Title: FAM83B Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in pulmonary adenocarcinoma

Title: FAM83B Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in pulmonary adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Title: FAM83B Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in pulmonary adenocarcinoma Jiajia Wang, Panpan Li, Limin Sun, Jing Zhang, Ke Yue, Yan Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3837359/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2024 Read the published version in Apoptosis → Version 1 posted 7 You are reading this latest preprint version Abstract Background: Chemotherapy is an effective therapeutic modality; nevertheless, a significant proportion of patients diagnosed with lung adenocarcinoma (LUAD) demonstrate resistance to chemotherapy. Therefore, it is crucial to understand the potential regulatory mechanisms to develop novel treatment strategies. Methods: Multiple assays, such as CCK8, wound healing, EdU, and transwell assays, were employed to confirm the augmented chemotherapy resistance, heightened cell proliferation, migration, and invasion caused by FAM83B overexpression in LUAD cells. Furthermore, MIMP, MTG, and ATP assays were utilized to quantify changes in mitochondria metabolism. In vitro functional assays were performed to evaluate the influence of FAM83B overexpression on the malignant progression and resistance mechanisms to chemotherapy in LUAD. Results: In the context of this study, it was determined that LUAD patients with increased FAM83B expression had shorter survival times, and tissue samples with FAM83B overexpression were more prone to metastasis compared to primary samples. As a result, FAM83B is identified as an adverse prognostic marker. The mechanistic analysis demonstrated that FAM83B impedes the translocation of calbindin 2 (CALB2) from the cytoplasm to the mitochondria, resulting in the inhibition of apoptosis and the promotion of mitochondrial activity. Consequently, this ultimately confers resistance to chemotherapy in LUAD. Furthermore, the administration of metformin, which blocks mitochondrial oxidative phosphorylation (OXPHOS), can restore sensitivity to drug resistance in LUAD. Conclusions: Taken together, these findings provide substantial evidence supporting the notion that FAM83B enhances chemotherapy resistance in LUAD through the upregulation of mitochondrial metabolism and the inhibition of apoptosis. FAM83B CALB2 mitochondrial metabolism apoptosis chemotherapy resistance lung adenocarcinoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights FAM83B is highly expressed in metastatic patients compared to the primary group and has been identified as an adverse prognostic marker in LUAD. High expression of FAM83B is closely related to chemotherapy resistance of lung adenocarcinoma. FAM83B promotes the emergence of LUAD chemotherapy resistance by enhancing mitochondrial metabolism and anti-apoptosis. 1. Introduction The non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers ( 1 , 2 ). The lung adenocarcinoma (LUAD) is the most prevalent pathological subtype among NSCLC. For LUAD, traditional chemotherapy has proven to be an effective treatment option ( 3 , 4 ). However, the development of chemoresistance poses a significant challenge, as it diminishes the efficacy of chemotherapeutic agents and ultimately leads to reduced survival rates in these individuals. Pemetrexed functions as a folate antagonist, and pemetrexed-based chemotherapy continues to be the established therapeutic approach for most patients with LUAD ( 5 , 6 ). Although pemetrexed has demonstrated effectiveness, the chemoresistance in LUAD is a challenge to its clinical efficacy. The FAM83 family, comprising eight members (A-H), exhibits conservative expression across vertebrates ( 7 ). FAM83B, a member of this family, has been identified as an oncogene that facilitates cell transformation through the activation of various signaling pathways. Okabe et al. have found the level of FAM83B protein was low in LUAD and it has no correction with overall survival (OS) ( 8 ); Sarah et al. revealed a negative correlation between the overexpression of FAM83B and the survival in NSCLC patients ( 9 ); Yamaura et al. identified FAM83B as a potential therapeutic target for EGFR wild type in LUAD ( 10 ). While the overexpression of FAM83B has been established as a prognostic marker in various malignant tumors, its role in LUAD remains a subject of controversy. Our previous study revealed that FAM83B exhibits elevated expression levels in primary LUAD compared to para-cancerous tissue. Furthermore, this heightened expression of FAM83B was found to be associated with lymph node metastasis ( 11 ). Calbindin-2 (CALB2) is a member of the EF-hand protein family, characterized by the presence of calcium-binding motifs consisting of two helices (E and F). The calcium-induced alterations in conformation observed in CALB2 indicate its probable classification as a calcium sensor protein within this protein family. Notably, CALB2 exhibits predominant expression in cells of the human nervous system ( 12 ). Calcium ions (Ca2+) have been recognized as a signaling molecule that engages in endoplasmic reticulum (ER)-mitochondria interactions to govern cellular apoptosis ( 13 ). Furthermore, the dynamics of mitochondrial Ca2 + are implicated in the regulation of cellular energy metabolism, wherein various calcium-binding proteins, including CALB2, may modulate cell apoptosis by influencing calcium release. The phenomenon of metabolic adaptation assumes significance in the emergence of drug resistance in cancer cells, as recent investigations have unveiled the ability of tumor cells to adapt their metabolic processes to acquire resistance ( 14 ). The potential impact of FAM83B overexpression on the chemosensitivity of LUAD through the modulation of mitochondria metabolism remains unexplored in the literature. Therefore, this study aims to examine the influence of FAM83B on LUAD and elucidate a plausible underlying mechanism. 2. Methods 2.1. Immunohistochemistry A total of 119 cases of primary LUAD and 109 cases of metastasis LUAD were collected from January 2011 to December 2018 at Qilu Hospital, Shandong University. The tissue specimens were fixed in formalin and embedded in paraffin were confirmed by pathologists, and the study received approval from the Ethics Committee of Shandong University. For antigen retrieval, the sections were autoclaved in EDTA buffer (pH 8.0). The slides were then incubated overnight at 4°C with a rabbit polyclonal anti-FAM83B antibody (NBP1-86764; Novus) at a dilution of 1:200. Subsequently, the slides underwent incubation in a two-step plus Poly HRP Anti-Mouse/Rabbit IgG Detection System (PV-9000, ZSGB-Bio) in accordance with protocol. The visualization was achieved using DAB (ZSGB-Bio), followed by rinsing in distilled water and counterstaining with hematoxylin. Subsequent evaluation of protein staining was conducted under a microscope, with positivity being determined when moderate or strong positive cells occupied more than 10% of the area. 2.2. Cell lines culture, lentiviral transfection and small interfering RNA (siRNA) treatment The human LUAD cell lines, H1299, A549, and PC9, were acquired from ATCC and cultured in RPMI-1640 with 10% FBS under a humidified atmosphere containing 5% CO2 at 37°C. Lentiviral particles carrying FAM83B cDNA or control sequences were from GeneChem (Shanghai GeneChem Co., Ltd, Shanghai, China). The interference sequences targeting FAM83B, with a specific sequence of 5'-TTCGTTCCTCTTTAGTATT-3', along with their respective negative controls, were from GeneChem. The transfection procedure was conducted utilizing the Lipofectamine 2000 reagent (Invitrogen) in accordance with the guidelines provided by the manufacturer. 2.3. Scratch Migration Assay Cell suspensions were prepared in 6-well plates, followed by the creation of scratch wounds using pipette tips and subsequent rinsing with PBS upon reaching 100% confluence. Subsequently, the cells were cultured in a medium supplemented with 10% serum and subjected to photographic documentation at both 0 and 24 hours. 2.4. Invasion assays in Boyden chambers A suspension of 5 × 104 LUAD cells was prepared in invasion medium and introduced into the upper compartment of Boyden chambers, along with 20 µg of BD MatrigelTM Basement Membrane Matrix (BD Biosciences). Invasion medium was subsequently added to the lower compartment of the chamber. Following a 24-hour incubation period, the filters were extracted from the chambers, fixed using 4% paraformaldehyde, and stained with a 0.1% crystal violet dye solution. The cells residing in the lower chamber were quantified within three discrete fields, and triplicate samples were subsequently subjected to analysis. 2.5. Identification and bioinformatics analysis of differential proteins with label-free quantitative proteomics method The investigation of FAM83B expression in LUAD and its influence on survival outcomes was carried out by utilizing datasets obtained from the GEO database ( http://www.ncbi.nlm.nih.gov/geo ). Quantitative protein analysis and significant difference analysis were performed on H1299/FAM83B OE and H1299/FAM83B NC cells. The process involved several steps, including protein extraction, protein quantification, SDS-PAGE, enzymatic hydrolysis of proteins, LC-MS/MS analysis, database query, quality control, and the utilization of high-resolution mass spectrometry to obtain the primary mass spectrum data. Enrichment analysis was performed by considering the presence or absence of differentially expressed proteins, as well as the combination of differentially expressed proteins with a fold change greater than 1.2 times (up-down) and a significance level of P<0.05. The differentially expressed genes underwent Gene Set Enrichment Analysis (GSEA) using the software provided by the Broad Institute ( http://software.broadinstitute.org/gsea/index.jsp ) and KOBAS 3.0 ( http://kobas.cbi.pku.edu.cn/ ). In order to explore the co-expression of FAM83B in LUAD, the cBioportal ( https://www.cbioportal.org/ ) and Coexpedia ( https://www.coexpedia.org/ ) databases were employed. 2.6. EdU and CCK-8 assay The sub-confluent LUAD cells underwent a 2-hour incubation with a concentration of 10 µM EdU. Following this, the evaluation of cellular proliferation was conducted using Andy FluorTM 488 Azide, which yielded red fluorescence. The nuclei were stained with Hoechst 33,342, resulting in blue fluorescence. The images were acquired utilizing a two-photon confocal laser microscope. The cells were seeded in a 96-well plate at a density of 3 × 103 cells/well and incubated overnight. Next day, the culture medium was substituted with RMPI1640 supplemented with different concentrations of pemetrexed. Cell viability was evaluated using the CCK-8 kit (Targetmol, Shanghai, China) following the protocol. 