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Background Intensive chemotherapeutic regimens with craniospinal irradiation have greatly improved survival in medulloblastoma patients. However, survival markedly differs among molecular subgroups and their biomarkers are unknown. Through unbiased screening, we found Schlafen family member 11 (SLFN11), which is known to improve response to DNA damaging agents in various cancers, to be one of the top prognostic markers in medulloblastomas. Hence, we explored the expression and functions of SLFN11 in medulloblastoma. Methods SLFN11 expression for each subgroup was assessed by immunohistochemistry in 98 medulloblastoma patient samples and by analyzing transcriptomic databases. We genetically or epigenetically modulated SLFN11 expression in medulloblastoma cell lines and determined cytotoxic response to the DNA damaging agents cisplatin and topoisomerase I inhibitor SN-38 in vitro and in vivo. Results High SLFN11 expressing cases exhibited significantly longer survival than low expressing cases. SLFN11 was highly expressed in the WNT-activated subgroup and in a proportion of the SHH-activated subgroup. While WNT activation was not a direct cause of the high expression of SLFN11, a specific hypomethylation locus on the SLFN11 promoter was significantly correlated with high SLFN11 expression. Overexpression or deletion of SLFN11 made medulloblastoma cells sensitive and resistant to cisplatin and SN-38, respectively. Pharmacological upregulation of SLFN11 by the brain-penetrant histone deacetylase-inhibitor RG2833 markedly increased sensitivity to cisplatin and SN-38 in SLFN11-negative medulloblastoma cells. Intracranial xenograft studies also showed marked sensitivity to cisplatin by SLFN11-overexpression in medulloblastoma cells. Conclusions High SLFN11 expression is one factor which renders favorable outcomes in WNT-activated and a subset of SHH-activated medulloblastoma possibly through enhancing response to cisplatin.

Gut microbiota and their metabolites are instrumental in regulating homeostasis at intestinal and extraintestinal sites. However, the complex effects of prenatal and early postnatal microbial exposure on adult health and disease outcomes remain incompletely understood. Here, we showed that mice raised under germ-free conditions until weaning and then transferred to specific pathogen-free (SPF) conditions harbored altered microbiota composition, augmented inflammatory cytokine and chemokine expression, and were hyper-susceptible to colitis-associated tumorigenesis later in adulthood. Increased number and size of colon tumors and intestinal epithelial cell proliferation in recolonized germ-free mice were associated with augmented intratumoral CXCL1, CXCL2, and CXCL5 expression and granulocytic myeloid-derived suppressor cell (G-MDSC) accumulation. Consistent with these findings, CXCR2 neutralization in recolonized germ-free mice completely reversed the exacerbated susceptibility to colitis-associated tumorigenesis. Collectively, our findings highlight a crucial role for early-life microbial exposure in establishing intestinal homeostasis that restrains colon cancer in adulthood.

Complete Supplementary Data File

Purpose:Uveal melanoma (UM) is the most prevalent and lethal intraocular malignancy in adults. Here, we examined the importance of hypoxia in UM growth and tested the antitumor effects of arylsulfonamide 64B, an inhibitor of the hypoxia-induced factor (HIF) pathway in animal models of UM and investigated the related mechanisms.Experimental Design:UM cells were implanted in the uvea of mice eyes and mice systemically treated with 64B. Drug effect on primary eye tumor growth, circulating tumor cells, metastasis formation in liver, and survival were examined. 64B effects on UM cell growth, invasion and hypoxia-induced expression of C-X-C chemokine receptor type 4 (CXCR4) and mesenchymal–epithelial transition factor (c-Met) were measured. Luciferase reporter assays, chromatin immunoprecipitation, co-immunoprecipitation, and cellular thermal shift assays were used to determine how 64B interferes with the HIF transcriptional complex.Results:Systemic administration of 64B had potent antitumor effects against UM in several orthotopic mouse models, suppressing UM growth in the eye (∼70% reduction) and spontaneous liver metastasis (∼50% reduction), and extending mice survival (P < 0.001) while being well tolerated. 64B inhibited hypoxia-induced expression of CXCR4 and c-Met, 2 key drivers of tumor invasion and metastasis. 64B disrupted the HIF-1 complex by interfering with HIF-1α binding to p300/CBP co-factors, thus reducing p300 recruitment to the MET and CXCR4 gene promoters. 64B could thermostabilize p300, supporting direct 64B binding to p300.Conclusions:Our preclinical efficacy studies support the further optimization of the 64B chemical scaffold toward a clinical candidate for the treatment of UM.

Purpose:Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide–Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells.Experimental Design:sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling.Results:We found sGal-3 preferentially binds to β1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-β1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival.Conclusions:We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin β1–dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

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Supplementary materials and methods and figures

Table of contents: 1. Abbreviations 2. Author Contributions 3. Extended Material and Methods 4. Supplementary Figure Legends 5. Supplementary References

Death receptor Fas-mediated apoptosis not only eliminates nonspecific and autoreactive B cells but also plays a major role in antitumor immunity. However, the possible mechanisms underlying impairment of Fas-mediated induction of apoptosis during lymphomagenesis remain unknown. In this study, we employed our developed syngeneic lymphoma model to demonstrate that downregulation of Fas is required for both lymphoma development and lymphoma cell survival to evade immune cytotoxicity. CD40 signal activation significantly restored Fas expression and thereby induced apoptosis after Fas ligand treatment in both mouse and human lymphoma cells. Nevertheless, certain human lymphoma cell lines were found to be resistant to Fas-mediated apoptosis, with Livin (melanoma inhibitor of apoptosis protein; ML-IAP) identified as a driver of such resistance. High expression of Livin and low expression of Fas were associated with poor prognosis in patients with aggressive non-Hodgkin's lymphoma. Livin expression was tightly driven by bromodomain and extraterminal (BET) proteins BRD4 and BRD2, suggesting that Livin expression is epigenetically regulated in refractory lymphoma cells to protect them from Fas-mediated apoptosis. Accordingly, the combination of CD40-mediated Fas restoration with targeting of the BET proteins–Livin axis may serve as a promising immunotherapeutic strategy for refractory B-cell lymphoma.Significance:These findings yield insights into identifying risk factors in refractory lymphoma and provide a promising therapy for tumors resistant to Fas-mediated antitumor immunity.

