Effects of pharmacological calcimimetics on colorectal cancer cells over-expressing the human calcium-sensing receptor

AbstractThe calcium-sensing receptor (CaSR) is a ubiquitously expressed multifunctional G protein-coupled receptor. Several studies reported that the CaSR plays an anti-inflammatory and anti-tumorigenic role in the intestine, and that it is down-regulated during colorectal carcinogenesis. We hypothesized that intestine-specific positive allosteric CaSR modulators (type II calcimimetics) could be used for the treatment of intestinal pathologies. Therefore, the aim of this study was to determine the effect of pharmacological stimulation of CaSR on gene expression in vitro and on tumor growth in vivo.We stably transduced two colon cancer cell lines (HT29 and Caco2) with lentiviral vectors containing either the CaSR fused to GFP or GFP only. Using RNA sequencing, RT-qPCR experiments and ELISA, we determined that CaSR over-expression itself had generally little effect on gene expression in these cells. However, treatment with 1μM of the calcimimetic NPS R-568 increased the expression of pro-inflammatory factors such as IL-23α and IL-8 and reduced the transcription of various differentiation markers in the cells over-expressing the CaSR. In vivo, neither the presence of the CaSR nor p.o. treatment of the animals with the calcimimetic cinacalcet affected tumor growth, tumor cell proliferation or tumor vascularization of murine HT29 xenografts.In summary, CaSR stimulation in CaSR over-expressing cells enhanced the expression of inflammatory markers in vitro, but was not able to repress colorectal cancer tumorigenicity in vivo. These findings suggest potential pro-inflammatory effects of the CaSR and type II calcimimetics in the intestine.

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RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽

10.1038/s41388-021-01877-4 ◽

2021 ◽

Author(s):

Jiuna Zhang ◽

Xiaoyu Jiang ◽

Jie Yin ◽

Shiying Dou ◽

Xiaoli Xie ◽

...

Keyword(s):

Colorectal Cancer ◽

Plasma Membrane ◽

Tumor Growth ◽

Cancer Progression ◽

Critical Role ◽

Ring Finger ◽

Immunohistochemical Analysis ◽

Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

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Peptide Blocking Self-Polymerization of Extracellular Calcium-Sensing Receptor Attenuates Hypoxia-Induced Pulmonary Hypertension

Hypertension ◽

10.1161/hypertensionaha.120.16712 ◽

2021 ◽

Author(s):

Rui Xiao ◽

Shengquan Luo ◽

Ting Zhang ◽

Yankai Lv ◽

Tao Wang ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Disulfide Bonds ◽

Acute Hypoxia ◽

Extracellular Domain ◽

Left Ventricular ◽

Extracellular Calcium ◽

Calcium Sensing Receptor ◽

Calcium Sensing

Activation of the CaSR (extracellular calcium-sensing receptor) has been recognized as a critical mediator of hypoxia-induced pulmonary hypertension. Preventive targeting of the early initiating phase as well as downstream events after CaSR activation remains unexplored. As a representative of the G protein-coupled receptor family, CaSR polymerizes on cell surface upon stimulation. Immunoblotting together with MAL-PEG technique identified a reactive oxygen species-sensitive CaSR polymerization through its extracellular domain in pulmonary artery smooth muscle cells upon exposure to acute hypoxia. Fluorescence resonance energy transfer screening employing blocking peptides determined that cycteine129/131 residues in the extracellular domain of CaSR formed intermolecular disulfide bonds to promote CaSR polymerization. The monitoring of intracellular Ca 2+ signal highlighted the pivotal role of CaSR polymerization in its activation. In contrast, the blockade of disulfide bonds formation using a peptide decreased both CaSR and hypoxia-induced mitogenic factor expression as well as other hypoxic-related genes in vitro and in vivo and attenuated pulmonary hypertension development in rats. The blocking peptide did not affect systemic arterial oxygenation in vivo but inhibited acute hypoxia-induced pulmonary vasoconstriction. Pharmacokinetic analyses revealed a more efficient lung delivery of peptide by inhaled nebulizer compared to intravenous injection. In addition, the blocking peptide did not affect systemic arterial pressure, body weight, left ventricular function, liver, or kidney function or plasma Ca 2+ level. In conclusion, a peptide blocking CaSR polymerization reduces its hypoxia-induced activation and downstream events leading to pulmonary hypertension and represents an attractive inhaled preventive alternative worthy of further development.

