Axin2 Expression Research Articles

Sonic Hedgehog and WNT Signaling Regulate a Positive Feedback Loop Between Intestinal Epithelial and Stromal Cells to Promote Epithelial Regeneration

Active intestinal stem cells are prone to injury by ionizing radiation. We previously showed that upon radiation-induced injury, normally quiescent reserve intestinal stem cells (rISCs) (marked by BMI1) are activated by Musashi-1 (MSI1) and exit from the quiescent state to regenerate the intestinal epithelium. This study aims to further establish the mechanism that regulates activation of Bmi1-CreER;Rosa26eYFP (Bmi1-CreER) rISCs following γ radiation-induced injury. Bmi1-CreER mice were treated with tamoxifen to initiate lineage tracing of BMI1 (eYFP+) cells and exposed to 12 Gy of total body γ irradiation or sham. Intestinal tissues were collected and analyzed by immunofluorescence, Western blot, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and chromatin immunoprecipitation real-time polymerase chain reaction. After irradiation, increased expression of Msi1 in eYFP+ cells was accompanied by increased expression of Axin2, a WNT marker. Promoter studies of the Msi1 gene indicated that Msi1 is a WNT target gene. Coculture of stromal cells isolated from irradiated mice stimulated Bmi1-CreER-derived organoid regeneration more effectively than those from sham mice. Expression of WNT ligands, including Wnt2b, Wnt4, Wnt5a, and Rspo3, was increased in irradiated stromal cells compared with sham-treated stromal cells. Moreover, expression of the Sonic hedgehog (SHH) effector Gli1 was increased in stromal cells from irradiated mice. This was correlated with an increased expression of SHH in epithelial cells postirradiation, indicating epithelial-stromal interaction. Finally, preinjury treatment with SHH inhibitor cyclopamine significantly reduced intestinal epithelial regeneration and Msi1 expression postirradiation. Upon ionizing radiation-induced injury, intestinal epithelial cells increase SHH secretion, stimulating stromal cells to secrete WNT ligands. WNT activators induce Msi1 expression in the Bmi1-CreER cells. This stromal-epithelial interaction leads to Bmi1-CreER rISCs induction and epithelial regeneration.

Alteration of Cellular Energy Metabolism through LPAR2-Axin2 Axis in Gastric Cancer.

Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and the malignant behavior of cancer cells through G-protein-coupled receptors. However, the role of LPA in β-catenin-mediated gastric cancer is unknown. Here, we have noted the high expression of LPAR2 in human gastric cancer tissues, and that LPA treatment significantly increased the proliferation, migration, and invasion of human gastric cancer cells. Results from our biochemical experiments showed that an LPA exposure increased the expression of β-catenin and its nuclear localization, increased the phosphorylation of glycogen synthase kinase 3β (GSK-3β), decreased the expression of Axin2, and increased the expression of the target genes of the β-catenin signaling pathway. The LPA2 receptor (LPAR2) antagonist significantly reduced the LPA-induced nuclear localization of β-catenin, the primary signaling event. The knockdown of LPAR2 in the gastric cancer cell lines robustly reduced the LPA-induced β-catenin activity. An LPA exposure increased the ATP production by both oxidative phosphorylation and glycolysis, and this effect was abrogated with the addition of an LPAR2 antagonist and XAV393, which stabilizes the Axin and inhibits the β-catenin signaling pathway. Based on our findings, the possibility that LPA contributes to gastric cancer initiation and progression through the β-catenin signaling pathway as well as by the dysregulation of the energy metabolism via the LPAR2 receptor and Axin2, respectively, provides a novel insight into the mechanism of and possible therapeutic targets of gastric cancer.

Toothpastes for children and their detergent contents affect molecular mechanisms of odontogenesis in zebrafish embryos

We aimed to evaluate how different types of toothpaste (TP) for children affected molecular mechanisms of odontogenesis in zebrafish embryos. Commercially available TPs were selected according to their detergent contents as the cocamidopropyl betaine (CAPB) containing TP (TP1) and sodium lauryl sulfate (SLS) containing TP (TP2). TP3 contained no detergent. Effects of SLS, and CAPB alone were also examined. TP and detergent concentrations affecting development were determined as 750 mg/L and 4 mg/L, respectively. Embryos were exposed to TP1, TP2, TP3, SLS, CAPB, and embryo medium (control) for 72 h post fertilization. Acetylcholinesterase (AChE) activity and oxidant–antioxidant parameters were analyzed spectrophotometrically. Expressions of tooth development genes were evaluated by reverse transcription PCR (RT-PCR). Intraocular distance, lower jaw, and ceratohyal cartilage length were displayed using Alcian Blue staining. axin2 and wnt10a expressions increased in SLS and TP2 groups. igf2a and eve1 expressions decreased in all groups except TP3. nrOb1 expression decreased in TP1, SLS, and CAPB groups. Oxidant–antioxidant balance was disturbed in all groups except TP3, evidenced by increased lipid peroxidation, nitric oxide. SLS, and CAPB groups were more affected in terms of AChE, glutathione-S-transferase, and superoxide dismutase; perturbations were observed in cartilage structures. Altered expression of tooth development gene axin2 correlated with wnt10a, and with changes in cartilage structures in SLS and TP2 groups. TP3 group presented no disruptions in oxidant–antioxidant balance. Our study shows the availability of externally developing zebrafish embryos in examining the effects of TP’ contents on embryogenesis.