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Supplementary Figure S4: (A,B) Viable AXT cells after the treatment of chemotherapeutic agents. (C) Representative flow cytometric analysis of γ-H2AX abundance in AXT cells as described in Fig.4E. (D) Immunofluorescence analysis of γ-H2AX in AXT cells.

Supplementary Table S1. Sequences of primers and predicted product sizes for real-time RT-PCR analysis

Supplementary Information. Supplementary Materials and Methods and Supplementary Figure Legends

Supplementary movie S1

The current standard-of-care treatment for glioblastoma includes DNA damaging agents, γ-irradiation (IR) and temozolomide (TMZ). These treatments fail frequently and there is limited alternative strategy. Therefore, identifying a new therapeutic target is urgently needed to develop a strategy that improves the efficacy of the existing treatments. Here, we report that tumor samples from GBM patients express a high level of SAMHD1, emphasizing SAMHD1’s importance. The depletion of SAMHD1 using virus-like particles containing Vpx, VLP(+Vpx), sensitized two independent GBM cell lines (LN-229 and U-87) to veliparib, a well-established PARP inhibitor, and slowed cell growth in a dose-dependent manner. In the mouse GBM xenograft model, Vpx-mediated SAMHD1 depletion reduced tumor growth and SAMHD1 knockout (KO) improved survival. In combination with IR or TMZ, SAMHD1 KO and exposure to 50% growth inhibitory dose (gID50) of VLP(+Vpx) displayed a synergistic effect, resulting in impaired HR, and improved LN-229 cells’ sensitivity to TMZ and IR. In conclusion, our finding demonstrates that SAMHD1 promotes GBM resistance to treatment, and it is a plausible therapeutic target to improve the efficacy of TMZ and IR in GBM. Furthermore, we show that Vpx could be a potential therapeutic tool that can be utilized to deplete SAMHD1 in GBM.

Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor in adults. GBM cells spread extensively in the brain and disseminate into the cerebrospinal fluid space, strongly restricting multimodal therapies. Acquiring a better knowledge of molecular defects underlying GBM invasion is essential for developing effective treatments.Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in the normal brain, but its expression is lost in the majority of GBM through epigenetic silencing and restoration of its expression can inhibit glioma growth. However, whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in GBM has not been investigated.Microarray analysis of the GBM TCGA dataset (restricted to IDHwt GBM; WHO 2021) revealed that low BAI1 mRNA expression correlates with elevated expression of many mesenchymal genes. Restoration of BAI1 expression in human GBM cells suppresses mesenchymal gene expression in culture and dramatically decreases brain tumor invasion in mice xenografts. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in GBM cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically suppresses cell invasion in the brain and reduces leptomeningeal dissemination of GBM cells to the spine in mouse xenografts.These experiments demonstrate that epigenetic silencing of BAI1 is important for activating the GBM invasive phenotype through TGFβ1 pathway activation. This new tumor-suppressive pathway can be epigenetically targeted to reactivate BAI1 expression in GBM patients.

BACKGROUND: Compelling evidence suggests that glioblastoma (GBM) recurrence results from the expansion of a subset of tumor cells with robust intrinsic or therapy-induced radioresistance. However, the mechanisms underlying GBM radioresistance and recurrence remain elusive. To overcome obstacles in radioresistance research, we present a novel preclinical model ideally suited for radiobiological studies. METHODS: With this model, we performed a screen and identified a radiation-tolerant persister (RTP) subpopulation. RNA sequencing was performed on RTP and parental cells to obtain mRNA and miRNA expression profiles. The regulatory mechanisms among NF-κB, YY1, miR-103a, XRCC3, and FGF2 were investigated by transcription factor activation profiling array analysis, chromatin immunoprecipitation, western blot analysis, luciferase reporter assays, and the MirTrap system. Transferrin-functionalized nanoparticles (Tf-NPs) were employed to improve blood–brain barrier permeability and RTP targeting. RESULTS: RTP cells drive radioresistance by preferentially activating DNA damage repair and promoting stemness. Mechanistic investigations showed that continual radiation activates the NF-κB signaling cascade and promotes nuclear translocation of p65, leading to enhanced expression of YY1, the transcription factor that directly suppresses miR-103a transcription. Restoring miR-103a expression under these conditions suppressed the FGF2–XRCC3 axis and decreased the radioresistance capability. Moreover, Tf-NPs improved radiosensitivity and provided a significant survival benefit. CONCLUSIONS: We suggest that the NF-κB–YY1–miR-103a regulatory axis is indispensable for the function of RTP cells in driving radioresistance and recurrence. Thus, our results identified a novel strategy for improving survival in patients with recurrent/refractory GBM.