2.7. Western blotting The antibodies utilized in this research as followed: FAM83B (NBP2-16423; Novus; diluted at 1:1000), CALB2 (96235; Cell Signaling Technology; diluted at 1:1000), GAPDH (GOODHERE, Hangzhou, China; AB-PR001; diluted at 1:1000), and TOMM40 (55959; Cell Signaling Technology; diluted at 1:1000). Subsequently, the membranes were subjected to incubation with HRP-conjugated goat anti-rabbit IgG H&L secondary antibodies (1:5000). The fluorescence signal was then detected employing an enhanced chemiluminescence kit (Millipore, Darmstadt, Germany), following the guidelines provided by the manufacturer. 2.8. MIMP, mitochondrial mass and ATP Assay using fluorescent molecular probes. The evaluation of the mitochondrial inner membrane potential (MIMP) was conducted using tetramethylrhodamine ester (TMRE) obtained from BestBio in China. To assess the MIMP, a negative control was established using CCCP. The evaluation of mitochondrial mass was carried out using Mito Tracker Red FM (MTG) obtained from Beyotime in China. Following the addition of the fluorophores (100 nM TMRE, 100 nM MTG) and incubation at 37°C for 30 min in the absence of light, the cells were collected in RMPI 1640 and promptly analyzed using an Envision instrument from Perkin Elmer. The ATP concentration was quantified through fluorescence production using an ATP Assay Kit (Beyotime) and a luminometer. The results were expressed as relative luciferase units (RLU) and further analyzed as fold RLU, which were found to be equivalent to those observed in the control group. 2.9. Statistical analysis The data analysis was conducted using GraphPad Prism version 6.0 and SPSS version 23.0. Chi-square and two-sided t-tests were employed to examine the correlation between FAM83B expression and the corresponding clinicopathological characteristics in patients with primary lung adenocarcinoma. Kaplan-Meier analysis was utilized to estimate overall survival (OS) and disease-free survival (DFS). Statistical significance was determined as follows: ns, P ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. 3. Results 3.1. The expression of FAM83B in LUAD To investigate the clinical relevance of FAM83B expression in LUAD, we conducted an analysis of FAM83B expression across various publicly available datasets. Specifically, we examined the mRNA sequencing data from the GSE19188 database. Our findings revealed a statistically significant increase in FAM83B expression in tumors compared to normal samples (P < 0.001). Furthermore, the expression of FAM83B was found to be significantly elevated in lung tumor samples compared to corresponding normal samples, as evidenced by various gene expression profile data sources, including GSE116959, GSE18842, and GSE101929 databases (Fig. 1 A). To establish the correlation between FAM83B expression and OS, we conducted an analysis from GSE101929, GSE19188, and GSE13213 datasets from the GEO database. Within these datasets, patients with high FAM83B expression exhibited a significantly decreased OS compared to those with low FAM83B expression (P = 0.0024, P < 0.0001, P < 0.0001) (Fig. 1 B). Subsequently, additional databases were employed to examine the impact of FAM83B expression on DFS. Our analysis revealed an association between elevated levels of FAM83B and a poor DFS outcome. Specifically, the datasets GSE50081 and GSE30219 demonstrated a noteworthy reduction in DFS among patients exhibiting high FAM83B expression compared to those with low FAM83B expression (P = 0.0089, 0.00068) (Fig. 1 C). Tumors exhibiting drug resistance demonstrate an increased propensity for recurrence or metastasis. In instances of advanced LUAD accompanied by distant metastasis, patients are generally deemed ineligible for surgical excision of the primary lesions. Within the confines of this investigation, a total of 109 instances of metastatic LUAD were meticulously selected, and the expression of FAM83B within these cases was scrutinized. The resultant positivity rate was estimated to be approximately 54.13% (59 out of 109), surpassing the positivity rate of 26.89% (32 out of 119) observed in 119 primary LUAD samples. (Fig. 1 D-E). 3.2. The overexpression of FAM83B conferred chemoresistance and facilitated proliferation, migration, invasion and metastasis in LUAD In our previous report, it was certified that the cell lines H1299 and A549 displayed low expression of FAM83B, while the PC9 cell line exhibited high expression. FAM83B overexpression cell lines (FAM83B OE) and their corresponding control lines (FAM83B NC) were established in H1299 and A549 cells, while a FAM83B silencing cell line (si FAM83B) and its control (si NC) were established in PC9 cells. A fluorescence microscopy analysis revealed that over 70% of the cells were infected with fluorescent green-labeled lentivirus, indicating a high transfection efficiency. To investigate the impact of FAM83B overexpression on the drug resistance of LUAD cells, we subjected H1299/FAM83B, A549/FAM83B, and PC9/si FAM83B cells to varying concentrations of pemetrexed and subsequently assessed cell viability after 72 hours. The findings revealed a cell survival rate of 66.3% in the H1299/FAM83B OE group, whereas it was 57.3% in the H1299/FAM83B NC cells (P<0.01). In the A549/FAM83B OE group, the survival rate was observed to be 80%, whereas in the control group it was 57.6% (P<0.01). Similarly, in the PC9/si FAM83B group, the survival rate was found to be 25.4%, while in the control group it was 35.1% (P<0.05). These results indicate a significantly higher drug resistance in FAM83B OE LUAD cells compared to the corresponding control group (Fig. 2 A-C). The EdU assay was employed to track cell proliferation, specifically investigating the involvement of FAM83B in cell proliferation in LUAD cells. The findings demonstrated that the upregulation of FAM83B substantially augmented the proportion of EdU-positive cells, whereas the downregulation of FAM83B resulted in a decrease in the proportion of EdU-positive cells compared to the control group (Fig. 2 D-F). The analysis of scratch distance indicated a notable increase in tumor migration speed within the FAM83B OE group compared to the FAM83B NC group. Conversely, the PC9/siFAM83B group exhibited a deceleration in migration rate, suggesting that FAM83B overexpression expedites the wound healing process. This observation is supported by Fig. 3 A-C. The effects of FAM83B on the migration and invasion of H1299/FAM83B OE, A549/FAM83B OE, and PC9/siFAM83B cells were investigated using Boyden chamber assays. The results depicted in Fig. 3 E-F demonstrate that the overexpression of FAM83B significantly increased the invasive capacity of H1299/FAM83B OE by approximately 28.5% compared to H1299/FAM83B NC, and A549/FAM83B OE by approximately 31.6% (P<0.0001) compared to A549/FAM83B NC (P<0.0001). In contrast, the invasive potential of PC9/siFAM83B exhibited a significant decrease of 27.8% when compared to PC9/FAM83B siNC (P<0.0001). 3.3. Identification of key candidate genes and biological pathways between H1299/FAM83B OE and H1299/FAM83B NC by proteomic sequencing analysis A proteomic analysis was conducted comparing H1299/FAM83B OE and H1299/FAM83B NC, resulting in the identification of 2801 genes (Supplementary Table S1 ) and 75 genes (Supplementary Table S2 ) that exhibited statistically significant differences in expression. These differences were determined based on sequencing results that met the criteria of a fold change greater than 1.2 (up or down) and a p-value less than 0.05, as determined by JiKai Gene. The proteomic heat map in (Fig. 4 A) displays the differential protein expressions associated with OXPHOS between the two groups. The GSEA analysis revealed that the FAM83B OE group exhibited a higher level of OXPHOS compared to the control group (Fig. 4 B). Additionally, the cBioPortal database revealed the identification of 1421 genes (Supplementary Table S3) that exhibited co-expression with FAM83B. The identification of differential protein expression and the associated metabolic pathway was conducted through GO and KEGG enrichment analyses using FunRich Software (v3.1.3), with subsequent analysis performed on the downloaded TXT files of the results. Furthermore, the Gene Co-expression website was utilized to identify 1,500 genes (Supplementary Table S4) and their network of interactions, while the GO and KEGG pathway functions on the KOBAS were employed to determine the involved metabolic pathway (Fig. 4 C and Supplementary Fig. S1 A-B). Moreover, a total of 113 genes exhibited co-expression with the target gene FAM83B, out of which 54 genes (Supplementary Table S5) demonstrated co-expression with NSCLC as documented in the Coexpedia database. By intersecting the metabolism-related genes obtained through sequencing analysis and the genes co-expressed with FAM83B, CALB2 and HMGA1 were isolated (Fig. 4 D and E). 3.4. FAM83B affected the distribution of CALB2 between cytoplasm and mitochondria and inhibited apoptosis Proteins isolated from H1299, A549, and PC9 cells were subjected to Western blotting analysis. The results revealed that the expression of CALB2 in H1299/FAM83B OE cells was significantly higher compared to H1299/FAM83B NC cells. However, the ratio of CALB2 in the mitochondria/cytoplasm was found to be significantly lower in H1299/FAM83B OE cells compared to NC cells (P = 0.007). Similarly, in the A549/FAM83B OE group, the expression of CALB2 was observed to be lower than A549/FAM83B NC cells, and the ratio of mitochondria/cytoplasm CALB2 was decreased compared to the NC cell group (P = 0.007) (Fig. 5 A, C and D). In contrast, the silencing of FAM83B resulted in an increased expression of CALB2 in PC9/siFAM83B cells compared to PC9/siNC cells. Additionally, the ratio of mitochondria/cytoplasm CALB2 was significantly higher in NC cells (P = 0.03) (Fig. 5 B and E). Western blot analysis was conducted to evaluate the levels of caspase 3 and B-cell lymphoma (Bcl)-2 expression. The levels of caspase 3 in the H1299/FAM83B NC, A549/FAM83B NC, and PC9/siFAM83B groups were significantly higher than those in the respective control group (Fig. 5 F). 3.5. FAM83B overexpression increased mitochondrial metabolism in LUAD cells The findings revealed a noteworthy elevation in the MIMP of H1299/FAM83B OE and A549/FAM83B OE cells in comparison to the parental cell lines (Fig. 6 A). To elucidate the underlying mechanism responsible for this increase in MIMP, measurements were taken for MTG and ATP production. The findings of the study indicate a notable increase in MTG in H1299/FAM83B OE and A549/FAM83B OE cells when compared to H1299/FAM83B NC and A549/FAM83B NC, respectively (Fig. 6 B). Additionally, the production of ATP was significantly elevated in H1299/FAM83B OE and A549/FAM83B OE cells compared to the control group (Fig. 6 C). The data presented in this study are the average values obtained from triple independent experiments. Statistical significance was determined using Student's t-test, with a threshold of P < 0.05 (*P<0.05, ** P<0.01, and *** P<0.001). 