Interleukin-35 Promote Osteogenesis and Inhibit Adipogenesis: Role of Wnt/β-Catenin and PPARγ Signaling Pathways.

Mesenchymal stem cells (MSCs) are multipotent stem cells that are able to differentiate into several cell types, including cartilage, fat, and bone. It has been reported that the decision process of MSCs into fat and bone cells is competing and reciprocal. Interleukin (IL)-35 is an important effector protein in the Wnt/β-catenin signaling pathway that acts as a bone metabolism regulator. However, it is unclear whether IL-35 is also important for regulating MSC differentiation to fat and bone. In the current study, we evaluated the role of IL-35 in C3H10T1/2 cells, which are a good cell model for investigating osteogenesis and adipogenesis in bone marrows. The role of IL-35 on osteoblast proliferation and apoptosis was assessed using cell counting kit-8 assay and flow cytometry, respectively. Extracellular matrix mineralization and lipid accumulation were measured by Alizarin Red S staining and Oil Red O staining, respectively. The most important transcription factor of the process of osteogenesis Runx2 and Wnt/β-catenin signaling pathway components β-catenin and Axin2 were investigated in response to IL-35 treatment. Furthermore, the adipogenic markers PPAR-γ and C/EBPα were also investigated. Our observations showed that IL-35 could promote the proliferation of MSCs and inhibit the apoptosis of MSCs. We found that IL-35 treatment resulted in a dramatic stimulation of osteogenesis and inhibition of adipogenesis. Moreover, IL-35 enhanced Wnt/β-catenin pathway key component β-catenin as well as Axin2 expression during MSCs differentiated to osteoblasts. Our findings suggested that IL-35 might control the balance between osteogenic and adipogenic differentiation of progenitor cells through the Wnt/β-catenin-PPARγ signaling pathway, suggesting its potential application in providing an intervention in osteoporosis and obesity.

Retinoic acid effects on in vitro palatal fusion and WNT signaling.

Retinoic acid is the main active vitamin A derivate and a key regulator of embryonic development. Excess of retinoic acid can disturb palate development in mice leading to cleft palate. WNT signaling is one of the main pathways in palate development. We evaluated the effects of retinoic acid on palate fusion and WNT signaling in in vitro explant cultures. Unfused palates from E13.5 mouse embryos were cultured for 4 days with 0.5μM, 2μM or without retinoic acid. Apoptosis, proliferation, WNT signaling and bone formation were analyzed by histology and quantitative PCR. Retinoic acid treatment with 0.5 and 2.0μM reduced palate fusion from 84% (SD 6.8%) in the controls to 56% (SD 26%) and 16% (SD 19%), respectively. Additionally, 2μM retinoic acid treatment increased Axin2 expression. Retinoic acid also increased the proliferation marker Pcna as well as the number of Ki-67-positive cells in the palate epithelium. At the same time, the WNT inhibitors Dkk1, Dkk3, Wif1 and Sfrp1 were downregulated at least two-fold. Retinoic acid also down-regulated Alpl and Col1a2 gene expression. Alkaline phosphatase (ALP) activity was notably reduced in the osteogenic areas of the retinoic acid- treated palates. Our data suggest that retinoic acid impairs palate fusion and bone formation by upregulation of WNT signaling.

Copper-olsalazine metal-organic frameworks as a nanocatalyst and epigenetic modulator for efficient inhibition of colorectal cancer growth and metastasis