Death receptor Fas-mediated apoptosis not only eliminates nonspecific and autoreactive B cells but also plays a major role in antitumor immunity. However, the possible mechanisms underlying impairment of Fas-mediated induction of apoptosis during lymphomagenesis remain unknown. In this study, we employed our developed syngeneic lymphoma model to demonstrate that downregulation of Fas is required for both lymphoma development and lymphoma cell survival to evade immune cytotoxicity. CD40 signal activation significantly restored Fas expression and thereby induced apoptosis after Fas ligand treatment in both mouse and human lymphoma cells. Nevertheless, certain human lymphoma cell lines were found to be resistant to Fas-mediated apoptosis, with Livin (melanoma inhibitor of apoptosis protein; ML-IAP) identified as a driver of such resistance. High expression of Livin and low expression of Fas were associated with poor prognosis in patients with aggressive non-Hodgkin's lymphoma. Livin expression was tightly driven by bromodomain and extraterminal (BET) proteins BRD4 and BRD2, suggesting that Livin expression is epigenetically regulated in refractory lymphoma cells to protect them from Fas-mediated apoptosis. Accordingly, the combination of CD40-mediated Fas restoration with targeting of the BET proteins–Livin axis may serve as a promising immunotherapeutic strategy for refractory B-cell lymphoma. Significance: These findings yield insights into identifying risk factors in refractory lymphoma and provide a promising therapy for tumors resistant to Fas-mediated antitumor immunity.

Medulloblastoma (MB) is the most aggressive pediatric brain tumor and a better understanding of this disease is warranted to develop new therapeutic approaches. Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is an orphan seven-transmembrane G protein-coupled receptor predominantly expressed in the brain and epigenetically silenced in MB formation. MDM2 is an E3 ubiquitin ligase that leads to the proteosomal degradation of multiple proteins. We previously found that BAI1 can prevent MDM2 nuclear activity by trapping it at the cell surface, and in the process suppress tumor formation by stabilizing p53. Since MDM2 also ubiquitinates cell surface proteins, we hypothesized that its membrane trapping might increase the degradation of oncogenic tyrosine kinase receptors. To explore this idea, we examined IGF1R, a known cell surface MDM2 target and found that BAI1 negatively regulates IGF1R at the protein level. In contrast, BAI1 expression did not change the protein expression levels of other RTKs (INSR, EGFR). Using co-immunoprecipitation assays we confirmed that MDM2 interacts with both BAI1 and IGF1R in MB cells. In addition, the interaction between IGF1R and β-Arrestin, which is crucial for ubiquitination and down-regulation of IGF1R by acting as an adaptor for MDM2, was increased with BAI1 expression. To examine the impact of BAI1 overexpression on IGF1 receptor signaling, we examined its downstream signaling mediators (Stat3, Akt, Erk). Stat3 and Akt phosphorylation were suppressed upon BAI1 expression, while Erk signaling was activated but through a mechanism independent from IGF1R expression. As IGF1R is known to drive radiation resistance, we examined whether BAI1 could radiosensitize the cells and found that this was indeed the case. Altogether, our data indicate that reactivating BAI1 in MB has dual anti-tumor effects by stabilizing p53 and blocking IGF1R signalling.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, and patients that develop metastases (~50%) survive

Finding novel treatment approaches for brain tumors is challenging, especially for malignant gliomas. A wealth of genetic and expression data on malignant glioma has accumulated in recent years; however, therapies targeting the underlying oncogenic pathways have not succeeded in substantially improving therapeutic responses or survival, owning to tumor resistance mechanisms to these therapies. Therefore, new therapeutic approaches are necessary to reduce the mortality rates in patients with glioma. This study examined current trends in preclinical research for malignant glioma to predict future therapies. This analysis revealed that current preclinical research mainly focused on the immune response and tumor microenvironment. Knowledge from these researches may lead to the development of new therapeutics for the management of malignant glioma.

Gut microbiota and their metabolites are instrumental in regulating homeostasis at intestinal and extraintestinal sites. However, the complex effects of prenatal and early postnatal microbial exposure on adult health and disease outcomes remain incompletely understood. Here, we showed that mice raised under germ-free conditions until weaning and then transferred to specific pathogen-free (SPF) conditions harbored altered microbiota composition, augmented inflammatory cytokine and chemokine expression, and were hyper-susceptible to colitis-associated tumorigenesis later in adulthood. Increased number and size of colon tumors and intestinal epithelial cell proliferation in recolonized germ-free mice were associated with augmented intratumoral CXCL1, CXCL2, and CXCL5 expression and granulocytic myeloid-derived suppressor cell (G-MDSC) accumulation. Consistent with these findings, CXCR2 neutralization in recolonized germ-free mice completely reversed the exacerbated susceptibility to colitis-associated tumorigenesis. Collectively, our findings highlight a crucial role for early-life microbial exposure in establishing intestinal homeostasis that restrains colon cancer in adulthood.

Many subtypes of brain tumors are highly malignant and resistant to chemo- and radio- therapy. Tumor cells can shift their phenotype in response to treatments, the so-called adaptive resistance. Adaptive resistance mechanisms in malignant brain tumors are still poorly understood, and effective treatments have not yet been developed. To unveil such mechanisms, we have developed unique new experimental models to identify the adaptive resistance mechanisms to fractionated radiation in malignant brain tumors. We performed repeated irradiation (2-5Gy every 3-4 days, 3-6 weeks) on 6 human Glioma stem cells (GSCs), 2 mouse GSCs and 4 medulloblastomas (MB) cells in vitro and examined how tumor cells adapt to repeated irradiation. Brain tumor cells demonstrated dynamic adaptation to fractionated irradiation. They rapidly altered cell proliferation, intercellular adhesion, and stemness and acquired strong radioresistance. To identify genes responsible for radio-resistance, we performed RNA-seq analysis and CRISPR library screening using primary and radioresistant cells. We found that N-cadherin was upregulated in the majority of radioresistant GSCs. Stably transfecting N-cadherin in parental GSC rendered them radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of b-catenin at the cell surface, which suppressed Wnt/b-catenin proliferative signaling, and reduced neural differentiation. Moreover, N-cadherin increased Clusterin secretion, which protected GSCs against apoptosis after radiation treatment. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, which caused an EMT-like phenotype change in GSCs. The N-cadherin-mediated radioresistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor. Adjuvant PPP combined with irradiation significantly extended the survival of orthotopically xenografted mice versus irradiation-only or drug-alone controls, supporting clinical translation. In conclusion, our data indicate that IGF1R inhibition can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive resistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/b-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability. These findings also confirmed that our radioresistant models effectively identify new adaptive resistance mechanisms in malignant brain tumors. (References: Osuka S, et. al., J Clin Invest. 2021;131(6):e136098) Citation Format: Satoru Osuka, Dan Zhu, Zhaobin Zhang, Chaoxi Li, Christian T. Stackhouse, Oltea Sampetrean, Jeffrey J. Olson, G. Yancey Gillespie, Hideyuki Saya, Christopher D. Willey, Erwin G. Van Meir. N-cadherin is a driver of adaptive radioresistance in malignant brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1430.