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Promotion of Tumor Growth by ADAMTS4 in Colorectal Cancer: Focused on Macrophages

Cellular Physiology and Biochemistry ◽

10.1159/000489245 ◽

2018 ◽

Vol 46 (4) ◽

pp. 1693-1703 ◽

Cited By ~ 10

Author(s):

Jianjun Chen ◽

Yang Luo ◽

Yong Zhou ◽

Shaolan Qin ◽

Yier Qiu ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Proliferation ◽

Tumor Growth ◽

Expression Profile ◽

Copy Number ◽

Copy Number Alterations ◽

Extracellular Proteases ◽

Proliferation And Invasion

Background/Aims: ADAMTSs (A disintegrin and metalloprotease domains with thrombospondins motifs) are a family of extracellular proteases that have been related to both oncogenic and tumor-suppressive functions. The aim of the present study was to investigate: 1) the mutation, copy-number alterations, and expression profile of ADAMTSs in colorectal cancer and 2) whether ADAMTSs participate in colorectal cancer (CRC) progression and invasion. Methods: The mutation, copy-number alterations, and expression profile of ADAMTSs in CRC were analyzed in the TCGA cohort using cBioportal. ADAMTS4 expression in tumor tissues and cell lines were determined by immunostaining and real-time quantitative PCR. The role of ADAMTS-4 in CRC progression and the underlying mechanisms were studied by using short hairpin RNA-mediated knockdown of ADAMTS4. The effects of ADAMTS4 in cell proliferation and invasion were determined by clone formation assay and transwell migration assay, respectively. Macrophages were depleted by liposomal clodronate in immune-competent BALB/c mice and tumor growth was analyzed. Results: ADAMTS4 was differentially expressed in CRC and predicted a poor prognosis. Elevated ADAMTS4 expression was closely associated with larger tumor size, enhanced TNM stage, and a poor clinical outcome in patients with CRC. ADAMTS4 knockdown had no inhibitory implications on cell proliferation and invasion in vitro, but significantly attenuated tumor growth in vivo. Mechanistically, we revealed that ADAMTS4 was associated macrophages infiltration and polarization in the tumor microenvironment of CRC. Macrophage depletion largely abolished the promotive effect of ADAMTS4 on tumor growth in the immune competent BALB/c mice. Conclusion: ADAMTS4 seemed to be a promising prognostic indicator in CRC. The novel link between ADAMTS4 and macrophages mirrors the potential regulatory roles of ADAMTSs in the inflammatory microenvironment of cancers.

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A specific Upregulated lncRNA in Colorectal Cancer Promotes Cancer Progression by Modulating miR-185-5p/CCND2 Axis

10.21203/rs.3.rs-923878/v1 ◽

2021 ◽

Author(s):

Junshu Li ◽

Yanhong Ji ◽

Na Chen ◽

Huiling Wang ◽

Chao Fang ◽

...

Keyword(s):