Despite the extensive explorations of nanoscale metal−organic frameworks (nanoMOFs) in drug delivery, the intrinsic bioactivity of nanoMOFs, such as anticancer activity, is severely underestimated owing to the overlooked integration of the hierarchical components including nanosized MOFs and molecular-level organic ligands and metal-organic complexes. Herein, we propose a de novo design of multifunctional bioactive nanoMOFs ranging from molecular to nanoscale level, and demonstrate this proof-of-concept by a copper−olsalazine (Olsa, a clinically approved drug for inflammatory bowel disease, here as a bioactive linker and DNA hypomethylating agent) nanoMOF displaying a multifaceted anticancer mechanism: (1) Cu-Olsa nanoMOF-mediated redox dyshomeostasis for enhanced catalytic tumor therapy, (2) targeting downregulation of cyclooxygenase-2 by the organic complex of Cu2+ and Olsa, and (3) Olsa-mediated epigenetic regulation. Cu-Olsa nanoMOF displayed an enzyme-like catalytic activity to generate cancericidal species ·OH and 1O2 from rich H2O2 in tumors, improved the expression of tumor suppressors TIMP3 and AXIN2 by epigenetic modulation, and fulfilled selective inhibition of colorectal cancer cells over normal cells. The hyaluronic acid-modified nanoMOF further verified the efficient suppression of CT26 colorectal tumor growth and metastasis in murine models. Overall, these results suggest that Olsa-based MOF presents a platform of epigenetic therapy-synergized nanomedicine for efficient cancer treatment and provides a powerful strategy for the design of intrinsically bioactive nanoMOFs. Statement of significanceMetal−organic frameworks (MOFs) with intrinsic bioactivities such as anticancer and antibacterial activity are of great interest. Herein, we reported a bioactive copper−olsalazine (Cu-Olsa) nanoMOF as a nanodrug for colorectal cancer treatment. This nanoMOF per se displayed enzyme-like catalytic activity to generate cancericidal species ·OH and 1O2 from rich H2O2 in tumors for nanocatalytic tumor therapy. Upon dissociation into small molecular copper-organic complex and olsalazine in cancer cells, COX-2 inhibition and epigenetic modulation were fulfilled for selective inhibition of colorectal cancer growth and metastasis.

Pharmacologic inhibition of PARP5, but not that of PARP1 or 2, promotes cytokine production and osteoclastogenesis through different pathways.

PARPs, which are members of the poly(ADP-ribose) polymerase superfamily, promote tumorigenesis and tumour-associated inflammation and are thus therapeutic targets for several cancers. The aim of the present study is to investigate the mechanistic insight into the roles PARPs for inflammation. Primary murine macrophages were cultured in the presence or absence of the PARP5 inhibitor NVP-TNKS656 to examine the role of PARP5 for cytokine production. In contrast to the roles of other PARPs for induction of inflammation, we found in the present study that pharmacologic inhibition of PARP5 induces production of inflammatory cytokines in primary murine macrophages. We found that treatment with the PARP5 inhibitor NVP-TNKS656 in macrophages enhanced steady-state and LPS-mediated cytokine production through degradation of IκBα and subsequent nuclear translocation of NF-κB. We also found that pharmacologic inhibition of PARP5 stabilises the adaptor protein 3BP2, a substrate of PARP5, and that accelerated cytokine production induced by PARP5 inhibition was rescued in 3BP2-deleted macrophages. Additionally, we found that LPS increases the expression of 3BP2 and AXIN1, a negative regulator of β-catenin, through suppression of PARP5 transcripts in macrophages, leading to further activation of cytokine production and inhibition of β-catenin-mediated cell proliferation, respectively. Lastly, we found that PARP5 inhibition in macrophages promotes osteoclastogenesis through stabilisation of 3BP2 and AXIN1, leading to activation of SRC and suppression of β-catenin, respectively. Our results show that pharmacologic inhibition of PARP5 against cancers unexpectedly induces adverse autoinflammatory side effects through activation of innate immunity, unlike inhibition of other PARPs.

Cannabidiol inhibits invasion and metastasis in colorectal cancer cells by reversing epithelial-mesenchymal transition through the Wnt/β-catenin signaling pathway.

Colorectal cancer (CRC) is the leading cause of cancer deaths worldwide, wherein distant metastasis is the main reason for death.The non-psychoactive phytocannabinoid cannabidiol (CBD) effectively induces the apoptosis of CRC cells. We investigated the role of CBD in the migration and metastasis of CRC cells. CBD significantly inhibited proliferation, migration, and invasion of colon cancer cells in a dose- or time-dependent manner. CBD could also inhibit epithelial-mesenchymal transition (EMT) by upregulating epithelial markers such as E-cadherin and downregulating mesenchymal markers such as N-cadherin, Snail, Vimentin, and HIF-1α. CBD could suppress the activation of the Wnt/β-catenin signaling pathway, inhibit the expression of β-catenin target genes such as APC and CK1, and increase the expression of Axin1. Compared to the control group, the volume and weight of orthotopic xenograft tumors significantly decreased after the CBD treatment. The results demonstrated that CBD inhibits invasion and metastasis in CRC cells. This was the first study elucidating the underlying molecular mechanism of CBD in inhibiting EMT and metastasis via the Wnt/β-catenin signaling pathway in CRC cells. The molecular mechanism by which CBD inhibits EMT and metastasis of CRC cells was shown to be through the Wnt/β-catenin signaling pathway for the first time.

DYNLT3 overexpression induces apoptosis and inhibits cell growth and migration via inhibition of the Wnt pathway and EMT in cervical cancer.

