RPL21 interacts with LAMP3 to promote colorectal cancer invasion and metastasis by regulating focal adhesion formation.

BACKGROUND: Metastasis is the leading cause of death among patients with colorectal cancer (CRC). Therefore, it is important to explore the molecular mechanisms of metastasis to develop effective therapeutic targets for CRC. In the present study, ribosomal protein L21 (RPL21) was considered as being involved in promoting CRC metastasis, yet the underlying mechanism requires further investigation. METHODS: Immunohistochemistry, western blotting, and quantitative reverse transcription polymerase chain reaction were performed to measure the expression of RPL21 and lysosome-associated membrane protein 3 (LAMP3) in CRC tissues and cells. Wound healing, transwell migration, and invasion assays were performed to study the migration and invasion of cultured CRC cells. An orthotopic CRC mouse model was developed to investigate the metastatic ability of CRC. Transcriptome sequencing was conducted to identify the genes related to RPL21. The dual-luciferase reporter gene assay was performed to determine the transcriptional activity of transcription factor EB (TFEB). The GST/His pull-down assay was performed to investigate the specific binding sites of RPL21 and LAMP3. The cell adhesion assay was performed to determine the adhesion ability of CRC cells. Immunofluorescence staining was performed to observe focal adhesions (FAs). RESULTS: RPL21 was highly expressed in CRC, contributing to tumor invasiveness and poor patient prognosis. Functionally, RPL21 promoted the migration and invasion of CRC cells in vitro and tumor metastasis in vivo. Moreover, LAMP3 was identified as being highly related to RPL21 and was essential in promoting the migration and invasion of CRC cells. Mechanistically, RPL21 activated the transcriptional function of TFEB to upregulate LAMP3 expression. RPL21 directly bound to the aa 341-416 domain of LAMP3 via its aa 1-40 and aa 111-160 segments. The combination of RPL21 and LAMP3 enhanced the stability of the RPL21 protein by suppressing the degradation of the ubiquitin-proteasome system. Furthermore, RPL21 and LAMP3 promoted the formation of immature FAs by activating the FAK/paxillin/ERK signaling pathway. CONCLUSIONS: RPL21 promoted invasion and metastasis by regulating FA formation in a LAMP3-dependent manner during CRC progression. The interaction between RPL21 and LAMP3 may function as a potential therapeutic target against CRC.

AKAP-9 promotes colorectal cancer development by regulating Cdc42 interacting protein 4.

Our previous studies have shown that PRKA kinase anchor protein 9 (AKAP-9) is involved in colorectal cancer (CRC) cell proliferation and migration in vitro. However, whether or not AKAP-9 is important for CRC development or metastasis in vivo remains unknown. In the present study, we found that AKAP-9 expression was significantly higher in human colorectal cancer tissues than the paired normal tissues. In fact, AKAP-9 level correlated with the CRC infiltrating depth and metastasis. Moreover, the higher AKAP-9 expression was associated with the lower survival rate in patients. In cultured CRC cells, knockdown of AKAP-9 inhibited cell proliferation, invasion, and migration. AKAP-9 deficiency also attenuated CRC tumor growth and metastasis in vivo. Mechanistically, AKAP-9 interacted with cdc42 interacting protein 4 (CIP4) and regulated its expression. CIP4 levels were interrelated to the AKAP-9 level in CRC cells. Functionally, AKAP-9 was essential for TGF-ß1-induced epithelial-mesenchymal transition of CRC cells, and CIP4 played a critical role in mediating the function of AKAP-9. Importantly, CIP4 expression was significantly up-regulated in human CRC tissues. Taken together, our results demonstrated that AKAP-9 facilitates CRC development and metastasis via regulating CIP4-mediated epithelial-mesenchymal transition of CRC cells.

MALAT1 promotes colorectal cancer cell proliferation/migration/invasion via PRKA kinase anchor protein 9.