Medulloblastoma (MB) is the most common malignant brain tumor in children. MB tends to metastasize to the brain meninges and subarachnoid space and the spinal cord. Leptomeningeal metastasis is frequently found at initial diagnosis and leads to tumor relapse after standard treatment. Leptomeningeal metastasis remains a major challenge and is related with poor outcome. Acquiring a better knowledge of molecular defects underlying metastatic disease is essential for the development of effective therapies. Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in normal brain, but its expression is lost in the majority of medulloblastoma through epigenetic silencing. We reported that BAI1 protects p53 from Mdm2-mediated degradation and regulate tumor growth in medulloblastoma (Zhu D. et al, Cancer Cell, 2018). However, it is unclear whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in MB. Microarray analysis of the published MB dataset revealed that low BAI1 mRNA expression correlates with poor outcome and with expression of many key mesenchymal genes, including Fibronectin1, SLUG, and TWIST1. Restoration of BAI1 expression in human MB cells suppresses mesenchymal gene expression in culture, and dramatically decreases brain tumor invasion. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in MB cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically reduced tumor cell invasion of MB cells. These experiments demonstrate that epigenetic silencing of BAI1 is important for activation of the MB invasive phenotype through TGFβ1 pathway activation. Epigenetic targeting of this process by KCC-07 can reduce MB invasion.

Highlights • Regucalcin expression is reduced in the metastatic tumors of prostate cancer patients. • The progression-free survival rate is prolonged in patients with a higher regucalcin expression. • Overexpressed regucalcin suppresses the growth and bone activity of human prostate cancer cells. • Overexpressed regucalcin regulates many proteins in cell signaling and transcription processes. • Regucalcin is suppressor in human prostate cancer, suggesting a novel therapeutic strategy., Prostate cancer, which is a bone metastatic cancer, is the second leading cause of cancer-related death in men. There is no effective treatment for metastatic prostate cancer. Regucalcin has been shown to contribute as a suppressor in various types of human cancers. In the present study, furthermore, we investigate an involvement of regucalcin in suppression of prostate cancer. Regucalcin expression was compared in 131 primary tumor tissues and 19 metastatic tumor tissues in prostate cancer patients. Regucalcin expression in the metastatic tumor was found to be reduced as compared with that in primary tumor. The progression-free survival rate was prolonged in patients with a higher regucalcin expression. Translationally, overexpression of regucalcin in bone metastatic human prostate cancer PC-3 and DU-145 cells suppressed colony formation and cell growth in vitro. Mechanistically, overexpressed regucalcin enhanced the levels of p53, Rb, and p21, and decreased the levels of Ras, PI3 kinase, Akt, and mitogen-activated protein kinase, leading to suppression of cell growth. Furthermore, higher regucalcin expression suppressed the levels of nuclear factor-κB p65, β-catenin, and signal transducer and activator of transcription 3, which regulate a transcription activity. Cell growth was promoted by culturing with the calcium agonist Bay K 8644. This effect was blocked by overexpression of regucalcin. Notably, overexpressed regucalcin suppressed bone metastatic activity of PC-3 and DU-145 cells when cocultured with preosteoblastic or preosteoclastic cells. Regucalcin may suppress the development of human prostate cancer, suggesting that gene delivery systems in which its expression is forced may be a novel therapeutic strategy.

Purpose: Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide–Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells. Experimental Design: sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling. Results: We found sGal-3 preferentially binds to β1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-β1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival. Conclusions: We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin β1–dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

Glioblastoma (GBM) is composed of a variety of tumor cell populations including those with stem cell properties, known as glioma stem cells (GSCs). GSCs are innately less sensitive to radiation than the tumor bulk and are believed to drive GBM formation and recurrence following repeated irradiation. However, it is unclear how GSCs adapt to avoid the toxicity of repeated irradiation used in clinical practice. We established radioresistant human and mouse GSCs by exposing them to repeated rounds of irradiation in order to uncover critical mediators of adaptive radioresistance. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs reduced their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of β-catenin at the cell surface, which decreased Wnt/ β-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. Restoration of wild type N-cadherin, but not mutant N-cad lacking β-catenin binding region, led to increased radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and β-catenin. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype can be reversed with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation. Moreover, the elevation of N-cad and Clusterin are related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibitor can block the N-cadherin-mediated resistance pathway. Our research provides a deeper understanding of adaptive radioresistance after repeated irradiation, and validates the IGF1/N-cadherin/β-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability.