3.6. Metabolic targeting with metformin in concomitance with chemicals to enhance the chemotherapy sensitivity in FAM83B overexpressed cells of the LUAD The inhibitory effect of metformin on mitochondrial activity has been established. Upon co-administration of pemetrexed and metformin in H1299/FAM83B OE, A549/FAM83B OE, and PC9 cells, a notable decrease in MIMP, MTG, and ATP production was observed compared to the use of pemetrexed alone (Fig. 6 D-G). H1299/FAM83B OE, A549/FAM83B OE, and PC9 cell lines were subjected to treatment with 10mM metformin in combination with varying concentrations of pemetrexed to investigate the impact of metformin on drug resistance in LUAD. Notably, in H1299/FAM83B OE cells, the survival rate of the pemetrexed combined with metformin group (59%) was significantly lower compared to the pemetrexed alone group (74.3%) (P = 0.0247) (Fig. 6 H); In the A549/FAM83B OE cell line, the survival rates of the group treated with pemetrexed combined with metformin and the group treated with pemetrexed alone were 65.7% and 74.3%, respectively (P = 0.0299) (Fig. 6 J). In the PC9 cell line, the survival rates of the group treated with pemetrexed alone and the group treated with pemetrexed combined with metformin were 54.3% and 27.6%, respectively (P = 0.0039) (Fig. 6 K). These findings indicate that metformin has the potential to enhance the chemosensitivity of LUAD. 4. Discussion Substantial evidence supports the upregulation of FAM83B mRNA and protein expression in various types of solid tumors ( 15 ). Our previous study showed that FAM83B exhibited elevated expression levels in primary LAUD and high level of FAM83B promotes local lymph node metastasis. In the present investigation, we expanded upon this finding by examining the expression of FAM83B in four distinct GEO databases pertaining to LUAD. The results further substantiated the significantly higher expression of FAM83B in cancerous tissues compared to para-cancerous tissues. Furthermore, we conducted an examination of the association between elevated levels of FAM83B and prognosis in an additional five GEO databases using Kaplan-Meier analysis. The study findings indicated a significant decrease in survival rates among patients exhibiting high levels of FAM83B expression in contrast to those with low expression levels. These findings suggest that FAM83B exhibits as a viable target for the chemotherapy of LUAD. Furthermore, we obtained samples of metastases from a cohort of 109 patients diagnosed with inoperable advanced LUAD and proceeded to analyze the expression levels of FAM83B. In comparison to the positive rate of FAM83B (26.89%) observed in primary LUAD cases, the metastatic LUAD cases exhibited a significantly higher rate of FAM83B expression (54.13%). These experimental findings indicate that LUAD cases characterized by an over-expression of FAM83B may possess a heightened metastatic capacity and a more unfavorable prognosis. In our prior investigation, we reported that FAM83B facilitated the migratory and invasive capabilities of H1299 cells. Here, we expanded our analysis to include additional LUAD cell lines to examine the potential impact of FAM83B overexpression on the malignant characteristics of LUAD cells. The results indicated that the upregulation of FAM83B contributes to drug resistance and augmented the proliferation, migration, and invasion capabilities of LUAD cells. However, the mechanisms underlying the regulatory role of FAM83B in these phenotypic changes remain inadequately elucidated. The analysis focused on the differentially expressed proteins between the H1299/FAM83B OE and H1299/FAM83B NC groups. GSEA enrichment analysis revealed that the H1299/FAM83B OE group exhibited a higher level of OXPHOS compared to the control. Metabolic abnormalities are recognized as indicators of cancer, yet the underlying regulatory mechanisms remain poorly understood. Warburg effect is a defining feature of cancer cells, as it pertains to their capacity to predominantly utilize glycolysis for the generation of ATP. However, recent evidence has indicated the significant involvement of OXPHOS in cancer progression. Cancer cells possess the ability to transition between anaerobic respiration and OXPHOS to obtain energy and facilitate tumor advancement. Recognizing this metabolic trait could potentially offer a therapeutic avenue for certain cancer types with OXPHOS inhibitors ( 16 , 17 ). The inhibition of hepatic glucose production by metformin has been shown to effectively reduce glycemia in patients with hyperglycemia ( 18 ). Emerging evidence indicates that metformin may also possess the potential to inhibit OXPHOS ( 19 ). Hirpra et al. have proposed that OXPHOS-dependent metabolism serves as the primary mechanism for acquired drug resistance in oncogene-addicted cancer cells ( 14 ). In order to investigate the role of FAM83B in drug resistance to pemetrexed, the evaluation of CCK8 and mitochondrial activity was conducted. The results indicate that FAM83B OE cells demonstrated a greater survival rate. Furthermore, the administration of metformin enhanced the effectiveness of chemotherapy in these cells. Additionally, it was observed that FAM83B OE cells exhibited higher levels of MIMP, MTG, and intracellular ATP in comparison to cells with suppressed expression of FAM83B. These results suggest that FAM83B may contribute to the enhancement of drug resistance in LUAD cells by augmenting mitochondrial OXPHOS. This finding aligns with prior research indicating that numerous forms of drug-resistant cancer cells heavily depend on mitochondrial OXPHOS as an energy source. Consequently, it is anticipated that various inhibitors of OXPHOS will augment the chemosensitivity of these tumors ( 18 , 19 ). Additionally, alternative studies have put forth the notion that heightened levels of OXPHOS in CSCs can foster resistance to chemotherapy ( 20 ). However, the specific mechanisms by which OXPHOS activity contributes to chemotherapy resistance and tumor growth remain unclear. Further analysis of protein expression has identified three proteins, namely CALB2, HMGA1, and SLC2A1, that exhibit differential expression between H1299/FAM83B OE and H1299/FAM83B NC cells. CALB2 encompasses calcium-binding motifs and is qualified as intracellular calcium buffer ( 21 ). Multiple studies have provided evidence indicating that calretinin expression in LUAD is limited and feeble ( 19 , 22 ). The interplay of calcium flux between the ER and mitochondria has the potential to influence several fundamental characteristics of cancer, such as evasion of apoptosis and cellular invasion. CALB2, acted as transitory calcium ion sinks/stores, affected the calcium ion-free or calcium ion-bound state in cytoplasm and mitochondria, thereby triggering the behavior of LUAD ( 13 , 23 ). It is plausible that CALB2 is a member of a cluster of calcium sensor proteins within this family ( 24 , 25 ). The involvement of mitochondrial calcium dynamics in the regulation of cellular energy metabolism is well-established. The process of mitochondrial calcium uptake plays a crucial role in governing mitochondrial metabolism ( 26 , 27 ). This study examined the influence of FAM83B on the subcellular localization of CALB2 and found that, irrespective of its expression level, the proportion of CALB2 entering mitochondria was markedly reduced in both H1299/FAM83B OE and A549/FAM83B OE. Conversely, in PC9 cells, CALB2 cytoplasm/mitochondrial translocation increased following FAM83B interference. Therefore, we hypothesized that the inhibition of CALB2 translocation to the mitochondria by FAM83B could lead to a relative increase in calcium binding to CALB2 and change the cellular calcium homeostasis. The reduction in free Ca2 + counteracted the effects of extrinsic apoptosis, ultimately leading to the suppression of apoptosis. Consequently, the overexpression of FAM83B aggravated the development of chemotherapy resistance in LUAD cells, as evidenced by the increased expression of Bcl-2 and decreased expression of cleaved caspase3 ( 28 ). According to previous studies, it has been observed that mitochondrial calcium signaling plays a role in regulating the differentiation of fibroblasts into myofibroblasts. It is hypothesized that CALB2 could serve as a significant mediator and targeting OXPHOS inhibition may hold promise as a novel therapeutic approach for drug resistant cancer cells ( 29 ). Valerio et al have discovered the downregulation of TRPC3 protein leads to the promotion of cytosolic and mitochondrial Ca2 + oscillations, as well as an increase in mitochondrial Ca2 + levels, mitochondrial oxygen consumption rate, and OXPHOS, which promotes the proliferation of cancerous epithelial cells and tumor development in vivo ( 30 ). In our investigation, we observed an inhibition of CALB2 translocation, which might disrupt cellular calcium homeostasis as the calcium buffer and induced cytosolic and mitochondrial Ca2 + oscillations. Consequently, this led to alterations in mitochondrial function, including an increase in mitochondrial metabolism, as demonstrated by the upregulation of MIMP, MTG, and ATP levels in FAM83B OE LUAD cells. Ruolan et al. have reported that metformin chemo sensitized the recipient acute myeloid leukemia cells by decreasing the transfer of mitochondria and inhibiting OXPHOS ( 31 ). Our investigation revealed that FAM83B OE cells displayed a more aggressive malignant phenotype, characterized by increased susceptibility to drug resistance, enhanced proliferation and metastatic capabilities. These phenomena could potentially be attributed to the overexpression of FAM83B, which may impede the translocation of CALB2 into the mitochondria, thereby leading to the cytosolic and mitochondrial Ca2 + oscillations. Additionally, the reduction of free Ca2 + in the cytoplasm impedes the progression of apoptotic cells by impeding the activity of caspase3 and substract cleavage. On the other hand, the changes of cellular calcium homeostasis have the capacity to elicit modifications in mitochondrial metabolism and OXPHOS. After the administration of metformin to block mitochondrial OXPHOS, the metabolic activity of LUAD cells was attenuated, resulting in an increased sensitivity of tumor cells to chemotherapy drugs. These findings suggest a potential role of FAM83B overexpression in the development of chemoresistance. Consequently, this phenomenon contributes to chemoresistance and fosters the development of the malignant phenotype in LUAD. In this experiment, we only detected the expression of Bcl-2 and additional experiments are required to ascertain the potential impact of altered calcium homeostasis on the activation of the intrinsic apoptosis pathway, specifically by examining the elevation of Ca2 + within the mitochondria. 