Our previous studies have shown that the 3' end of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is involved in colorectal cancer (CRC) cell proliferation and migration/invasion in vitro. The role and mechanism of MALAT1 in CRC metastasis in vivo, however, remain largely unknown. In the present study, we found that MALAT1 was up-regulated in human primary CRC tissues with lymph node metastasis. Overexpression of MALAT1 via RNA activation promoted CRC cell proliferation, invasion and migration in vitro, and stimulated tumor growth and metastasis in mice in vivo. Conversely, knockdown of MALAT1 inhibited CRC tumor growth and metastasis. MALAT1 regulated at least 243 genes in CRC cells in a genome-wide expression profiling. Among these genes, PRKA kinase anchor protein 9 (AKAP-9) was significantly up-regulated at both mRNA and protein levels. AKAP-9 was highly expressed in CRC cells with metastatic potential and human primary CRC tissues with lymph node metastasis, but not in normal cells or tissues. Importantly, knockdown of AKAP-9 blocked MALAT1-mediated CRC cell proliferation, migration and invasion. These data indicate that MALAT1 may promote CRC tumor development via its target protein AKAP-9.

NDRG1 attenuates epithelial-mesenchymal transition of nasopharyngeal cancer cells via blocking Smad2 signaling.

N-myc downstream-regulated gene 1 (NDRG1) has been implicated in tumorigenesis and metastasis in different cancers. However, its role in nasopharyngeal carcinoma remains unknown. We found that NDRG1 expression level was high in nasopharyngeal cancer 5-8F cells but low in 5-8F-LN cells with lymphatic metastasis potential. Knockdown of NDRG1 by shRNA promoted 5-8F cell proliferation, migration, and invasion in vitro and its tumorigenesis in vivo. Moreover, NDRG1 deficiency induced an epithelial-mesenchymal transition (EMT) of 5-8F cells as shown by an attenuation of E-cadherin and an induction of N-cadherin and vimentin expression. NDRG1 knockdown also enhanced Smad2 expression and phosphorylation. Smad2 signaling was attenuated in 5-8F cells but was significantly activated in 5-8F-LN cells. Knockdown of Smad2 restored E-cadherin but attenuated N-cadherin expression in NDRG1-deficient 5-8F cells, suggesting a reduction of EMT. Consistently, blockade of Smad2 in 5-8F-LN cells increased E-cadherin while diminishing N-cadherin and vimentin expression. These data indicate that Smad2 mediates the NDRG1 deficiency-induced EMT of 5-8F cells. In tumors derived from NDRG1-deficient 5-8F cells, E-cadherin expression was inhibited while vimentin and Smad2 were increased in a large number of cancer cells. Most importantly, NDRG1 expression was attenuated in human nasopharyngeal carcinoma tissues, resulted in a lower survival rate in patients. The NDRG1 was further decreased in the detached nasopharyngeal cancer cells, which was associated with a further reduced survival rate in patients with lymphatic metastasis. Taken together, these results demonstrated that NDRG1 prevents nasopharyngeal tumorigenesis and metastasis via inhibiting Smad2-mediated EMT of nasopharyngeal cells.

Smad2 and myocardin-related transcription factor B cooperatively regulate vascular smooth muscle differentiation from neural crest cells.

RATIONALE: Vascular smooth muscle cell (VSMC) differentiation from neural crest cells (NCCs) is critical for cardiovascular development, but the mechanisms remain largely unknown. OBJECTIVE: Transforming growth factor-ß (TGF-ß) function in VSMC differentiation from NCCs is controversial. Therefore, we determined the role and mechanism of a TGF-ß downstream signaling intermediate Smad2 in NCC differentiation to VSMCs. METHODS AND RESULTS: By using Cre/loxP system, we generated a NCC tissue-specific Smad2 knockout mouse model and found that Smad2 deletion resulted in defective NCC differentiation to VSMCs in aortic arch arteries during embryonic development and caused vessel wall abnormality in adult carotid arteries where the VSMCs are derived from NCCs. The abnormalities included 1 layer of VSMCs missing in the media of the arteries with distorted and thinner elastic lamina, leading to a thinner vessel wall compared with wild-type vessel. Mechanistically, Smad2 interacted with myocardin-related transcription factor B (MRTFB) to regulate VSMC marker gene expression. Smad2 was required for TGF-ß-induced MRTFB nuclear translocation, whereas MRTFB enhanced Smad2 binding to VSMC marker promoter. Furthermore, we found that Smad2, but not Smad3, was a progenitor-specific transcription factor mediating TGF-ß-induced VSMC differentiation from NCCs. Smad2 also seemed to be involved in determining the physiological differences between NCC-derived and mesoderm-derived VSMCs. CONCLUSIONS: Smad2 is an important factor in regulating progenitor-specific VSMC development and physiological differences between NCC-derived and mesoderm-derived VSMCs.