Glioblastoma (GBM) is composed of heterogeneous tumor cell populations including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance, we generated radioresistant human and mouse GSCs by exposing them to repeated cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties and N-cadherin expression. Stably transfecting N-cadherin in parental GSC rendered them radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of b-catenin at the cell surface, which suppressed Wnt/b-catenin proliferative signaling, and reduced neural differentiation. Transfection of wild type N-cadherin, but not mutant N-cadherin lacking the b-catenin binding region, restored radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and b-catenin. Moreover, N-cadherin increased Clusterin secretion, which protected GSCs against apoptosis after radiation treatment. N-cadherin knockout decreased Clusterin secretion and sensitized the cells to radiation therapy. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, which induced an EMT-like phenotype change in GSCs. The N-cadherin-mediated radioresistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor. Adjuvant PPP combined with irradiation significantly extended the survival of orthotopically xenografted mice versus irradiation-only or drug alone controls, supporting clinical translation. Moreover, elevated N-cadherin and Clusterin mRNA expression is related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibition can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive radioresistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/β-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability. Citation Format: Satoru Osuka, Dan Zhu, Zhaobin Zhang, Chaoxi Li, Christian T. Stackhouse, Oltea Sampetrean, Jeffrey J. Olson, G. Yancey Gillespie, Hideyuki Saya, Christopher D. Willey, Erwin G. Van Meir. IGF1/N-cadherin/b-catenin/Clusterin signaling axis can mediates adaptive radioresistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3078.

Glioblastoma (GBM) is the most common type of malignant brain tumor and has a very poor prognosis. Most patients relapse within 12 months despite aggressive treatment and patient outcome after recurrent is extremely worse. This study was designed to clarify the change of the molecular expression, including programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), on the initial and secondary resected tumor specimens and to address the influence of these expressions for patient outcome after second surgery of glioblastoma. We investigated 16 patients, ranging in age from 14 to 65 years, with histologically verified WHO grade IV GBM, whose original tumor was resected between 2008 and 2014, and treated with fractionated radiotherapy and temozolomide. Four patients who were treated with immunotherapy using autologous formalin-fixed tumor vaccine were enrolled. All of the patients underwent secondary resection after tumor recurrence within 24 months. We carried out an immunohistochemical examination of the initial and secondary resected tumors from patients using a panel of immune system molecular markers, and assessed whether marker expression correlated with clinical outcomes. CD3, CD8 and PD-1 on tumor-infiltrating lymphocytes was significantly increased in secondary resected specimens compared with initially resected specimens (p ≤ 0.05). All patients expressed PD-L1 on tumor cells in initial and secondary resection specimens. Patients were divided into high or low expression group by median IHC score of PD-1 on initial or secondary resected specimens. No significant differences in patient outcomes were observed between high and low PD-1 or PD-L1 groups of initially resected specimens. In high expression group of secondary resected specimens, most patients score had increased which compared with initial resected tumor specimens. The PD-1 high expression score group of secondary resected specimens was associated with long progression-free survival and short survival after recurrence. PD-L1 expression was detected in almost all initial and secondary specimens. Patients with high PD-1 expression of secondary specimen had bad prognosis after secondary resection. PD-1/PD-L1 pathway may be associated with patient outcome after second surgery of glioblastoma.

Regulatory and effector T helper (Th) cells are abundant at mucosal surfaces, especially in the intestine, where they control the critical balance between tolerance and inflammation. However, the key factors that reciprocally dictate differentiation along these specific lineages remain incompletely understood. Here we report that the interleukin-1 (IL-1) family member IL-36γ signals through IL-36 receptor, myeloid differentiation primary response gene 88, and nuclear factor-κBp50 in CD4+ T cells to potently inhibit Foxp3-expressing induced regulatory T cell (Treg) development, while concomitantly promoting the differentiation of Th9 cells via a IL-2-STAT5- (signal transducer and activator of transcription factor 5) and IL-4-STAT6-dependent pathway. Consistent with these findings, mice deficient in IL-36γ were protected from Th cell-driven intestinal inflammation and exhibited increased colonic Treg cells and diminished Th9 cells. Our findings thus reveal a fundamental contribution for the IL-36/IL-36R axis in regulating the Treg-Th9 cell balance with broad implications for Th cell-mediated disorders, such as inflammatory bowel diseases and particularly ulcerative colitis.

Regucalcin plays a crucial role as a suppressor of transcription signaling, and its diminished expression or activity may play a key role in human carcinogenesis. Higher regucalcin expression has been demonstrated to prolong survival of the patients of pancreatic cancer, breast cancer, and hepatocellular carcinoma. Moreover, we investigated an involvement of regucalcin in human lung cancer. Human non-small cell lung cancer (NSCLC) accounts for over 80% in human lung cancer and is one of the leading causes of malignancy-related mortality with fewer than 16% patients surviving beyond 5 years. In this study, gene expression and survival data of 204 lung adenocarcinoma patients were obtained through the gene expression omnibus database (GSE31210) for outcome analysis. Gene expression data demonstrated that prolonged survival in lung cancer patients is associated with higher regucalcin gene expression. Overexpression of regucalcin suppressed the proliferation, cell death, and migration of human lung adenocarcinoma NSCLC A549 cells in vitro. Mechanistically, regucalcin induced G1 and G2/M phase cell cycle arrest of A549 cells through suppression of multiple signaling pathways including Ras, Akt, MAP kinase, and SAPK/JNK. Moreover, overexpression of regucalcin caused decreases in the oncogenes c-fos and c-myc and elevation of the tumor suppressers p53 and Rb. These findings suggest that regucalcin may play a potential role as a suppressor of human lung cancer, and that downregulation of regucalcin expression may predispose patients to development of lung cancer. Overexpression of regucalcin using gene delivery may constitute a novel therapeutic approach to treating lung cancer.