5. Conclusions In conclusion, our research has presented empirical evidence substantiating the role of FAM83B in the augmentation of chemotherapy resistance in LUAD by impeding the translocation of CALB2 to mitochondria. Consequently, this interference impacts cellular calcium homeostasis, a crucial factor in cell viability and mitochondrial functionality, thereby fostering chemoresistance. This discovery introduces a potential therapeutic target for the treatment of LUAD. Declarations Author Contributions The study was conceptualized by Xiaojuan Wu and the manuscript was revised by her. Cell culture was conducted by Jiajia Wang, while IHC was performed by Jing Zhang and Yan Wang. Molecular biology experiments were carried out by Panpan Li and Limin Sun. Data analysis was performed by Ke Yue, and Jiajia Wang edited the manuscript. The final manuscript and its published versions were approved by all authors. Funding information This research was supported by the National Natural Science Foundation of China (NSFC, grant no. 81402181) and the National Natural Science Foundation of Shandong Province (grant no. ZR2023MH105). Conflict of Interest The authors declare no conflicts of interest related to this work. Ethical Statement These procedures were performed in accordance with the ethical standards of the Committee on Human Experimentation of Shandong University Medical Research Ethics Committee. Data Availability Statement The data presented in this study are available on request from the corresponding author. 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Sci Signal 11 Okabe N et al (2015) FAM83B is a novel biomarker for diagnosis and prognosis of lung squamous cell carcinoma. Int J Oncol 46:999–1006 Richtmann S et al (2019) FAM83A and FAM83B as Prognostic Biomarkers and Potential New Therapeutic Targets in NSCLC. Cancers (Basel) 11 Yamaura T et al (2018) Family with sequence similarity 83, member B is a predictor of poor prognosis and a potential therapeutic target for lung adenocarcinoma expressing wild-type epidermal growth factor receptor. Oncol Lett 15:1549–1558 Zhang J et al (2023) FAM83B promotes the invasion of primary lung adenocarcinoma via PI3K/AKT/NF-kappaB pathway. BMC Pulm Med 23:32 Bertschy S, Genton CY, Gotzos V (1998) Selective immunocytochemical localisation of calretinin in the human ovary. Histochem Cell Biol 109:59–66 Boehning D et al (2003) Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nat Cell Biol 5:1051–1061 Hirpara J et al (2019) Metabolic reprogramming of oncogene-addicted cancer cells to OXPHOS as a mechanism of drug resistance. Redox Biol 25:101076 Cipriano R et al (2012) FAM83B mediates EGFR- and RAS-driven oncogenic transformation. J Clin Invest 122:3197–3210 Yu L et al (2017) Modeling the Genetic Regulation of Cancer Metabolism: Interplay between Glycolysis and Oxidative Phosphorylation. Cancer Res 77:1564–1574 Chen CL, Lin CY, Kung HJ (2021) Targeting Mitochondrial OXPHOS and Their Regulatory Signals in Prostate Cancers. Int J Mol Sci 22 Bosc C, Selak MA, Sarry JE (2017) Resistance Is Futile: Targeting Mitochondrial Energetics and Metabolism to Overcome Drug Resistance in Cancer Treatment. Cell Metab 26:705–707 Matsuda M et al (2020) Calretinin-expressing lung adenocarcinoma: Distinct characteristics of advanced stages, smoker-type features, and rare expression of other mesothelial markers are useful to differentiate epithelioid mesothelioma. Pathol Res Pract 216:152817 Jones CL et al (2018) Inhibition of Amino Acid Metabolism Selectively Targets Human Leukemia Stem Cells. Cancer Cell 34:724–740e724 Schwaller B (2010) Cytosolic Ca2 + buffers. Cold Spring Harb Perspect Biol 2:a004051 Comin CE et al (2014) Expression of thrombomodulin, calretinin, cytokeratin 5/6, D2-40 and WT-1 in a series of primary carcinomas of the lung: an immunohistochemical study in comparison with epithelioid pleural mesothelioma. Tumori 100:559–567 Mattson MP, Chan SL (2003) Calcium orchestrates apoptosis. Nat Cell Biol 5:1041–1043 Hack NJ, Wride MC, Charters KM, Kater SB, Parks TN (2000) Developmental changes in the subcellular localization of calretinin. J Neurosci 20:RC67 Schwaller B, Durussel I, Jermann D, Herrmann B, Cox JA (1997) Comparison of the Ca2+-binding properties of human recombinant calretinin-22k and calretinin. J Biol Chem 272:29663–29671 Dong H et al (2022) Mitochondrial calcium uniporter promotes phagocytosis-dependent activation of the NLRP3 inflammasome. Proc Natl Acad Sci U S A 119:e2123247119 Stevenson L et al (2011) Calbindin 2 (CALB2) regulates 5-fluorouracil sensitivity in colorectal cancer by modulating the intrinsic apoptotic pathway. PLoS ONE 6:e20276 Wu Y, Fabritius M, Ip C (2009) Chemotherapeutic sensitization by endoplasmic reticulum stress: increasing the efficacy of taxane against prostate cancer. Cancer Biol Ther 8:146–152 Lombardi AA et al (2019) Mitochondrial calcium exchange links metabolism with the epigenome to control cellular differentiation. Nat Commun 10:4509 Farfariello V et al (2022) TRPC3 shapes the ER-mitochondria Ca(2+) transfer characterizing tumour-promoting senescence. Nat Commun 13:956 You R et al (2022) Metformin sensitizes AML cells to chemotherapy through blocking mitochondrial transfer from stromal cells to AML cells. Cancer Lett 532:215582 Additional Declarations No competing interests reported. Supplementary Files SupplementaryAppendix.docx OriginalImagesforBlots.pdf Supportinginformation.xlsx Cite Share Download PDF Status: Published Journal Publication published 13 Mar, 2024 Read the published version in Apoptosis → Version 1 posted Editorial decision: Revision requested 29 Jan, 2024 Reviews received at journal 25 Jan, 2024 Reviewers agreed at journal 17 Jan, 2024 Reviewers invited by journal 07 Jan, 2024 Submission checks completed at journal 06 Jan, 2024 Editor assigned by journal 06 Jan, 2024 First submitted to journal 05 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3837359","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265833568,"identity":"14767bb4-8e3a-44f4-8439-4ead6f9c371b","order_by":0,"name":"Jiajia Wang","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jiajia","middleName":"","lastName":"Wang","suffix":""},{"id":265833569,"identity":"0c8e0658-a771-400a-a8ae-e8cdb56005b0","order_by":1,"name":"Panpan Li","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Panpan","middleName":"","lastName":"Li","suffix":""},{"id":265833570,"identity":"e10ac5ae-cc11-4cf0-8e2a-f235da01ccd7","order_by":2,"name":"Limin Sun","email":"","orcid":"","institution":"Shandong provincial third hospital","correspondingAuthor":false,"prefix":"","firstName":"Limin","middleName":"","lastName":"Sun","suffix":""},{"id":265833571,"identity":"f302c496-d772-4606-85f5-56f58d36334a","order_by":3,"name":"Jing Zhang","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":265833572,"identity":"72b7eb3f-7c99-4695-a54e-0f7f8c7e8367","order_by":4,"name":"Ke Yue","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Yue","suffix":""},{"id":265833573,"identity":"dd18c665-ec48-44fb-a534-34b629b6d543","order_by":5,"name":"Yan Wang","email":"","orcid":"","institution":"Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Wang","suffix":""},{"id":265833574,"identity":"cfd0ab52-5407-4a24-a92e-71e776f5b533","order_by":6,"name":"Xiaojuan Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIie3PsYrCMBjA8YQP4vJJ1wR8iByCcCDqo7QUnOQWQQQHC4G4+AAWn8LF1UrhupS6Flzu8AXictx4jdsNRseDyx8CCXw/PkKIz/cXg+bw242uvsN5H4NW8jSBjJty3BHr7Ol9LBSpzvuyHrnnZAGfH6968Cbriey29QlJTai5Tu4ToVhXCh1PLYmxOiPdJiDS/X0SAOlxoSHaNyTH2RmhkzFoOwiD1ldDlpa8KGQVMh66SQBot+QNGcd0ozPER0QonEpeFdGhvLwTU8bI8aicf5GnYnfhs0WUriJNwvlgOCzU0VwdxAb895sm7nk7Yh6O+Hw+37/uB4iTT+YB9DrZAAAAAElFTkSuQmCC","orcid":"","institution":"Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-01-05 13:30:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3837359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3837359/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10495-024-01944-7","type":"published","date":"2024-03-13T20:06:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49382535,"identity":"99774267-d35e-4a04-ad46-4b448a78dc51","added_by":"auto","created_at":"2024-01-09 19:29:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3606810,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAM83B expression in LUAD patients. \u003c/strong\u003e(A) The data from GSE 19188, GSE116959, GSE18842, and GSE101929. (B) Survival curves for OS were generated from GSE 101929, GSE19188, and GSE13213database, with sample sizes of 65, 82, and 117, respectively. The best separation cut off was used. (C) Survival curves for DFS were generated from GSE 50081 and GSE30219, with sample sizes of 181 and 278, respectively. The best separation cut off was used. (D, E) Representative photographs of immunohistochemistry staining for FAM83B expression were shown in primary LUAD and metastatic patients. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, ***P \u0026lt; 0.001 based on Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/ba019b1e47145c01858c646a.png"},{"id":49382339,"identity":"329860e7-cbb1-4ccd-9bb4-9c745e0bbc0a","added_by":"auto","created_at":"2024-01-09 19:21:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1998888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCK-8 assay analysis and EDU assays.\u003c/strong\u003e (A, B and C) CCK-8 assay analysis shows the cell drug resistance of FAM83B in LUAD cells by chemicals. (D, E and F) The impact of FAM83B on the proliferation of LUAD cells was investigated through EdU staining * P \u0026lt; 0.05, ** P \u0026lt; 0.01, ***P \u0026lt; 0.001 based on Student’s t-test. The data shown are the means ± SD of triplicate wells and are representative of at least three replicate experiments.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/8467b8a5d9eab3a0a6a5f061.png"},{"id":49382532,"identity":"30ba36de-40a9-4105-9a55-251a0e89e92b","added_by":"auto","created_at":"2024-01-09 19:29:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4448559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe wound healing assays. \u003c/strong\u003e(A and B) The images depicted a significant decrease in the distance between wound edges in cells overexpressing FAM83B compared to control cells. (C) The assay demonstrated a higher rate of wound healing due to reduced migration in siFAM83B cells compared to siNC cells. Histogram plots displayed the total number of wound healing events in LUAD cells. Migration and invasion assays were conducted. (D and E) Overexpression of FAM83B promoted migration and invasion in H1299 and A549 cells. (F) The results indicated that knock-down of FAM83B suppressed migration and invasion in PC9 cells. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, and ***P \u0026lt; 0.001, based on Student’s t-test. Each experiment was repeated at least three times.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/cb9cc3b457443088ab4daa1a.png"},{"id":49382338,"identity":"a589d5b6-2319-487e-86ec-18a3ca9d302e","added_by":"auto","created_at":"2024-01-09 19:21:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":622891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe potential hub genes expression between H1299/FAM83B OE and H1299/FAM83B NC.\u003c/strong\u003e(A) A heatmap illustrating the co-expression of differentially expressed genes with FAM83B, consisting of 37 upregulated and 39 down-regulated genes from two distinct groups. (B) Gene Set Enrichment Analysis (GSEA) was performed to compare the OXPHOS signature in FAM83B over-expression groups with control cells. (C) The GO and KEGG enrichment of the differentially ex-pressed genes were analyzed with KOBAS. (D and E) Three distinct expression genes co-expressed with FAM83B were identified using online tools and Venn diagram software.