Comparison of 18 F-FAPI and 18 F-FDG PET/CT in a Patient With Fibrous Dysplasia.

ABSTRACT: A 16-year-old woman presented with an acute headache on the left side. A head CT scan revealed bone destruction in the skull. Subsequent 18 F-FDG and 18 F-FAPI PET/CT scans were performed within a week. The 18 F-FDG PET/CT indicated mild uptake in the regions of bone destruction, whereas the 18 F-FAPI PET/CT displayed significant tracer accumulation. The patient was ultimately diagnosed with fibrous dysplasia.

Assessing osteoporosis and bone mineral density through 18F-NaF uptake at lumbar spine.

OBJECTIVES: The use of 18F-Sodium fluoride (NaF) PET/CT is established in the detection of metastatic bone disease, yet its utility in osteoporosis remains underexplored. This research aims to assess the variations in 18F-NaF uptake among individuals with differing bone mineral density (BMD) and to examine the relationship between 18F-NaF uptake and BMD. METHODS: In this retrospective study, 199 patients (average age 56 ± 6, comprising 52 males and 147 females) with a history of cancer were analyzed. Each participant underwent both 18F-NaF PET/CT and lumbar dual-energy X-ray absorptiometry (DXA) scans within a span of 7 days. Based on DXA outcomes, patients and their lumbar vertebrae were categorized into normal BMD, osteopenia, and osteoporosis groups. The lumbar 18F-NaF uptake across these groups were compared, and to explore the association between lumbar standardized uptake values (SUV) values and BMD. The efficacy of 18F-NaF uptake in diagnosing osteoporosis or osteopenia was also evaluated. Analysis was conducted using Mann-Whitney U tests, Spearman regression, and receiver operating characteristic (ROC) curve analysis through GraphPad Prism 10.0. RESULTS: A total of 796 lumbar vertebrae from 199 patients were measured. It was observed that osteoporotic patients had significantly lower 18F-NaF uptake than those with osteopenia and normal BMD across the L1-L4 lumbar vertebrae (P < 0.0001). In a vertebra-based analysis, normal BMD vertebrae exhibited the highest maximum SUV(SUVmax) compared to osteopenic (8.13 ± 1.28 vs. 6.61 ± 1.01, P < 0.0001) and osteoporotic vertebrae (8.13 ± 1.28 vs. 4.82 ± 1.01, P < 0.0001). There was a positive correlation between lumbar 18F-NaF uptake and BMD across all vertebrae, with correlation coefficients exceeding 0.5 (range: 0.57-0.8). The area under the ROC curve values were notably high, at 0.96 for osteoporosis and 0.83 for osteopenia diagnosis. CONCLUSION: This study demonstrates distinct 18F-NaF uptake patterns among individuals with varying BMD levels, with a positive correlation between 18F-NaF uptake and BMD. These findings highlight the potential of 18F-NaF PET/CT as a supportive diagnostic method in the management of osteoporosis.

Prognosis and immunotherapy response prediction based on M2 macrophage-related genes in colon cancer.

BACKGROUND: M2 macrophage were revealed to play a crucial role in immune evasion and immunotherapies. This study aims to explore the potential significance of M2 macrophage-related genes in colon adenocarcinoma (COAD) by analysizing the transcriptome data in a comprehensive way. METHODS: We collected RNA-sequencing (RNA-seq) data of COAD from The Cancer Genome Atlas (TCGA) and Gene Expression Ominibus (GEO) databases. We calculated the immune infiltration scores of every sample using CIBERSORT algorithm. Through weighted gene co-expression network analysis (WGCNA), we picked out M2 macrophage-related genes. With these genes we screened out prognosis related genes which were utilized to construct a signature to assess the prognosis of patients. To extend the potential application of the signature, we also calculated the correlations with immune infiltration. Finally, we applied techniques such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunoblotting (Western Blotting) to validate the RNF32 gene in cellular in vitro assays. RESULTS: Seven M2 macrophage-related genes signature was constructed, which was an excellent prognostic predictor in two independent groups. The high-risk group showed lower immune infiltration and poorer response to immunotherapies than those of the low-risk group. The cell vitro experiments showed that the expression level of RNF32 was upregulated in colon cancer cell lines compared with normal cell lines. Moreover, we found that RNF32 may promote the proliferation, migration and invasion of cancer cells in vitro by inhibiting apoptosis. CONCLUSION: A novel M2 macrophage-related gene signature affects the prognosis and immune characteristics of colon cancer.