Glioblastoma is the most common and lethal primary malignant brain tumor in adults. Patients die from recurrent tumors that have become resistant to therapy. New strategies are needed to design future therapies that target resistant cells. Recent genomic studies have unveiled the complexity of tumor heterogeneity in glioblastoma and provide new insights into the genomic landscape of tumor cells that survive and initiate tumor recurrence. Resistant cells also co-opt developmental pathways and display stem-like properties; hence we propose to name them recurrence-initiating stem-like cancer (RISC) cells. Genetic alterations and genomic reprogramming underlie the innate and adaptive resistance of RISC cells, and both need to be targeted to prevent glioblastoma recurrence.

Medulloblastoma (MB) are the most aggressive primary malignant brain tumors in children and are classified into four major molecular subgroups. WNT-MB is characterized by an activated WNT signature and has relatively better prognosis with treatment. However, young WNT-MB patients who survive after conventional treatment suffer from long-term irreversible neurocognitive and endocrine side effects leading to poor quality of life. ADGRB3 is a member of the ADGRB1-3 subfamily of adhesion GPCR transmembrane proteins, which are highly expressed in the cerebellum and hippocampal neurons. Our recent analysis of RNA-seq data from a published panel of MB tumor samples and RT-PCR experiments with tumor samples showed that ADGRB3 mRNA expression is selectively repressed in WNT-MB tumors compared to the other molecular subgroups and normal human cerebellum. We detected hypermethylation of the ADGRB3 promoter exclusively in WNT-MB samples. ChIP assays revealed enrichment of repressive methyl CpG binding MBD2 protein and trimethylated histone H3K9me3 in the ADGRB3 promoter region. We found that ATP dependent chromatin remodeler protein BRG-1 regulates ADGRB3 expression through epigenetic reprogramming at the gene promoter. Lentiviral reconstitution of ADGRB3 inhibited growth and WNT signaling in UW288-1, and PFSK-1 cells in culture. ADGRB3 reconstitution in UW288-1 MB cells causes a significant reduction in tumor growth and WNT target gene activation in orthotopic xenografts in nude mice. Furthermore, we found that ADGRB3 reactivation could be achieved using a blood brain barrier permeable MBD2 antagonist and an EZH2 inhibitor and this can significantly reduce cell growth and inhibit WNT signaling targets. Altogether, our findings define an epigenetic mechanism for ADGRB3 silencing in WNT-MB and demonstrated that reactivation of ADGRB3 can prevent WNT-MB tumor growth by suppressing WNT signaling. Our study supports translating epigenetic reactivation of ADGRB3 as a novel therapeutic intervention for children affected with WNT-MB.

Purpose: Uveal melanoma (UM) is the most prevalent and lethal intraocular malignancy in adults. Here, we examined the importance of hypoxia in UM growth and tested the antitumor effects of arylsulfonamide 64B, an inhibitor of the hypoxia-induced factor (HIF) pathway in animal models of UM and investigated the related mechanisms. Experimental Design: UM cells were implanted in the uvea of mice eyes and mice systemically treated with 64B. Drug effect on primary eye tumor growth, circulating tumor cells, metastasis formation in liver, and survival were examined. 64B effects on UM cell growth, invasion and hypoxia-induced expression of C-X-C chemokine receptor type 4 (CXCR4) and mesenchymal–epithelial transition factor (c-Met) were measured. Luciferase reporter assays, chromatin immunoprecipitation, co-immunoprecipitation, and cellular thermal shift assays were used to determine how 64B interferes with the HIF transcriptional complex. Results: Systemic administration of 64B had potent antitumor effects against UM in several orthotopic mouse models, suppressing UM growth in the eye (∼70% reduction) and spontaneous liver metastasis (∼50% reduction), and extending mice survival (P < 0.001) while being well tolerated. 64B inhibited hypoxia-induced expression of CXCR4 and c-Met, 2 key drivers of tumor invasion and metastasis. 64B disrupted the HIF-1 complex by interfering with HIF-1α binding to p300/CBP co-factors, thus reducing p300 recruitment to the MET and CXCR4 gene promoters. 64B could thermostabilize p300, supporting direct 64B binding to p300. Conclusions: Our preclinical efficacy studies support the further optimization of the 64B chemical scaffold toward a clinical candidate for the treatment of UM.

Original Article, Background: Glioma stem cells (GSCs) have an important role in tumor recurrence after treatment. We recently showed that, in a mouse model of glioma, GSCs adapt to repeated radiation exposures by changing their secretory profile and modulating survival signaling. Irradiated GSCs also reduce their rate of proliferation. In the present study we examined whether these changes require a specific adaptation of GSC energy metabolism. Methods: Initial tumors were established in mice by orthotopic implantation of Ink4a/Arf-null neural stem cells transduced with human HRASV12. GSCs isolated from the tumors were exposed (or not) to fractionated radiation (12 fractions of 5 Gy), and five clones in each group of cells were assessed for glucose consumption, lactate production, and content of intracellular metabolites. Results: Rates of glucose consumption and lactate production were lower in clones exposed to radiation than in nonirradiated clones, which suggests a reduction in glycolysis. Among intracellular metabolites, the high concentrations of intermediates of glycolysis and nucleoside metabolism were reduced in irradiated clones, whereas levels of essential amino acids were largely unchanged. Conclusions: Repeated radiation changes the metabolic preferences of GSCs, in conjunction with their slowed proliferation. Future studies should thus investigate intra- and extracellular metabolites, to identify potential diagnostic and therapeutic targets for glioma recurrence.