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/75c6212f931fe6f9ca90b34e.png"},{"id":49382346,"identity":"abe091de-aada-4562-977a-f2e4b1fdc6b8","added_by":"auto","created_at":"2024-01-09 19:21:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":698282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAM83B affected the mitochondria vs cytoplasm distribution of CALB2 and induced apoptosis in LUAD cells. \u003c/strong\u003e(A) Following transfection with FAM83B NC and FAM83B OE, the total-cell lysates were extracted from H1299 and A549 cells, respectively. (B) The whole-cell lysates were extracted from PC9 cells after transfection with siNC and siFAM83B. (C, D and E) After 72 hours of transfection, the mitochondria and cytoplasmic lysates were isolated and extracted from H1299, A549, and PC9 cells. (F) The total-cell lysates were extracted from H1299, A549, and PC9 cells after transfection with the corresponding lentiviral particles. The indicated antibodies were utilized for Western blot analysis. Data are the mean ± SD of experiments performed in triplicate. * P \u0026lt; 0.05, **P \u0026lt; 0.01, and *** P \u0026lt; 0.001. Each experiment was repeated at least three times.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/51e83afc05deda068007895e.png"},{"id":49382345,"identity":"2b2cd905-bd00-420d-b3e9-aa487f21a7dd","added_by":"auto","created_at":"2024-01-09 19:21:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":340573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAM83B modulated the metabolic reprograming in LUAD cells.\u003c/strong\u003e (A and D) Flow cytometry was used to measure the mitochondrial inner membrane potential using the fluorescent probe TMRE. (B and E) Mitochondrial mass was measured using Mito tracker Red. (C and F) The intra-cellular ATP level was measured using fluorescence. (G) MIMP, MTG, and ATP level of PC9 cells were measured after treatment with pemetrexed alone or in combination with metformin for 72 hours. (H-K) The viability of H1299, A549, and PC9 cells was assessed after exposure to various concentrations of pemetrexed alone or in combination with metformin for 72 hours. * P \u0026lt; 0.05, **P \u0026lt; 0.01, and *** P \u0026lt; 0.001. Each experiment was repeated at least three times.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/56809dadb2f57af819632c86.png"},{"id":52823878,"identity":"9ef26d53-99fd-475c-b39e-05adcd6dd3dc","added_by":"auto","created_at":"2024-03-16 20:06:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3920322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/fcfc3fdf-7a36-457c-9cc0-e58c91f50540.pdf"},{"id":49382533,"identity":"ecf9ee42-2b63-4d27-97ad-7bf4ef8d155e","added_by":"auto","created_at":"2024-01-09 19:29:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":163224,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryAppendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/ff2424064217cd221800d514.docx"},{"id":49382927,"identity":"28978cf9-2345-4c51-94d2-26387a651d5c","added_by":"auto","created_at":"2024-01-09 19:37:09","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":442615,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalImagesforBlots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/f3d13e1ebb575623b70f52a9.pdf"},{"id":49382343,"identity":"8edfa589-98cc-4213-8af1-4891e24b1da2","added_by":"auto","created_at":"2024-01-09 19:21:09","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":83653,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3837359/v1/220f4bfd322a4d3967078e9f.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Title: FAM83B Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in pulmonary adenocarcinoma","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eFAM83B is highly expressed in metastatic patients compared to the primary group and has been identified as an adverse prognostic marker in LUAD.\u003c/li\u003e\n \u003cli\u003eHigh expression of FAM83B is closely related to chemotherapy resistance of lung adenocarcinoma.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFAM83B promotes the emergence of LUAD chemotherapy resistance by enhancing mitochondrial metabolism and anti-apoptosis.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eThe non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The lung adenocarcinoma (LUAD) is the most prevalent pathological subtype among NSCLC. For LUAD, traditional chemotherapy has proven to be an effective treatment option (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, the development of chemoresistance poses a significant challenge, as it diminishes the efficacy of chemotherapeutic agents and ultimately leads to reduced survival rates in these individuals. Pemetrexed functions as a folate antagonist, and pemetrexed-based chemotherapy continues to be the established therapeutic approach for most patients with LUAD (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Although pemetrexed has demonstrated effectiveness, the chemoresistance in LUAD is a challenge to its clinical efficacy. The FAM83 family, comprising eight members (A-H), exhibits conservative expression across vertebrates (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). FAM83B, a member of this family, has been identified as an oncogene that facilitates cell transformation through the activation of various signaling pathways. Okabe et al. have found the level of FAM83B protein was low in LUAD and it has no correction with overall survival (OS) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e); Sarah et al. revealed a negative correlation between the overexpression of FAM83B and the survival in NSCLC patients (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e); Yamaura et al. identified FAM83B as a potential therapeutic target for EGFR wild type in LUAD (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). While the overexpression of FAM83B has been established as a prognostic marker in various malignant tumors, its role in LUAD remains a subject of controversy. Our previous study revealed that FAM83B exhibits elevated expression levels in primary LUAD compared to para-cancerous tissue. Furthermore, this heightened expression of FAM83B was found to be associated with lymph node metastasis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCalbindin-2 (CALB2) is a member of the EF-hand protein family, characterized by the presence of calcium-binding motifs consisting of two helices (E and F). The calcium-induced alterations in conformation observed in CALB2 indicate its probable classification as a calcium sensor protein within this protein family. Notably, CALB2 exhibits predominant expression in cells of the human nervous system (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Calcium ions (Ca2+) have been recognized as a signaling molecule that engages in endoplasmic reticulum (ER)-mitochondria interactions to govern cellular apoptosis (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Furthermore, the dynamics of mitochondrial Ca2\u0026thinsp;+\u0026thinsp;are implicated in the regulation of cellular energy metabolism, wherein various calcium-binding proteins, including CALB2, may modulate cell apoptosis by influencing calcium release. The phenomenon of metabolic adaptation assumes significance in the emergence of drug resistance in cancer cells, as recent investigations have unveiled the ability of tumor cells to adapt their metabolic processes to acquire resistance (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The potential impact of FAM83B overexpression on the chemosensitivity of LUAD through the modulation of mitochondria metabolism remains unexplored in the literature. Therefore, this study aims to examine the influence of FAM83B on LUAD and elucidate a plausible underlying mechanism.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eA total of 119 cases of primary LUAD and 109 cases of metastasis LUAD were collected from January 2011 to December 2018 at Qilu Hospital, Shandong University. The tissue specimens were fixed in formalin and embedded in paraffin were confirmed by pathologists, and the study received approval from the Ethics Committee of Shandong University. For antigen retrieval, the sections were autoclaved in EDTA buffer (pH 8.0). The slides were then incubated overnight at 4\u0026deg;C with a rabbit polyclonal anti-FAM83B antibody (NBP1-86764; Novus) at a dilution of 1:200. Subsequently, the slides underwent incubation in a two-step plus Poly HRP Anti-Mouse/Rabbit IgG Detection System (PV-9000, ZSGB-Bio) in accordance with protocol. The visualization was achieved using DAB (ZSGB-Bio), followed by rinsing in distilled water and counterstaining with hematoxylin. Subsequent evaluation of protein staining was conducted under a microscope, with positivity being determined when moderate or strong positive cells occupied more than 10% of the area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell lines culture, lentiviral transfection and small interfering RNA (siRNA) treatment\u003c/h2\u003e \u003cp\u003eThe human LUAD cell lines, H1299, A549, and PC9, were acquired from ATCC and cultured in RPMI-1640 with 10% FBS under a humidified atmosphere containing 5% CO2 at 37\u0026deg;C. Lentiviral particles carrying FAM83B cDNA or control sequences were from GeneChem (Shanghai GeneChem Co., Ltd, Shanghai, China). The interference sequences targeting FAM83B, with a specific sequence of 5'-TTCGTTCCTCTTTAGTATT-3', along with their respective negative controls, were from GeneChem. The transfection procedure was conducted utilizing the Lipofectamine 2000 reagent (Invitrogen) in accordance with the guidelines provided by the manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Scratch Migration Assay\u003c/h2\u003e \u003cp\u003eCell suspensions were prepared in 6-well plates, followed by the creation of scratch wounds using pipette tips and subsequent rinsing with PBS upon reaching 100% confluence. Subsequently, the cells were cultured in a medium supplemented with 10% serum and subjected to photographic documentation at both 0 and 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Invasion assays in Boyden chambers\u003c/h2\u003e \u003cp\u003eA suspension of 5 \u0026times; 104 LUAD cells was prepared in invasion medium and introduced into the upper compartment of Boyden chambers, along with 20 \u0026micro;g of BD MatrigelTM Basement Membrane Matrix (BD Biosciences). Invasion medium was subsequently added to the lower compartment of the chamber. Following a 24-hour incubation period, the filters were extracted from the chambers, fixed using 4% paraformaldehyde, and stained with a 0.1% crystal violet dye solution. The cells residing in the lower chamber were quantified within three discrete fields, and triplicate samples were subsequently subjected to analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Identification and bioinformatics analysis of differential proteins with label-free quantitative proteomics method\u003c/h2\u003e \u003cp\u003eThe investigation of FAM83B expression in LUAD and its influence on survival outcomes was carried out by utilizing datasets obtained from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Quantitative protein analysis and significant difference analysis were performed on H1299/FAM83B OE and H1299/FAM83B NC cells. The process involved several steps, including protein extraction, protein quantification, SDS-PAGE, enzymatic hydrolysis of proteins, LC-MS/MS analysis, database query, quality control, and the utilization of high-resolution mass spectrometry to obtain the primary mass spectrum data. Enrichment analysis was performed by considering the presence or absence of differentially expressed proteins, as well as the combination of differentially expressed proteins with a fold change greater than 1.2 times (up-down) and a significance level of P\u003c0.05. The differentially expressed genes underwent Gene Set Enrichment Analysis (GSEA) using the software provided by the Broad Institute (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://software.broadinstitute.org/gsea/index.jsp\u003c/span\u003e\u003cspan address=\"http://software.broadinstitute.org/gsea/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and KOBAS 3.0\u003c/p\u003e \u003cp\u003e(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://kobas.