The role of the dynein light chain Tctex-type 3 (DYNLT3) protein in the biological behavior of cervical cancer and its relative molecular mechanisms were investigated. Immunohistochemical staining was used to detect DYNLT3 protein expression in cervical cancer tissues. Cell proliferation and apoptosis rates and invasiveness and migratory capacities were determined by CCK-8 assays, BrdU staining assays and colony formation assays, fluorescence activated cell sorting (FACS), wound healing assays, and Transwell invasion assays of cervical cancer cells after DYNLT3 modulation. The expression levels of Wnt signaling pathway- and EMT-related proteins were examined by Western blotting. Furthermore, the effects of DYNLT3 on the tumorigenicity and metastasis of cervical cancer in nude mice were analyzed by performing immunohistochemistry, and we found that the expression level of the DYNLT3 protein was higher in human normal cervical tissues than in cervical cancer tissues. Overexpression of DYNLT3 obviously attenuated the proliferation, migration and invasion of CaSki and SiHa cells, and promoted cell apoptosis. Upregulation of DYNLT3 expression markedly decreased the expression of Wnt signaling pathway-related proteins (Dvl2, Dvl3, p-LRP6, Wnt3a, Wnt5a/b, Naked1, Naked2, β-catenin and C-Myc) and EMT-related proteins (N-cadherin, SOX2, OCT4, vimentin and Snail), and increased the expression of E-cadherin and Axin1. However, the opposite results were observed after down-regulation of DYNLT3 expression. Up-regulation of DYNLT3 expression significantly inhibited tumor growth in a nude mouse model, while downregulation of DYNLT3 showed the opposite results. In addition, the major metastatic site of cervical cancer cells in mice was the lung, and downregulation of DYNLT3 expression increased cancer metastasis in vivo. DYNLT3 exerted inhibitory effects on cervical cancer by inhibiting cell proliferation, migration and invasion, promoting cell apoptosis in vitro, and inhibiting tumor growth and metastasis in vivo, possibly by suppressing the Wnt signaling pathway and the EMT.

Risk Factors of Microscopically Tumor-Free Surgical Margins for Recurrence and Survival of Oral Squamous Cell Carcinoma Patients.

ObjectivesThe concept of adequate surgical margins remains controversial in oral squamous cell carcinoma (OSCC) surgery. This study aimed to identify surgical margin-related indicators that might impact recurrence and survival of OSCC patients.Materials and MethodsHistopathological examination was performed using hematoxylin-eosin-stained surgical margin tissue sections in 235 OSCC patients. Axin2 and Snail expression at the surgical margin was detected by immunohistochemistry. The impact of the Axin2-Snail cascade on tumorigenesis of the immortalized human oral keratinocyte (IHOK) line was investigated in vivo.ResultsThe width and dysplasia of surgical margins were not significantly associated with the outcome of OSCC patients. In a multivariate analysis using variable clinicopathologic factors and with Axin2 and Snail expression as cofactors, higher age (hazard ratio [HR]:1.050; P=0.047), Axin2 (HR:6.883; P=0.014), and Snail abundance (HR:5.663; P=0.009) had independent impacts on worsened overall survival. Similarly, lesion site in retromolar trigone (HR:4.077; P=0.010), upper (HR:4.332; P=0.005) and lower gingiva (HR:3.545; P=0.012), presence of extranodal extension (HR:9.967; P<0.001), perineural invasion (HR:3.627; P=0.024), and Snail abundance (HR:3.587; P<0.001) had independent impacts on worsened recurrence-free survival. Furthermore, Axin2 knockdown induced decreased Snail expression and attenuated tumorigenesis in the IHOK line.ConclusionHistopathological examination of surgical margins may not be reliable to predict OSCC patient outcome. Molecular analysis may provide a more accurate risk assessment of surgical margins in OSCC. In particular, Axin2 and Snail are potential predictive biomarkers for the risk assessment of surgical margins in OSCC.

Riluzole regulates pancreatic cancer cell metabolism by suppressing the Wnt-β-catenin pathway

Most cancer cells rely on aerobic glycolysis to support uncontrolled proliferation and evade apoptosis. However, pancreatic cancer cells switch to glutamine metabolism to survive under hypoxic conditions. Activation of the Wnt/β-catenin pathway induces aerobic glycolysis by activating enzymes required for glucose metabolism and regulating the expression of glutamate transporter and glutamine synthetase. The results demonstrate that riluzole inhibits pancreatic cancer cell growth and has no effect on human pancreatic normal ductal epithelial cells. RNA-seq experiments identified the involvement of Wnt and metabolic pathways by riluzole. Inhibition of Wnt-β-catenin/TCF-LEF pathway by riluzole suppresses the expression of PDK, MCT1, cMyc, AXIN, and CyclinD1. Riluzole inhibits glucose transporter 2 expression, glucose uptake, lactate dehydrogenase A expression, and NAD + level. Furthermore, riluzole inhibits glutamate release and glutathione levels, and elevates reactive oxygen species. Riluzole disrupts mitochondrial homeostasis by inhibiting Bcl-2 and upregulating Bax expression, resulting in a drop of mitochondrial membrane potential. Finally, riluzole inhibits pancreatic cancer growth in KPC (Pdx1-Cre, LSL-Trp53R172H, and LSL-KrasG12D) mice. In conclusion, riluzole can inhibit pancreatic cancer growth by regulating glucose and glutamine metabolisms and can be used to treat pancreatic cancer.