Human breast cancer is highly metastatic to bone and drives bone turnover. Breast cancer metastases cause osteolytic lesions and skeletal damage that leads to bone fractures. Regucalcin, which plays a pivotal role as an inhibitor of signal transduction and transcription activity, has been suggested to act as a suppressor of human cancer. In the present study, we compared the clinical outcome between 44 breast cancer patients with higher regucalcin expression and 43 patients with lower regucalcin expression. Prolonged relapse-free survival was identified in the patients with increased regucalcin gene expression. We further demonstrated that overexpression of full length, but not alternatively spliced variants of regucalcin, induces G1 and G2/M phase cell cycle arrest, suppressing the proliferation of MDA-MB-231 cells, a commonly used in vitro model of human breast cancer that metastasize to bone causing osteolytic lesions. Overexpression of regucalcin was found to suppress multiple signaling pathways including Akt, MAP kinase and SAPK/JNK, and NF-κB p65 and β-catenin along with increased p53, a tumor suppressor, and decreased K-ras, c-fos and c-jun. Moreover, we found that co-culture of regucalcin-overexpressing MDA-MB-231 cells with mouse bone marrow cells prevented enhanced osteoclastogenesis and suppressed mineralization in mouse bone marrow cells in vitro. Taken together, the present study suggests that regucalcin may have important anticancer properties in human breast cancer patients. Mechanistically, these effects are likely mediated through suppression of multiple signaling pathways, upregulation of p53 and downregulation of oncogenes leading to anti-proliferative effects and reduced metastases to bone, a phenotype associated with poor clinical outcome.

Approximately 90% of all pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is a highly aggressive malignancy and is one of the deadliest. This poor clinical outcome is due to the prominent resistance of pancreatic cancer to drug and radiation therapies. Regucalcin plays a pivotal role as a suppressor protein in signal transduction in various types of cells including tumor tissues. We demonstrated that the prolonged survival is induced in PDAC patients with increased regucalcin gene expression using a dataset of PDAC obtained from GEO database (GSE17891) together with the clinical annotation data file. Moreover, overexpression of regucalcin with full length was demonstrated to suppress the proliferation, cell death and migration in human pancreatic cancer MIA PaCa-2 (K-ras mutated) cells that possess resistance to drug and radiation therapies. Suppressive effects of regucalcin on cell proliferation and death were not seen in the cells overexpressed with regucalcin cDNA alternatively spliced variants (deleted exon 4 or deleted exon 4 and 5). Regucalcin was suggested to induce G1 and G2/M phase cell cycle arrest in MIA PaCa-2 cells. Suppressive effects of regucalcin on cell proliferation were independent of cell death. Overexpression of regucalcin was found to suppress signaling pathways including Akt, MAP kinase and SAPK/JNK, to increase the protein levels of p53, a tumor suppresser, and to decrease K-ras, c-fos and c-jun, a oncogene, by suppressing signaling pathways that are related to signaling of K-ras. Regucalcin may play a potential role as a suppressor protein in human pancreatic cancer.

Neutrophils loaded with cationic liposomal paclitaxel migrate across the blood/brain barrier to deliver chemotherapeutic nanoparticles in the inflamed post-surgical tumour margin.

Glioblastoma (GBM) is composed of heterogeneous tumor cell populations including those with stem cell properties, termed glioma stem cells (GSCs). GSCs are innately less radiation sensitive than the tumor bulk and are believed to drive GBM formation and recurrence after repeated irradiation. However, it is unclear how GSCs adapt to escape the toxicity of repeated irradiation used in clinical practice. To identify important mediators of adaptive radioresistance, we generated radioresistant human and mouse GSCs by exposing them to repeat cycles of irradiation. Surviving subpopulations acquired strong radioresistance in vivo, which was accompanied by increased cell-cell adhesion, slower proliferation, an elevation of stemness properties and N-cadherin expression. Increasing N-cadherin expression rendered parental GSCs radioresistant, reduced their proliferation, and increased their stemness and intercellular adhesive properties. Conversely, radioresistant GSCs lost their acquired phenotypes upon CRISPR/Cas9-mediated knockout of N-cadherin. Mechanistically, elevated N-cadherin expression resulted in the accumulation of b-catenin at the cell surface, which suppressed Wnt/ b-catenin proliferative signaling, reduced neural differentiation, and protected against apoptosis through Clusterin secretion. Restoration of wild type N-cadherin, but not mutant N-cad lacking b-catenin binding region, led to induce radioresistance in N-cadherin knockout GSCs, indicating the importance of the binding between N-cadherin and b-catenin. We also demonstrated that N-cadherin upregulation was induced by radiation-induced IGF1 secretion, and the radiation resistance phenotype could be reverted with picropodophyllin (PPP), a clinically applicable blood-brain-barrier permeable IGF1 receptor inhibitor, supporting clinical translation. Moreover, the elevation of N-cad and Clusterin are related to prognosis of GBM in the TCGA dataset. In conclusion, our data indicate that IGF1R inhibitor can block the N-cadherin-mediated resistance pathway. Our study deepens our understanding of adaptive radioresistance during repeated irradiation in GBM, and validates the IGF1/N-cadherin/b-catenin/Clusterin signaling axis as a novel target for radio-sensitization, which has direct therapeutic applicability.