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://kobas.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In order to explore the co-expression of FAM83B in LUAD, the cBioportal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cbioportal.org/\u003c/span\u003e\u003cspan address=\"https://www.cbioportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Coexpedia (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.coexpedia.org/\u003c/span\u003e\u003cspan address=\"https://www.coexpedia.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) databases were employed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. EdU and CCK-8 assay\u003c/h2\u003e \u003cp\u003eThe sub-confluent LUAD cells underwent a 2-hour incubation with a concentration of 10 \u0026micro;M EdU. Following this, the evaluation of cellular proliferation was conducted using Andy FluorTM 488 Azide, which yielded red fluorescence. The nuclei were stained with Hoechst 33,342, resulting in blue fluorescence. The images were acquired utilizing a two-photon confocal laser microscope. The cells were seeded in a 96-well plate at a density of 3 \u0026times; 103 cells/well and incubated overnight. Next day, the culture medium was substituted with RMPI1640 supplemented with different concentrations of pemetrexed. Cell viability was evaluated using the CCK-8 kit (Targetmol, Shanghai, China) following the protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Western blotting\u003c/h2\u003e \u003cp\u003eThe antibodies utilized in this research as followed: FAM83B (NBP2-16423; Novus; diluted at 1:1000), CALB2 (96235; Cell Signaling Technology; diluted at 1:1000), GAPDH (GOODHERE, Hangzhou, China; AB-PR001; diluted at 1:1000), and TOMM40 (55959; Cell Signaling Technology; diluted at 1:1000). Subsequently, the membranes were subjected to incubation with HRP-conjugated goat anti-rabbit IgG H\u0026amp;L secondary antibodies (1:5000). The fluorescence signal was then detected employing an enhanced chemiluminescence kit (Millipore, Darmstadt, Germany), following the guidelines provided by the manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. MIMP, mitochondrial mass and ATP Assay using fluorescent molecular probes.\u003c/h2\u003e \u003cp\u003eThe evaluation of the mitochondrial inner membrane potential (MIMP) was conducted using tetramethylrhodamine ester (TMRE) obtained from BestBio in China. To assess the MIMP, a negative control was established using CCCP. The evaluation of mitochondrial mass was carried out using Mito Tracker Red FM (MTG) obtained from Beyotime in China. Following the addition of the fluorophores (100 nM TMRE, 100 nM MTG) and incubation at 37\u0026deg;C for 30 min in the absence of light, the cells were collected in RMPI 1640 and promptly analyzed using an Envision instrument from Perkin Elmer. The ATP concentration was quantified through fluorescence production using an ATP Assay Kit (Beyotime) and a luminometer. The results were expressed as relative luciferase units (RLU) and further analyzed as fold RLU, which were found to be equivalent to those observed in the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data analysis was conducted using GraphPad Prism version 6.0 and SPSS version 23.0. Chi-square and two-sided t-tests were employed to examine the correlation between FAM83B expression and the corresponding clinicopathological characteristics in patients with primary lung adenocarcinoma. Kaplan-Meier analysis was utilized to estimate overall survival (OS) and disease-free survival (DFS). Statistical significance was determined as follows: ns, P\u0026thinsp;\u0026ge;\u0026thinsp;0.05; *, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. The expression of FAM83B in LUAD\u003c/h2\u003e \u003cp\u003eTo investigate the clinical relevance of FAM83B expression in LUAD, we conducted an analysis of FAM83B expression across various publicly available datasets. Specifically, we examined the mRNA sequencing data from the GSE19188 database. Our findings revealed a statistically significant increase in FAM83B expression in tumors compared to normal samples (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, the expression of FAM83B was found to be significantly elevated in lung tumor samples compared to corresponding normal samples, as evidenced by various gene expression profile data sources, including GSE116959, GSE18842, and GSE101929 databases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To establish the correlation between FAM83B expression and OS, we conducted an analysis from GSE101929, GSE19188, and GSE13213 datasets from the GEO database. Within these datasets, patients with high FAM83B expression exhibited a significantly decreased OS compared to those with low FAM83B expression (P\u0026thinsp;=\u0026thinsp;0.0024, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, additional databases were employed to examine the impact of FAM83B expression on DFS. Our analysis revealed an association between elevated levels of FAM83B and a poor DFS outcome. Specifically, the datasets GSE50081 and GSE30219 demonstrated a noteworthy reduction in DFS among patients exhibiting high FAM83B expression compared to those with low FAM83B expression (P\u0026thinsp;=\u0026thinsp;0.0089, 0.00068) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTumors exhibiting drug resistance demonstrate an increased propensity for recurrence or metastasis. In instances of advanced LUAD accompanied by distant metastasis, patients are generally deemed ineligible for surgical excision of the primary lesions. Within the confines of this investigation, a total of 109 instances of metastatic LUAD were meticulously selected, and the expression of FAM83B within these cases was scrutinized. The resultant positivity rate was estimated to be approximately 54.13% (59 out of 109), surpassing the positivity rate of 26.89% (32 out of 119) observed in 119 primary LUAD samples. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The overexpression of FAM83B conferred chemoresistance and facilitated proliferation, migration, invasion and metastasis in LUAD\u003c/h2\u003e \u003cp\u003eIn our previous report, it was certified that the cell lines H1299 and A549 displayed low expression of FAM83B, while the PC9 cell line exhibited high expression. FAM83B overexpression cell lines (FAM83B OE) and their corresponding control lines (FAM83B NC) were established in H1299 and A549 cells, while a FAM83B silencing cell line (si FAM83B) and its control (si NC) were established in PC9 cells. A fluorescence microscopy analysis revealed that over 70% of the cells were infected with fluorescent green-labeled lentivirus, indicating a high transfection efficiency.\u003c/p\u003e \u003cp\u003eTo investigate the impact of FAM83B overexpression on the drug resistance of LUAD cells, we subjected H1299/FAM83B, A549/FAM83B, and PC9/si FAM83B cells to varying concentrations of pemetrexed and subsequently assessed cell viability after 72 hours. The findings revealed a cell survival rate of 66.3% in the H1299/FAM83B OE group, whereas it was 57.3% in the H1299/FAM83B NC cells (P\u003c0.01). In the A549/FAM83B OE group, the survival rate was observed to be 80%, whereas in the control group it was 57.6% (P\u003c0.01). Similarly, in the PC9/si FAM83B group, the survival rate was found to be 25.4%, while in the control group it was 35.1% (P\u003c0.05). These results indicate a significantly higher drug resistance in FAM83B OE LUAD cells compared to the corresponding control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe EdU assay was employed to track cell proliferation, specifically investigating the involvement of FAM83B in cell proliferation in LUAD cells. The findings demonstrated that the upregulation of FAM83B substantially augmented the proportion of EdU-positive cells, whereas the downregulation of FAM83B resulted in a decrease in the proportion of EdU-positive cells compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003eThe analysis of scratch distance indicated a notable increase in tumor migration speed within the FAM83B OE group compared to the FAM83B NC group. Conversely, the PC9/siFAM83B group exhibited a deceleration in migration rate, suggesting that FAM83B overexpression expedites the wound healing process. This observation is supported by Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C. The effects of FAM83B on the migration and invasion of H1299/FAM83B OE, A549/FAM83B OE, and PC9/siFAM83B cells were investigated using Boyden chamber assays. The results depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F demonstrate that the overexpression of FAM83B significantly increased the invasive capacity of H1299/FAM83B OE by approximately 28.5% compared to H1299/FAM83B NC, and A549/FAM83B OE by approximately 31.6% (P\u003c0.0001) compared to A549/FAM83B NC (P\u003c0.0001). In contrast, the invasive potential of PC9/siFAM83B exhibited a significant decrease of 27.8% when compared to PC9/FAM83B siNC (P\u003c0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3. Identification of key candidate genes and biological pathways between H1299/FAM83B OE and H1299/FAM83B NC by proteomic sequencing analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA proteomic analysis was conducted comparing H1299/FAM83B OE and H1299/FAM83B NC, resulting in the identification of 2801 genes (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and 75 genes (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) that exhibited statistically significant differences in expression. These differences were determined based on sequencing results that met the criteria of a fold change greater than 1.2 (up or down) and a p-value less than 0.05, as determined by JiKai Gene. The proteomic heat map in (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) displays the differential protein expressions associated with OXPHOS between the two groups. The GSEA analysis revealed that the FAM83B OE group exhibited a higher level of OXPHOS compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, the cBioPortal