Excess vitamin a might contribute to submucous clefting by inhibiting WNT-mediated bone formation.

Cleft lip and/or palate (CLP) is a common craniofacial birth defect caused by genetic as well as environmental factors. The phenotypic spectrum of CLP also includes submucous clefts with a defect in the palatal bone. To elucidate the contribution of vitamin A, we evaluated the effects of the vitamin A metabolite all-trans retinoic acid (ATRA) on the osteogenic differentiation and mineralization of mouse embryonic palatal mesenchymal cells (MEPM). MEPM cells were isolated from the prefusion palates of E13 mouse embryos from three different litters. MEPM cells were cultured with and without 0.5μM ATRA in osteogenic medium. Differentiation was analysed by the expression of osteogenic marker genes and alkaline phosphatase (ALP) activity after 1, 2, and 7 days. The expression of Wnt marker genes was also analysed. Mineralization was assessed by alizarin red staining after 7, 14, 21, and 28 days. The bone marker genes Sp7, Runx2, Alpl, and Col1a1 were inhibited 10% ± 2%, 59% ± 7%, 79% ± 12% and 57% ± 20% (P < .05) at day 7. ALP activity was inhibited at days 1 and 7 by 35 ± 0% (P < .05) and 23 ± 6% (P < .001). ATRA also inhibited mineralization at 3 and 4 weeks. Finally, expression of the universal Wnt marker gene Axin2 was strongly reduced, by 31 ± 18% (P < .001), at day 7. Our data indicate that ATRA (vitamin A) inhibits bone formation by reducing Wnt signalling. This might contribute to the molecular aetiology of submucous clefting.

MiRNA-222-3p enhances the proliferation and suppresses the apoptosis of acute myeloid leukemia cells by targeting Axin2 and modulating the Wnt/β-catenin pathway

MicroRNA (miRNA)-222-3p is overexpressed in numerous tumors, where it acts as an oncogene. Although miRNA-222 is highly expressed in acute myeloid leukemia (AML), its functions and the mechanisms underlying these functions have not yet been fully elucidated. This study aimed to investigate the regulatory roles of miRNA-222-3p in AML and the molecular mechanisms underlying these roles. In this study, we observed that miRNA-222-3p increased the viability and suppressed the apoptosis of AML cells. Axin2 was demonstrated to be a direct target of miRNA-222-3p, which when overexpressed, inhibited Axin2 expression and stimulated the Wnt/β-catenin pathway. In contrast, upregulation of Axin2 expression levels reduced the viability and enhanced the apoptosis of AML cells. Moreover, it partially reversed the effects of the miRNA-222-3p mimic on the proliferation and apoptosis of, and modulation of the Wnt/β-catenin pathway in, AML cells. Taken together, this study provides strong evidence that miRNA-222-3p can serve as a molecular target for AML treatment.

Abstract 5843: Truncated APC &amp; AXIN2 expression contribute to maintaining β-catenin/WNT signaling at the “Just Right” level for CRC cell proliferation

Abstract Adenomatous polyposis coli (APC) mutation leads to increased WNT signaling and stem cell overpopulation, which drives development of colorectal cancer (CRC). APC mutations often encode a truncated protein that has loss of APC’s domains that mediate APC interaction with AXIN2. A substantial body of scientific evidence indicates that truncated APC is essential for CRC cell proliferation by maintaining beta-catenin levels and WNT signaling at the “just right” level (known as the “Just Right” Hypothesis). Thus, AXIN2 is of particular interest because both wild-type APC and AXIN2 are part of the beta-catenin destruction complex, which is essential for tumor-suppressing ability. Hypothesis: the degree of AXIN2 expression, in the context of truncated APC, is critical to maintain WNT-signaling activity at the “just right” level which is optimal for CRC cell proliferation. Accordingly, we evaluated the effect of AXIN2 levels in CRC cells that contain APC protein-truncating mutations on WNT signaling (Top Flash assay), expression of WNT-targeted genes, and CRC cell proliferation and differentiation. We found that modulating AXIN2 expression altered WNT signaling, expression of WNT-targeted genes, and CRC cell proliferation in CRC cell lines with different APC-truncation causing mutations. Indeed, western blot results showed that knock down of AXIN2 gene expression increased truncated-APC and AXIN1 protein expression, and decreased expression of several WNT-target genes (e.g. LGR5, CD44, c-Jun, c-Myc, Cyclin D1), particularly genes that have reported roles in cell cycle regulation. These findings were consistent with our independent bioinformatics analyses of NCI TCGA RNA-seq data on CRC patients. Overall, our study suggests that i) The beta-catenin destruction complex is still functional at a certain level despite APC-truncating mutations; ii) Perturbing AXIN2 expression leads to changes in WNT-signaling activity that deviate from a level that is “just right” for malignant cell proliferation, likely by affecting the function of the destruction complex; iii) Deviation from “just right” WNT signaling levels alters WNT-target gene expression and diminishes the ability of APC-mutant CRC cells to maintain uncontrolled proliferation. Citation Format: Chi Zhang, Caroline O. Facey, Katherine Funk, Lynn M. Opdenaker, Bruce M. Boman. Truncated APC &amp; AXIN2 expression contribute to maintaining β-catenin/WNT signaling at the “Just Right” level for CRC cell proliferation [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 5843.