Adhesion G-protein coupled receptors (ADGRs) are transmembrane proteins involved in cell-cell/matrix interactions. We show ADGRB1 gene, which encodes Brain-specific angiogenesis inhibitor 1 (BAI1), is epigenetically silenced in human medulloblastomas through a methyl-CpG binding protein 2 (MBD2)-dependent mechanism. Knockout of Adgrb1 in mice augments proliferation of cerebellar granule neuron precursors (GNPs), and increases medulloblastoma penetrance and accelerated death in Ptch1(+/-) mice. BAI1 prevents Mdm2-mediated p53 polyubiquitination, and its loss substantially reduces p53 levels. Reactivation of BAI1/p53 signaling axis by targeting MBD2 pathway suppresses human medulloblastoma growth in orthotopic xenograft models. Our findings highlight the importance of BAI1 loss in medulloblastoma and demonstrate that epigenetic restoration of its expression with a new brain-permeable MBD2 inhibitor has therapeutic potential. Revealing BAI1 as a physiological tumor suppressor in medulloblastoma unveils a direct crosstalk between ADGRs and p53 signaling, and provides a causal relationship between ADGRs and cancer. The discovery of a novel upstream regulator of the p53 tumor suppressor is highly significant because of this pathway’s involvement in many cancers. Disruption of the BAI1/mdm2/p53 signaling axis through BAI1 silencing reveals a vulnerability in cancer, and offers an opportunity for therapeutic exploitation through epigenetic reactivation. We provide proof-of-principle that this can be achieved with a chemical scaffold targeting MBD2, and this lead molecule is actionable for translation into a first-in-class therapeutic intervention against medulloblastoma, and possibly other cancers (Zhu D et al, Cancer Cell, in press).

Adhesion G-protein coupled receptors (ADGRs) are transmembrane proteins involved in cell-cell/matrix interactions. We found that the ADGRB1 gene, which encodes Brain-specific angiogenesis inhibitor 1 (BAI1), is epigenetically silenced in human medulloblastomas through a methyl-CpG binding protein 2 (MBD2)-dependent mechanism. We revealed that BAI1 acts as a tumor suppressor by preventing Mdm2-mediated p53 polyubiquitination, and its loss substantially reduces p53 levels. We found BAI1 directly binds to mdm2 and sequesters it from p53. Reactivation of BAI1/p53 signaling axis by targeting the MBD2 pathway with a new brain-permeable MBD2 inhibitor suppressed human medulloblastoma growth in orthotopic xenograft models. Our findings highlight the importance of BAI1 loss in medulloblastoma and demonstrate that epigenetic restoration of its expression has therapeutic potential. Disruption of the BAI1/mdm2/p53 signaling axis through BAI1 silencing reveals a vulnerability in cancer, and offers an opportunity for therapeutic exploitation through epigenetic reactivation. We provide proof-of-principle that this can be achieved with a chemical scaffold targeting MBD2, and this lead molecule is actionable for translation into a first-in-class therapeutic intervention against medulloblastoma, and possibly other cancers (Zhu D et al, Cancer Cell, in press).

Renal cell carcinoma (RCC), which is a type of cancer found in the kidney tubule, is among the 10 most frequently occurring human cancers. Regucalcin plays a potential role as a regulator of transcriptional activity, and its downregulated expression or activity may contribute to the promotion of human cancers. In this study, we investigated the involvement of regucalcin in human RCC. Regucalcin expression was compared in 23 normal and 29 tumor samples of kidney cortex tissues of patients with clear cell RCC obtained through the Gene Expression Omnibus (GEO) database (GSE36895). Regucalcin expression was downregulated in the tumor tissues. The prolonged survival of patients with clear cell RCC was demonstrated to be associated with a higher regucalcin gene expression in the TCGA dataset. The overexpression of regucalcin suppressed the colony formation, proliferation and the death of human clear cell RCC A498 cells in vitro. Mechanistically, the overexpression of regucalcin induced the G1 and G2/M phase cell cycle arrest of A498 cells through the suppression of multiple signaling components, including Ras, PI3 kinase, Akt and mitogen-activated protein (MAP) kinase. Importantly, the overexpression of regucalcin led to an elevation in the levels of the tumor suppressors, p53, Rb and the cell cycle inhibitor, p21. The levels of the transcription factors, c-fos, c-jun, nuclear factor-κB p65, β-catenin and signal transducer and activator of transcription 3, were suppressed by regucalcin overexpression. On the whole, the findings of this study suggest that regucalcin plays a suppressive role in the promotion of human RCC. The overexpression of regucalcin by gene delivery systems may thus prove to be a novel therapeutic strategy for RCC.

Regucalcin plays a crucial role as a regulator of transcriptional signaling activity, and its decreased expression or activity may contribute to the promotion of human carcinogenesis. A higher regucalcin expression in the tumor tissues has been demonstrated to prolong the survival of patients with various types of cancer, including pancreatic cancer, breast cancer, liver cancer and lung adenocarcinoma. The involvement of regucalcin in human colorectal cancer was investigated in the current study. Regucalcin gene expression and the survival data of 62 patients with colorectal cancer were obtained though the Gene Expression Omnibus (GEO) database (GSE12945) for outcome analysis. The data of gene expression revealed that the prolonged survival of patients with colorectal cancer was associated with a higher regucalcin gene expression in tumor tissues. The overexpression of regucalcin suppressed colony formation and proliferation, and induced the death of human colorectal carcinoma RKO cells cultured in a medium containing fetal bovine serum in vitro. Mechanistically, the overexpression of regucalcin induced the G1 and G2/M phase cell cycle arrest of the RKO cells through the suppression of multiple signaling pathways, including Ras, Akt, mitogen-activated protein (MAP) kinase and SAPK/JNK. Of note, the overexpression of regucalcin induced an increase in the levels of the tumor suppressors, p53 and Rb, and the cell cycle inhibitor, p21. Moreover, the levels of the transcription factors, c‑fos, c‑jun, nuclear factor (NF)‑κB p65, β-catenin and signal transducer and activator of transcription 3 (Stat3), were suppressed by the overexpression of regucalcin. On the whole, the findings of this study suggest that regucalcin plays a crucial role as a suppressor in human colorectal cancer, and that the suppressed expression of the regucalcin gene may predispose patients to the promotion of colorectal cancer. The overexpression of regucalcin by gene delivery may thus prove to be a novel therapeutic strategy for colorectal cancer.