Colorectal Cancer ◽

Network Analysis ◽

Tumor Growth ◽

Cancer Progression ◽

Gene Mutations ◽

Disease Free Survival ◽

Luciferase Reporter ◽

Colorectal Cancer Patients

Abstract BackgroundAdenomatous polyposis coli (APC) gene mutations were found in most colorectal cancer patients and functioned as an important inducer of tumorigenesis. Long non-coding RNA (lncRNA) plays a crucial role in the pathogenesis of various diseases, including colorectal cancer (CRC). Here we investigated the role of SURC which is specific upregulated in CRC progression. MethodsBased on the previous microarray results, weighted correlation network analysis (WGCNA) and lncRNA-mRNA co-expression network analysis were used to identify a lncRNA (SURC) and found it was specific up-regulated in CRC patients by qPCR and FISH staining. Chromatin immunoprecipitation (ChIP) assay was used to demonstrate the regulatory effect and mechanism of APC mutation on SURC expression. The effects of SURC on proliferation and cell cycle were determined by in vitro and in vivo experiments. Chromatin Isolation by RNA Purification (CHIRP) and luciferase reporter assay were carried out to illustrate the interaction between SURC, miR-185-5p and CCND2.ResultsWe found that SURC was specific up-regulated in CRC, but not in other solid tumor, when compared with normal adjacent tissues. High expression of SURC correlates with poorer disease-free survival and overall survival of CRC patients. Mutated APC protein resulted in stabilization of β-catenin in CRC, which promotes the transcription of SURC via binding to its promoter. Knockdown of SURC impaired CRC cell proliferation, colony formation and CRC tumor growth. Mechanistically, after transcription, SURC was transferred to cytoplasm and inhibits miR-185-5p expression via binding to miR-185-5p and inhibiting the synthesis of miR-185-5p from pri-miR-185-5p, which results in CCND2 expression.ConclusionCollectively, these results indicated that SURC promoted CRC tumor growth via interacting with miR-185-5p and regulating the activity of miR-185-5p/CCND2 axis which would be a novel diagnosis and prognosis prediction target for CRC.

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Targeting OLFML3 in Colorectal Cancer Suppresses Tumor Growth and Angiogenesis, and Increases the Efficacy of Anti-PD1 Based Immunotherapy

Cancers ◽

10.3390/cancers13184625 ◽

2021 ◽

Vol 13 (18) ◽

pp. 4625

Author(s):

Jimmy Stalin ◽

Beat A. Imhof ◽

Oriana Coquoz ◽

Rachel Jeitziner ◽

Philippe Hammel ◽

...

Keyword(s):

Colorectal Cancer ◽

Tumor Growth ◽

Molecular Subtype ◽

Human Cancer ◽

Tumor Development ◽

Tumor Grade ◽

Tissue Expression ◽

In Vivo Studies

The role of the proangiogenic factor olfactomedin-like 3 (OLFML3) in cancer is unclear. To characterize OLFML3 expression in human cancer and its role during tumor development, we undertook tissue expression studies, gene expression analyses of patient tumor samples, in vivo studies in mouse cancer models, and in vitro coculture experiments. OLFML3 was expressed at high levels, mainly in blood vessels, in multiple human cancers. We focused on colorectal cancer (CRC), as elevated expression of OLFML3 mRNA correlated with shorter relapse-free survival, higher tumor grade, and angiogenic microsatellite stable consensus molecular subtype 4 (CMS4). Treatment of multiple in vivo tumor models with OLFML3-blocking antibodies and deletion of the Olfml3 gene from mice decreased lymphangiogenesis, pericyte coverage, and tumor growth. Antibody-mediated blockade of OLFML3 and deletion of host Olfml3 decreased the recruitment of tumor-promoting tumor-associated macrophages and increased infiltration of the tumor microenvironment by NKT cells. Importantly, targeting OLFML3 increased the antitumor efficacy of anti-PD-1 checkpoint inhibitor therapy. Taken together, the results demonstrate that OLFML3 is a promising candidate therapeutic target for CRC.

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TRPC1 promotes the genesis and progression of colorectal cancer via activating CaM-mediated PI3K/AKT signaling axis

Oncogenesis ◽

10.1038/s41389-021-00356-5 ◽

2021 ◽

Vol 10 (10) ◽

Author(s):