Abstract CT514: A phase I, open-label, dose-escalation study investigating a low-density lipoprotein receptor-related protein (LRP) 5/6 inhibitor, BI 905677, in patients with advanced solid tumors

Abstract Background: Ligand-dependent Wnt signaling, mediated by LRP5/6, is highly activated in a subset of solid tumors. Activation leads to accumulation of intracellular β-catenin and expression of β-catenin-dependent genes that promote cancer cell proliferation and treatment resistance. BI 905677 is a bi-paratopic antibody that binds LRP5/6 and blocks binding of Wnt ligands. This study (NCT03604445) aimed to identify the maximum tolerated dose (MTD) and determine the recommended dose for BI 905677 in patients with advanced solid tumors. Pharmacokinetics and efficacy will also be evaluated. Methods: This is a Phase I, open-label, non-randomized study of BI 905677, administered intravenously, in patients with advanced, unresectable and/or metastatic solid tumors. Patients were refractory to or not eligible for standard therapy. Patients were treated at increasing intravenous doses of BI 905677 (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 2.8 and 3.6 mg/kg every 3 weeks [q3w]) until progression. Subsequent patients within a cohort were treated ≥ 72 hours apart to allow adequate monitoring for cytokine release syndrome and implementation of preventive measures if required. Results: As of November 23, 2021, 37 patients have received BI 905677. The median age was 56 years (range 32-77) and most were male (65%) and had ECOG PS 1 (51%). At 3.6 mg/kg, 3/3 patients experienced dose-limiting toxicities (DLTs) and dose was reduced to 2.8 mg/kg, which was determined as the MTD. Patients received a median of 2.0 cycles (range 1-6). Across all dose levels, 19 patients (51%) experienced Grade ≥ 3 adverse events (AEs). The most common Grade ≥ 3 AEs were vomiting (11%), hyponatremia (8%), anemia (5%), diarrhea (5%), abdominal pain (5%), nausea (5%), hypokalemia (5%), pain (5%) and increased alkaline phosphatase (5%). No related Grade ≥ 3 AEs were reported for doses ≤ 1.6 mg/kg, and BI 905677 was considered well tolerated ≤ 2.8 mg/kg. There were two Grade 5 events not related to study drug (tumor lysis syndrome and disease progression). DLTs observed at 3.6 mg/kg were hyponatremia, increased β-CTX expression (&amp;gt; 2-fold versus baseline), diarrhea, hyperbilirubinemia and vomiting. Exposure to BI 905677 increased in an approximately dose-proportional manner over the complete dose groups tested. No drug accumulation between Cycle 1 and Cycle 2 was observed for any cohort. Only six patients presented with anti-drug antibodies (ADAs), including one with pre-existing ADAs. Axin2 expression by reverse transcription PCR was reduced in paired skin biopsies but no dose dependency was observed. Across all doses, the best response was stable disease (n = 13; 35%). Conclusions: BI 905677 was well tolerated, with the MTD established as 2.8 mg/kg q3w. An expansion cohort to evaluate the activity of BI 905677 in a molecularly selected population for Wnt ligand dependence is planned. Citation Format: Elena Élez, Heinz-Josef Lenz, Maja de Jonge, Rona Yaeger, Toshihiko Doi, Linda Pronk, Michael Teufel, Kristell Marzin, Josep Tabernero. A phase I, open-label, dose-escalation study investigating a low-density lipoprotein receptor-related protein (LRP) 5/6 inhibitor, BI 905677, in patients with advanced solid 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 CT514.