database revealed the identification of 1421 genes (Supplementary Table S3) that exhibited co-expression with FAM83B. The identification of differential protein expression and the associated metabolic pathway was conducted through GO and KEGG enrichment analyses using FunRich Software (v3.1.3), with subsequent analysis performed on the downloaded TXT files of the results. Furthermore, the Gene Co-expression website was utilized to identify 1,500 genes (Supplementary Table S4) and their network of interactions, while the GO and KEGG pathway functions on the KOBAS were employed to determine the involved metabolic pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B). Moreover, a total of 113 genes exhibited co-expression with the target gene FAM83B, out of which 54 genes (Supplementary Table S5) demonstrated co-expression with NSCLC as documented in the Coexpedia database. By intersecting the metabolism-related genes obtained through sequencing analysis and the genes co-expressed with FAM83B, CALB2 and HMGA1 were isolated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. FAM83B affected the distribution of CALB2 between cytoplasm and mitochondria and inhibited apoptosis\u003c/h2\u003e \u003cp\u003eProteins isolated from H1299, A549, and PC9 cells were subjected to Western blotting analysis. The results revealed that the expression of CALB2 in H1299/FAM83B OE cells was significantly higher compared to H1299/FAM83B NC cells. However, the ratio of CALB2 in the mitochondria/cytoplasm was found to be significantly lower in H1299/FAM83B OE cells compared to NC cells (P\u0026thinsp;=\u0026thinsp;0.007). Similarly, in the A549/FAM83B OE group, the expression of CALB2 was observed to be lower than A549/FAM83B NC cells, and the ratio of mitochondria/cytoplasm CALB2 was decreased compared to the NC cell group (P\u0026thinsp;=\u0026thinsp;0.007) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C and D). In contrast, the silencing of FAM83B resulted in an increased expression of CALB2 in PC9/siFAM83B cells compared to PC9/siNC cells. Additionally, the ratio of mitochondria/cytoplasm CALB2 was significantly higher in NC cells (P\u0026thinsp;=\u0026thinsp;0.03) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and E). Western blot analysis was conducted to evaluate the levels of caspase 3 and B-cell lymphoma (Bcl)-2 expression. The levels of caspase 3 in the H1299/FAM83B NC, A549/FAM83B NC, and PC9/siFAM83B groups were significantly higher than those in the respective control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5. FAM83B overexpression increased mitochondrial metabolism in LUAD cells\u003c/h2\u003e \u003cp\u003eThe findings revealed a noteworthy elevation in the MIMP of H1299/FAM83B OE and A549/FAM83B OE cells in comparison to the parental cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To elucidate the underlying mechanism responsible for this increase in MIMP, measurements were taken for MTG and ATP production. The findings of the study indicate a notable increase in MTG in H1299/FAM83B OE and A549/FAM83B OE cells when compared to H1299/FAM83B NC and A549/FAM83B NC, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Additionally, the production of ATP was significantly elevated in H1299/FAM83B OE and A549/FAM83B OE cells compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The data presented in this study are the average values obtained from triple independent experiments. Statistical significance was determined using Student's t-test, with a threshold of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*P\u003c0.05, ** P\u003c0.01, and *** P\u003c0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6. Metabolic targeting with metformin in concomitance with chemicals to enhance the chemotherapy sensitivity in FAM83B overexpressed cells of the LUAD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe inhibitory effect of metformin on mitochondrial activity has been established. Upon co-administration of pemetrexed and metformin in H1299/FAM83B OE, A549/FAM83B OE, and PC9 cells, a notable decrease in MIMP, MTG, and ATP production was observed compared to the use of pemetrexed alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-G). H1299/FAM83B OE, A549/FAM83B OE, and PC9 cell lines were subjected to treatment with 10mM metformin in combination with varying concentrations of pemetrexed to investigate the impact of metformin on drug resistance in LUAD. Notably, in H1299/FAM83B OE cells, the survival rate of the pemetrexed combined with metformin group (59%) was significantly lower compared to the pemetrexed alone group (74.3%) (P\u0026thinsp;=\u0026thinsp;0.0247) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH); In the A549/FAM83B OE cell line, the survival rates of the group treated with pemetrexed combined with metformin and the group treated with pemetrexed alone were 65.7% and 74.3%, respectively (P\u0026thinsp;=\u0026thinsp;0.0299) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). In the PC9 cell line, the survival rates of the group treated with pemetrexed alone and the group treated with pemetrexed combined with metformin were 54.3% and 27.6%, respectively (P\u0026thinsp;=\u0026thinsp;0.0039) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). These findings indicate that metformin has the potential to enhance the chemosensitivity of LUAD.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSubstantial evidence supports the upregulation of FAM83B mRNA and protein expression in various types of solid tumors (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Our previous study showed that FAM83B exhibited elevated expression levels in primary LAUD and high level of FAM83B promotes local lymph node metastasis. In the present investigation, we expanded upon this finding by examining the expression of FAM83B in four distinct GEO databases pertaining to LUAD. The results further substantiated the significantly higher expression of FAM83B in cancerous tissues compared to para-cancerous tissues. Furthermore, we conducted an examination of the association between elevated levels of FAM83B and prognosis in an additional five GEO databases using Kaplan-Meier analysis. The study findings indicated a significant decrease in survival rates among patients exhibiting high levels of FAM83B expression in contrast to those with low expression levels. These findings suggest that FAM83B exhibits as a viable target for the chemotherapy of LUAD. Furthermore, we obtained samples of metastases from a cohort of 109 patients diagnosed with inoperable advanced LUAD and proceeded to analyze the expression levels of FAM83B. In comparison to the positive rate of FAM83B (26.89%) observed in primary LUAD cases, the metastatic LUAD cases exhibited a significantly higher rate of FAM83B expression (54.13%). These experimental findings indicate that LUAD cases characterized by an over-expression of FAM83B may possess a heightened metastatic capacity and a more unfavorable prognosis.\u003c/p\u003e \u003cp\u003eIn our prior investigation, we reported that FAM83B facilitated the migratory and invasive capabilities of H1299 cells. Here, we expanded our analysis to include additional LUAD cell lines to examine the potential impact of FAM83B overexpression on the malignant characteristics of LUAD cells. The results indicated that the upregulation of FAM83B contributes to drug resistance and augmented the proliferation, migration, and invasion capabilities of LUAD cells. However, the mechanisms underlying the regulatory role of FAM83B in these phenotypic changes remain inadequately elucidated.\u003c/p\u003e \u003cp\u003eThe analysis focused on the differentially expressed proteins between the H1299/FAM83B OE and H1299/FAM83B NC groups. GSEA enrichment analysis revealed that the H1299/FAM83B OE group exhibited a higher level of OXPHOS compared to the control. Metabolic abnormalities are recognized as indicators of cancer, yet the underlying regulatory mechanisms remain poorly understood. Warburg effect is a defining feature of cancer cells, as it pertains to their capacity to predominantly utilize glycolysis for the generation of ATP. However, recent evidence has indicated the significant involvement of OXPHOS in cancer progression. Cancer cells possess the ability to transition between anaerobic respiration and OXPHOS to obtain energy and facilitate tumor advancement. Recognizing this metabolic trait could potentially offer a therapeutic avenue for certain cancer types with OXPHOS inhibitors (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The inhibition of hepatic glucose production by metformin has been shown to effectively reduce glycemia in patients with hyperglycemia (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Emerging evidence indicates that metformin may also possess the potential to inhibit OXPHOS (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Hirpra et al. have proposed that OXPHOS-dependent metabolism serves as the primary mechanism for acquired drug resistance in oncogene-addicted cancer cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In order to investigate the role of FAM83B in drug resistance to pemetrexed, the evaluation of CCK8 and mitochondrial activity was conducted. The results indicate that FAM83B OE cells demonstrated a greater survival rate. Furthermore, the administration of metformin enhanced the effectiveness of chemotherapy in these cells. Additionally, it was observed that FAM83B OE cells exhibited higher levels of MIMP, MTG, and intracellular ATP in comparison to cells with suppressed expression of FAM83B. These results suggest that FAM83B may contribute to the enhancement of drug resistance in LUAD cells by augmenting mitochondrial OXPHOS. This finding aligns with prior research indicating that numerous forms of drug-resistant cancer cells heavily depend on mitochondrial OXPHOS as an energy source. Consequently, it is anticipated that various inhibitors of OXPHOS will augment the chemosensitivity of these tumors (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Additionally, alternative studies have put forth the notion that heightened levels of OXPHOS in CSCs can foster resistance to chemotherapy (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, the specific mechanisms by which OXPHOS activity contributes to chemotherapy resistance and tumor growth remain unclear. Further analysis of protein expression has identified three proteins, namely CALB2, HMGA1, and SLC2A1, that exhibit differential expression between H1299/FAM83B OE and H1299/FAM83B NC cells.