Yang Sun ◽

Chen Ye ◽

Wen Tian ◽

Wen Ye ◽

Yuan-Yuan Gao ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Cycle ◽

Tumor Growth ◽

Cell Cycle Progression ◽

Akt Signaling ◽

Cycle Progression ◽

Colorectal Tumorigenesis ◽

Signaling Axis

AbstractTransient receptor potential canonical (TRPC) channels are the most prominent nonselective cation channels involved in various diseases. However, the function, clinical significance, and molecular mechanism of TRPCs in colorectal cancer (CRC) progression remain unclear. In this study, we identified that TRPC1 was the major variant gene of the TRPC family in CRC patients. TRPC1 was upregulated in CRC tissues compared with adjacent normal tissues and high expression of TRPC1 was associated with more aggressive tumor progression and poor overall survival. TRPC1 knockdown inhibited cell proliferation, cell-cycle progression, invasion, and migration in vitro, as well as tumor growth in vivo; whereas TRPC1 overexpression promoted colorectal tumor growth and metastasis in vitro and in vivo. In addition, colorectal tumorigenesis was significantly attenuated in Trpc1-/- mice. Mechanistically, TRPC1 could enhance the interaction between calmodulin (CaM) and the PI3K p85 subunit by directly binding to CaM, which further activated the PI3K/AKT and its downstream signaling molecules implicated in cell cycle progression and epithelial-mesenchymal transition. Silencing of CaM attenuated the oncogenic effects of TRPC1. Taken together, these results provide evidence that TRPC1 plays a pivotal oncogenic role in colorectal tumorigenesis and tumor progression by activating CaM-mediated PI3K/AKT signaling axis. Targeting TRPC1 represents a novel and specific approach for CRC treatment.

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AVIDIO as a novel oncolytic immunotherapy platform for the treatment of colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e15210 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e15210-e15210

Author(s):

Bijan Almassian ◽

Bhaskara R Madina ◽

Ju Chen ◽

Xiaoyang Ye ◽

Marie M Krady ◽

...

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

Tumor Growth ◽

Semliki Forest Virus ◽

Tumor Model ◽

Oncolytic Viruses ◽

Immunomodulatory Agents ◽

Oncolytic Activity

e15210 Background: Colorectal cancer is the third deadliest of all cancers causing more than 50,000 deaths per year in the U.S. Oncolytic viruses have seen limited use for the treatment of cancers, and further improvement of these methods with immune-modulating activities may prove crucial for the effectiveness of these agents in the treatment of human malignancies. To this end, we developed an artificial virus for infectious diseases and immuno-oncology (AVIDIO) platform that employs virus-like vesicles (VLV) for both the delivery of immunomodulatory agents to tumors and oncolytic activity. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Both unarmed VLVs and VLVs armed with the p35 subunit of IL-12 (VLV-IL12p35), an immunomodulatory cytokine that can induce Th1-mediated immunity, were tested for oncolytic activity against various cancer cell lines, including MC38 colorectal cancer cells, in vitro. Using the MC38 syngeneic murine tumor model, we evaluated the antitumor activity of VLV-IL-12p35 in vivo. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with VLV-IL-12p35 to monitor its antitumor and immunomodulatory activities, respectively. Results: VLV-IL-12p35 showed robust oncolytic activity against MC38 cells in vitro, killing over 80% of cells within 24 h. Treatment of intradermal MC38 tumors by intra-tumoral delivery of VLV-IL-12p35 resulted in more than 65% suppression of tumor growth within 2 weeks ( p< 0.05). VLV-IL-12p35-treated tumors also harbored significantly more CD8+ T cells, IFN-gamma-producing CD4+ T cells, and reduced numbers of Foxp3+ regulatory T cells. Conclusions: Our results show that VLV-IL-12p35 derived from the AVIDIO platform has oncolytic activity in vitro and antitumor and immunomodulatory activities in vivo. Therefore, AVIDIO is a promising platform for the delivery of immunomodulatory agents to tumors. Further optimization of the platform, including the addition of other immunomodulatory agents, is in progress to advance the AVIDIO platform to clinical applications for colorectal cancer.

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Calcium-Sensing Receptor and Aquaporin 2 Interplay in Hypercalciuria-Associated Renal Concentrating Defect in Humans. An In Vivo and In Vitro Study

PLoS ONE ◽

10.1371/journal.pone.0033145 ◽

2012 ◽

Vol 7 (3) ◽

pp. e33145 ◽

Cited By ~ 45

Author(s):

Giuseppe Procino ◽

Lisa Mastrofrancesco ◽

Grazia Tamma ◽

Domenica Rita Lasorsa ◽

Marianna Ranieri ◽

...