Abstract 484: Molecular drivers of tumor progression in microsatellite stable APC mutation-negative colorectal cancers

Abstract Background: The tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC). Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify regulatory mechanisms underlying APC mutation-negative (APCmut-) CRCs. Methods: We analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut- and APC mutation-positive (APCmut+) microsatellite stable CRCs. We also constructed and compared regulatory networks between APCmut- and APCmut+ CRCs by integrating transcription factor (TF) binding motifs, protein-protein interactions, and gene co-expression using the algorithms PANDA and LIONESS. Single sample networks were used to identify each tumor’s contribution to the differences in transcriptional regulation observed between the APCmut- and APCmut+ groups. Results: APCmut- CRC expression profiles clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut- CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3. Consistent with this, APCmut-CRC regulatory networks showed strong activation of immune pathways relative to APCmut+ CRCs. The second most prominently targeted pathway was RAS signaling, with the TFs TCF15 and RUNX2 regulating key pathway members including RAF1, GEFs, and GAPs. Single sample networks revealed that tumors with the strongest transcriptional regulation of RAS genes tend to lack a KRAS mutation and exhibit a slight skew towards earlier onset. Conclusions: APCmut- CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways. APCmut- tumors exhibit increased regulation of RAS pathway genes compared to APCmut+ tumors. The samples contributing most to this regulatory signature tend to lack a KRAS mutation, suggesting that APCmut- tumors have developed ways to modulate RAS pathway activity through non-mutational mechanisms. Focal emphasis on the RAF1 gene suggests these tumors may be more vulnerable to inhibition of the downstream MAPK pathway. Network analysis implicates TCF15 and RUNX2 as regulators of RAS pathway genes, suggesting a possible WNT-RAS crosstalk occurring in APCmut- CRC that warrants further investigation. Citation Format: Megha Padi, Dante Bellomo, Adam Grant, Rosa M. Xicola, Curtis Thorne, Bodour Salhia, Xavier Llor, Nathan Ellis. Molecular drivers of tumor progression in microsatellite stable APC mutation-negative colorectal cancers [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 484.

GATA4 and estrogen receptor alpha bind at SNPs rs9921222 and rs10794639 to regulate AXIN1 expression in osteoblasts.

Osteoporosis is a serious public health problem that affects 200 million people worldwide. Genome-wide association studies have revealed the association between several single nucleotide polymorphisms (SNPs) near WNT/β-catenin signaling genes and bone mineral density (BMD). The activation of β-catenin by WNT ligands is required for osteoblast differentiation. SNP rs9921222 is an intronic variant of AXIN1 (a scaffold protein in the destruction complex that regulates β-catenin signaling) that correlates with BMD. However, the biological mechanism of SNP rs9921222 has never been reported. Here, we show that the genotype of SNP rs9921222 correlates with the expression of AXIN1 in human osteoblasts. RNA and genomic DNA were analyzed from primary osteoblasts from 111 different individuals. Homozygous TT at rs9921222 correlates with a higher expression of AXIN1 than homozygous CC. Regional association analysis showed that rs9921222 is in high linkage disequilibrium (LD) with SNP rs10794639. In silico transcription factor analysis predicted that rs9921222 is within a GATA4 motif and rs10794639 is adjacent to an estrogen receptor alpha (ERα) motif. Mechanistically, GATA4 and ERα bind at SNPs rs9921222 and rs10794639 as detected by ChIP-qPCR. Luciferase assays demonstrate that rs9921222 is the causal SNP to alter ERα and GATA4 binding. GATA4 promoted the expression, and in contrast, ERα suppressed the expression of AXIN1 via the histone deacetylase complex member SIN3A. Functionally, the level of AXIN1 negatively correlates with the level of transcriptionally active β-catenin. In summary, we have discovered a molecular mechanism of the SNP rs9921222 to regulate AXIN1 through GATA4 and ERα binding in human osteoblasts.

Characterization and impact of canonical Wnt Signaling Pathway (WSP) alterations on outcomes of metastatic prostate cancer.

