Breast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets. METHODS: Transcriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target. RESULTS: Integrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours. CONCLUSION: Our findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

Gli1+ Progenitors Mediate Glucocorticoid-Induced Osteoporosis In Vivo.

For a wide range of chronic autoimmune and inflammatory diseases in both adults and children, synthetic glucocorticoids (GCs) are one of the most effective treatments. However, besides other adverse effects, GCs inhibit bone mass at multiple levels, and at different ages, especially in puberty. Although extensive studies have investigated the mechanism of GC-induced osteoporosis, their target cell populations still be obscure. Here, our data show that the osteoblast subpopulation among Gli1+ metaphyseal mesenchymal progenitors (MMPs) is responsive to GCs as indicated by lineage tracing and single-cell RNA sequencing experiments. Furthermore, the proliferation and differentiation of Gli1+ MMPs are both decreased, which may be because GCs impair the oxidative phosphorylation(OXPHOS) and aerobic glycolysis of Gli1+ MMPs. Teriparatide, as one of the potential treatments for GCs in bone mass, is sought to increase bone volume by increasing the proliferation and differentiation of Gli1+ MMPs in vivo. Notably, our data demonstrate teriparatide ameliorates GC-caused bone defects by targeting Gli1+ MMPs. Thus, Gli1+ MMPs will be the potential mesenchymal progenitors in response to diverse pharmaceutical administrations in regulating bone formation.