A new method for predicting the prognosis of colorectal cancer patients through a combination of multiple tumor-associated macrophage markers at the invasive front.

Tumor-associated macrophages (TAMs) are closely related to tumorigenesis and metastasis of multiple cancer types. The infiltration of TAMs is used for predicting the prognosis of cancers, including colorectal cancer (CRC). However, the density and prognostic significance of M1 and M2 TAM phenotypes in the intratumor versus the invasive front (IF) are largely unknown in CRC. In this study, CD68 was selected as a general marker of TAMs, CD11c, NOS2 and CXCL10 as markers for M1 phenotype and CD163, CD206, CD115 as markers for M2 phenotype. Firstly, immunohistochemistry staining and double-labeling immunofluorescence staining showed that M1 molecular markers (NOS2, CXCL10, CD11c) were lowly expressed at both IF and intratumor, while M2 molecular markers (CD163, CD206, CD115) were highly expressed mainly at IF. Moreover, we also demonstrated that three M1 molecular markers including NOS2, CXCL10 and CD11c were correlated to each other. Meanwhile, three M2 molecular markers including CD163, CD206, and CD115 were also correlated to each other. Patients with low expression of three M1 molecular markers (NOS2/CXCL10/CD11c) exhibited low overall survival (OS) rate, whereas patients with high expression of three M2 molecular markers (CD163/CD206/CD115) exhibited low OS rate. We also observed that the prognostic value of treble markers combination (NOS2/CXCL10/CD11c or CD163/CD206/CD115) was superior to that of single marker. Together, our results reveal the combination of treble TAMs markers (NOS2/CXCL10/CD11c or CD163/CD206/CD115) could better evaluate the prognosis of CRC patients, which might be used as a more comprehensive method for predicting the prognosis of CRC patients.

Comparative analysis of two timepoints on [18F]FAPI-42 PET/CT in various cancers.

PURPOSE: This study aimed to assess the biodistribution, detection rate, and uptake of the [18F]FAPI-42 at two distinct time intervals. METHODS: This prospective study enrolled 60 consecutive patients (median age 59; range 35-74) referred to [18F]FAPI-42 PET/CT. [18F]FAPI-42 PET/CT was performed early and late timepoint after tracer injection for staging or restaging. Positive lesions specified for anatomic locations (primary or recurrent tumor, LN metastasis and other metastasis) by visual analysis at both timepoints. Semiquantitative analysis of the tracer activity in lesions as well as normal tissues at both time points were measured and compared. In a subgroup analysis, eleven patients underwent 2-[18F]FDG PET/CT within 1 week, the detection rate and uptake of lesion were compared between early [18F]FAPI-42 and 2-[18F]FDG. RESULTS: Uptake of [18F]FAPI-42 in the late timepoint was significantly lower than the early timepoint in most organs (all p < 0.05), except for bone (SUVmean 0.88 vs. 0.85; p = 0.218). Tracer retention at biliary system showed less frequent at early timepoint than late timepoint. A total of 194 lesions were detected in 60 patients. One lesion was only seen at early timepoint but not at late timepoint. Lesions on early [18F]FAPI-42 PET/CT had higher visual score than that of late image(23 vs. 6). The uptake of lesion decreased significantly from early to late timepoint (all p < 0.05). In subgroup analysis, early [18F]FAPI-42 illustrated higher detection rate, visual score, and uptake of lesion than that of 2-[18F]FDG PET/CT. CONCLUSION: Early [18F]FAPI-42 PET/CT provided consistent detection rates and lesion uptake, but less tracer retention in the biliary system compared to late images. Therefore, acquisition at early timepoint could be a feasible strategy for improving acquisition protocols of [18F]FAPI-42 PET/CT. TRIAL REGISTRATION: ChiCTR2200063441. Registered 28 September 2022-Retrospectively registered, https://www.chictr.org.cn/bin/project/edit?pid=149714 .