5053 Background: Recurrent alterations in the WSP have been identified in patients with advanced prostate cancer. Aberrant Wnt signaling has been implicated in disease progression and hormonal resistance in prostate cancer. We utilized a multi-institutional real-world dataset to characterize molecular alterations in the canonical WSP in men with prostate cancer, and correlate that with overall survival (OS). Methods: Prostate cancer patients who underwent tissue-based DNA and RNA sequencing utilizing a commercially available CLIA-certified assay (Caris Life Sciences) were investigated. Next generation sequencing (NGS)/ whole exome (WES) and transcriptome sequencing (WTS) was performed on prostate cancer tissue derived from prostatic and/or metastatic sites. Patients with somatic activating mutations in CTNNB, pathogenic fusions in RSPO2, or inactivating mutations in APC or RNF43 were characterized as having aberrant canonical Wnt signaling (WSP-MT). A subset analysis was conducted in metastatic prostate cancer (mPCa) samples with microsatellite stable (MSS) tumors excluding RNF43 (G569fs*) mutations. Comparative analyses were done using Fisher-Exact or X2 tests, and significance was determined by adjusted p value using Benjamini-Hochberg correction (q &lt; 0.05). OS was obtained from insurance claims data and calculated using Kaplan-Meier estimates. Enriched mRNA transcripts were identified as those with an adjusted p value &lt; 0.001, logFC &gt; 1.5, and (-)log10 FDR &gt; 20. Results: Overall, 21.8% (n = 715/3283) of samples were classified as WSP-MT, of which 297 (41.5%) were from primary tumor samples and 418 (58.5%) were from metastatic sites. WSP-MT were highest in metastases to CNS (54.5%, n = 12/22), liver (43.8%, n = 91/208) and lung (42.3%, n = 91/208). Compared to WSP wild-type, WSP-MT tumors had a greater frequency of dMMR/MSI-H status (18% vs. 2%, q &lt; 0.001). In the subset of mPCa, MSS tumors (WSP-MT excluding RNF43 (G569fs*) mutations, n = 318; WSP wild-type n = 951), WSP-MT were enriched for mutations in SPOP (14.3% vs. 6.9%, q = 0.004). In these tumors, WSP-MT samples were most enriched for mRNA expression of CST1, NKD1, AXIN2, and ZNRF3 (all known canonical WSP activators). OS was inferior in mPCa WSP-MT patients compared to WSP wild-type (HR 0.49, 95% CI 0.37-0.64, p &lt; 0.0001). Relative to WSP wild-type cases, OS was shorter from time of enzalutamide/abiraterone/apalutamide start (HR 0.25, 95% CI 0.15-0.43, p = 0.0001), and from first-line taxane chemotherapy (HR 0.47, 95% CI 0.21-1.02, p = 0.052) in pts with WSP-MT. Conclusions: Canonical WSP alterations are enriched in metastatic tumors, with highest prevalence in visceral (CNS, liver, lung) metastases. Clinical outcomes in WSP-mutant cancers are inferior with both hormonal and chemotherapies, heralding an urgent need to develop novel therapeutic strategies for such patients.

Immunohistochemical expression of Axin-2, as an implication of the role of stem cell in scar pathogenesis and prognosis.

Wound healing is a multi-phased process. A disruption in these phases could result in a persistent wound or an atypical scar. Wounding activates wingless proteins (Wnt) signaling, which aids in the healing process. Axis inhibition protein-2 regulates a variety of cellular activities through the Wnt and other pathways. To assess the role of Axin-2 in patients with abnormal scars, using immunohistochemical study. This case-control study enrolled a total of 60 participants: 30 patients with abnormal scars (12 hypertrophic scars, 13 atrophic scars, and 5 keloid scars) and 30 age, sex, and site matched, apparently healthy controls. For immunohistochemistry examination of Axin-2 expression, skin samples were obtained from (i) lesional and (ii) perilesional skin of patients with aberrant scars, as well as (iii) normal control's skin. Epidermal Axin-2 expression positivity, cellular topography, intensity, and H score showed significant differences between the groups (p < 0.05). In the dermis (fibroblast/myofibroblast), there were significant differences in Axin-2 expression positivity, location, intensity, and H score (p < 0.001 for all). The epidermal Axin-2 H score and the Manchester scale had a significant positive correlation (r=0.832, p=0.001). The epidermal Axin-2 H score and age (r=-0.576, p=0.001), and the Stony Brook scale (r=-0.419, p=0.021), had significant negative correlations. Axin-2 overexpression might be accused in pathogenesis of abnormal scar and clinical worse scar outcome. In order to deprive scars of their regenerative cell pools, future scar therapies may target Axin-2 as a stem cell marker.

Multi-omics analysis revealed TEK and AXIN2 are potential biomarkers in multifocal papillary thyroid cancer

BackgroundPapillary thyroid carcinoma (PTC), the most common endocrine cancer, accounts for 80–85% of all malignant thyroid tumors. This study focused on identifying targets that affect the multifocality of PTC. In a previous study, we determined 158 mRNAs related to multifocality in BRAF-mutated PTC using The Cancer Genome Atlas.MethodsWe used multi-omics data (miRNAs and mRNAs) to identify the regulatory mechanisms of the investigated mRNAs. miRNA inhibitors were used to determine the relationship between mRNAs and miRNAs. We analyzed the target protein levels in patient sera using ELISA and immunohistochemical staining of patients’ tissues.ResultsWe identified 44 miRNAs that showed a negative correlation with mRNA expression. Using in vitro experiments, we identified four miRNAs that inhibit TEK and/or AXIN2 among the target mRNAs. We also showed that the downregulation of TEK and AXIN2 decreased the proliferation and migration of BRAF ( +) PTC cells. To evaluate the diagnostic ability of multifocal PTC, we examined serum TEK or AXIN2 in unifocal and multifocal PTC patients using ELISA, and showed that the serum TEK in multifocal PTC patients was higher than that in the unifocal PTC patients. The immunohistochemical study showed higher TEK and AXIN2 expression in multifocal PTC than unifocal PTC.ConclusionsBoth TEK and AXIN2 play a potential role in the multifocality of PTC, and serum TEK may be a diagnostic marker for multifocal PTC.