Keyword(s):

In Vitro Study ◽

Vitro Study ◽

Calcium Sensing Receptor ◽

Aquaporin 2 ◽

Calcium Sensing ◽

Concentrating Defect

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G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618706114 ◽

2017 ◽

Vol 114 (27) ◽

pp. 7077-7082 ◽

Cited By ~ 46

Author(s):

Francesco Casciello ◽

Fares Al-Ejeh ◽

Greg Kelly ◽

Donal J. Brennan ◽

Shin Foong Ngiow ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Tumor Growth ◽

Cellular Proliferation ◽

Gene Signature ◽

Breast Cancer Patients ◽

Hypoxic Response ◽

And Migration

G9a is an epigenetic regulator that methylates H3K9, generally causing repression of gene expression, and participates in diverse cellular functions. G9a is genetically deregulated in a variety of tumor types and can silence tumor suppressor genes and, therefore, is important for carcinogenesis. Although hypoxia is recognized to be an adverse factor in tumor growth and metastasis, the role of G9a in regulating gene expression in hypoxia has not been described extensively. Here, we show that G9a protein stability is increased in hypoxia via reduced proline hydroxylation and, hence, inefficient degradation by the proteasome. This inefficiency leads to an increase in H3K9me2 at its target promoters. Blocking the methyltransferase activity of G9a inhibited cellular proliferation and migration in vitro and tumor growth in vivo. Furthermore, an increased level of G9a is a crucial factor in mediating the hypoxic response by down-regulating the expression of specific genes, includingARNTL,CEACAM7,GATA2,HHEX,KLRG1, andOGN. This down-regulation can be rescued by a small molecule inhibitor of G9a. Based on the hypothesis that the changes in gene expression would influence patient outcomes, we have developed a prognostic G9a-suppressed gene signature that can stratify breast cancer patients. Together, our findings provide an insight into the role G9a plays as an epigenetic mediator of hypoxic response, which can be used as a diagnostic marker, and proposes G9a as a therapeutic target for solid cancers.

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Hsp90-stabilized MIF supports tumor progression via macrophage recruitment and angiogenesis in colorectal cancer

Cell Death and Disease ◽

10.1038/s41419-021-03426-z ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Luisa Klemke ◽

Tiago De Oliveira ◽

Daria Witt ◽

Nadine Winkler ◽

Hanibal Bohnenberger ◽

...

Keyword(s):

Colorectal Cancer ◽

Epithelial Cells ◽

Tumor Growth ◽

Tumor Progression ◽

Hsp90 Inhibitor ◽

Growth Defects ◽

Macrophage Recruitment ◽

Upstream Regulator

AbstractMacrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity, but its expression is increased in some cancers via stabilization with HSP90-associated chaperones. Here, we show that MIF stabilization is tumor-specific in an acute colitis-associated colorectal cancer (CRC) mouse model, leading to tumor-specific functions and selective therapeutic vulnerabilities. Therefore, we demonstrate that a Mif deletion reduced CRC tumor growth. Further, we define a dual role for MIF in CRC tumor progression. Mif deletion protects mice from inflammation-associated tumor initiation, confirming the action of MIF on host inflammatory pathways; however, macrophage recruitment, neoangiogenesis, and proliferative responses are reduced in Mif-deficient tumors once the tumors are established. Thus, during neoplastic transformation, the function of MIF switches from a proinflammatory cytokine to an angiogenesis promoting factor within our experimental model. Mechanistically, Mif-containing tumor cells regulate angiogenic gene expression via a MIF/CD74/MAPK axis in vitro. Clinical correlation studies of CRC patients show the shortest overall survival for patients with high MIF levels in combination with CD74 expression. Pharmacological inhibition of HSP90 to reduce MIF levels decreased tumor growth in vivo, and selectively reduced the growth of organoids derived from murine and human tumors without affecting organoids derived from healthy epithelial cells. Therefore, novel, clinically relevant Hsp90 inhibitors provide therapeutic selectivity by interfering with tumorigenic MIF in tumor epithelial cells but not in normal cells. Furthermore, Mif-depleted colonic tumor organoids showed growth defects compared to wild-type organoids and were less susceptible toward HSP90 inhibitor treatment. Our data support that tumor-specific stabilization of MIF promotes CRC progression and allows MIF to become a potential and selective therapeutic target in CRC.

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