\u003c/p\u003e \u003cp\u003eCALB2 encompasses calcium-binding motifs and is qualified as intracellular calcium buffer (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Multiple studies have provided evidence indicating that calretinin expression in LUAD is limited and feeble (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The interplay of calcium flux between the ER and mitochondria has the potential to influence several fundamental characteristics of cancer, such as evasion of apoptosis and cellular invasion. CALB2, acted as transitory calcium ion sinks/stores, affected the calcium ion-free or calcium ion-bound state in cytoplasm and mitochondria, thereby triggering the behavior of LUAD (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). It is plausible that CALB2 is a member of a cluster of calcium sensor proteins within this family (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The involvement of mitochondrial calcium dynamics in the regulation of cellular energy metabolism is well-established. The process of mitochondrial calcium uptake plays a crucial role in governing mitochondrial metabolism (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). This study examined the influence of FAM83B on the subcellular localization of CALB2 and found that, irrespective of its expression level, the proportion of CALB2 entering mitochondria was markedly reduced in both H1299/FAM83B OE and A549/FAM83B OE. Conversely, in PC9 cells, CALB2 cytoplasm/mitochondrial translocation increased following FAM83B interference. Therefore, we hypothesized that the inhibition of CALB2 translocation to the mitochondria by FAM83B could lead to a relative increase in calcium binding to CALB2 and change the cellular calcium homeostasis. The reduction in free Ca2\u0026thinsp;+\u0026thinsp;counteracted the effects of extrinsic apoptosis, ultimately leading to the suppression of apoptosis. Consequently, the overexpression of FAM83B aggravated the development of chemotherapy resistance in LUAD cells, as evidenced by the increased expression of Bcl-2 and decreased expression of cleaved caspase3 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). According to previous studies, it has been observed that mitochondrial calcium signaling plays a role in regulating the differentiation of fibroblasts into myofibroblasts. It is hypothesized that CALB2 could serve as a significant mediator and targeting OXPHOS inhibition may hold promise as a novel therapeutic approach for drug resistant cancer cells (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Valerio et al have discovered the downregulation of TRPC3 protein leads to the promotion of cytosolic and mitochondrial Ca2\u0026thinsp;+\u0026thinsp;oscillations, as well as an increase in mitochondrial Ca2\u0026thinsp;+\u0026thinsp;levels, mitochondrial oxygen consumption rate, and OXPHOS, which promotes the proliferation of cancerous epithelial cells and tumor development in vivo (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In our investigation, we observed an inhibition of CALB2 translocation, which might disrupt cellular calcium homeostasis as the calcium buffer and induced cytosolic and mitochondrial Ca2\u0026thinsp;+\u0026thinsp;oscillations. Consequently, this led to alterations in mitochondrial function, including an increase in mitochondrial metabolism, as demonstrated by the upregulation of MIMP, MTG, and ATP levels in FAM83B OE LUAD cells. Ruolan et al. have reported that metformin chemo sensitized the recipient acute myeloid leukemia cells by decreasing the transfer of mitochondria and inhibiting OXPHOS (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Our investigation revealed that FAM83B OE cells displayed a more aggressive malignant phenotype, characterized by increased susceptibility to drug resistance, enhanced proliferation and metastatic capabilities. These phenomena could potentially be attributed to the overexpression of FAM83B, which may impede the translocation of CALB2 into the mitochondria, thereby leading to the cytosolic and mitochondrial Ca2\u0026thinsp;+\u0026thinsp;oscillations. Additionally, the reduction of free Ca2\u0026thinsp;+\u0026thinsp;in the cytoplasm impedes the progression of apoptotic cells by impeding the activity of caspase3 and substract cleavage. On the other hand, the changes of cellular calcium homeostasis have the capacity to elicit modifications in mitochondrial metabolism and OXPHOS. After the administration of metformin to block mitochondrial OXPHOS, the metabolic activity of LUAD cells was attenuated, resulting in an increased sensitivity of tumor cells to chemotherapy drugs. These findings suggest a potential role of FAM83B overexpression in the development of chemoresistance. Consequently, this phenomenon contributes to chemoresistance and fosters the development of the malignant phenotype in LUAD. In this experiment, we only detected the expression of Bcl-2 and additional experiments are required to ascertain the potential impact of altered calcium homeostasis on the activation of the intrinsic apoptosis pathway, specifically by examining the elevation of Ca2\u0026thinsp;+\u0026thinsp;within the mitochondria.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, our research has presented empirical evidence substantiating the role of FAM83B in the augmentation of chemotherapy resistance in LUAD by impeding the translocation of CALB2 to mitochondria. Consequently, this interference impacts cellular calcium homeostasis, a crucial factor in cell viability and mitochondrial functionality, thereby fostering chemoresistance. This discovery introduces a potential therapeutic target for the treatment of LUAD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conceptualized by Xiaojuan Wu and the manuscript was revised by her. Cell culture was conducted by Jiajia Wang, while IHC was performed by Jing Zhang and Yan Wang. Molecular biology experiments were carried out by Panpan Li and Limin Sun. Data analysis was performed by Ke Yue, and Jiajia Wang edited the manuscript. The final manuscript and its published versions were approved by all authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (NSFC, grant no. 81402181) and the National Natural Science Foundation of Shandong Province (grant no. ZR2023MH105).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest related to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese procedures were performed in accordance with the ethical standards of the Committee on Human Experimentation of Shandong University Medical Research Ethics Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede Castro J, Rodriguez MC, Martinez-Zorzano VS, Sanchez-Rodriguez P, Sanchez-Yague J (2014) Erythrocyte fatty acids as potential biomarkers in the diagnosis of advanced lung adenocarcinoma, lung squamous cell carcinoma, and small cell lung cancer. 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Redox Biol 25:101076\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCipriano R et al (2012) FAM83B mediates EGFR- and RAS-driven oncogenic transformation. J Clin Invest 122:3197\u0026ndash;3210\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu L et al (2017) Modeling the Genetic Regulation of Cancer Metabolism: Interplay between Glycolysis and Oxidative Phosphorylation. Cancer Res 77:1564\u0026ndash;1574\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen CL, Lin CY, Kung HJ (2021) Targeting Mitochondrial OXPHOS and Their Regulatory Signals in Prostate Cancers. Int J Mol Sci 22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBosc C, Selak MA, Sarry JE (2017) Resistance Is Futile: Targeting Mitochondrial Energetics and Metabolism to Overcome Drug Resistance in Cancer Treatment. Cell Metab 26:705\u0026ndash;707\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuda M et al (2020) Calretinin-expressing lung adenocarcinoma: Distinct characteristics of advanced stages, smoker-type features, and rare expression of other mesothelial markers are useful to differentiate epithelioid mesothelioma. Pathol Res Pract 216:152817\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones CL et al (2018) Inhibition of Amino Acid Metabolism Selectively Targets Human Leukemia Stem Cells. Cancer Cell 34:724\u0026ndash;740e724\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwaller B (2010) Cytosolic Ca2\u0026thinsp;+\u0026thinsp;buffers. Cold Spring Harb Perspect Biol 2:a004051\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eComin CE et al (2014) Expression of thrombomodulin, calretinin, cytokeratin 5/6, D2-40 and WT-1 in a series of primary carcinomas of the lung: an immunohistochemical study in comparison with epithelioid pleural mesothelioma. Tumori 100:559\u0026ndash;567\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMattson MP, Chan SL (2003) Calcium orchestrates apoptosis. Nat Cell Biol 5:1041\u0026ndash;1043\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHack NJ, Wride MC, Charters KM, Kater SB, Parks TN (2000) Developmental changes in the subcellular localization of calretinin. J Neurosci 20:RC67\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwaller B, Durussel I, Jermann D, Herrmann B, Cox JA (1997) Comparison of the Ca2+-binding properties of human recombinant calretinin-22k and calretinin. J Biol Chem 272:29663\u0026ndash;29671\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong H et al (2022) Mitochondrial calcium uniporter promotes phagocytosis-dependent activation of the NLRP3 inflammasome. Proc Natl Acad Sci U S A 119:e2123247119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStevenson L et al (2011) Calbindin 2 (CALB2) regulates 5-fluorouracil sensitivity in colorectal cancer by modulating the intrinsic apoptotic pathway. PLoS ONE 6:e20276\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y, Fabritius M, Ip C (2009) Chemotherapeutic sensitization by endoplasmic reticulum stress: increasing the efficacy of taxane against prostate cancer. Cancer Biol Ther 8:146\u0026ndash;152\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLombardi AA et al (2019) Mitochondrial calcium exchange links metabolism with the epigenome to control cellular differentiation. Nat Commun 10:4509\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarfariello V et al (2022) TRPC3 shapes the ER-mitochondria Ca(2+) transfer characterizing tumour-promoting senescence. Nat Commun 13:956\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYou R et al (2022) Metformin sensitizes AML cells to chemotherapy through blocking mitochondrial transfer from stromal cells to AML cells. Cancer Lett 532:215582\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"apoptosis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"appt","sideBox":"Learn more about [Apoptosis](http://link.springer.com/journal/10495)","snPcode":"10495","submissionUrl":"https://submission.nature.com/new-submission/10495/3","title":"Apoptosis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"FAM83B, CALB2, mitochondrial metabolism, apoptosis, chemotherapy resistance, lung adenocarcinoma","lastPublishedDoi":"10.21203/rs.3.rs-3837359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3837359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Chemotherapy is an effective therapeutic modality; nevertheless, a significant proportion of patients diagnosed with lung adenocarcinoma (LUAD) demonstrate resistance to chemotherapy. Therefore, it is crucial to understand the potential regulatory mechanisms to develop novel treatment strategies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eMultiple assays, such as CCK8, wound healing, EdU, and transwell assays, were employed to confirm the augmented chemotherapy resistance, heightened cell proliferation, migration, and invasion caused by FAM83B overexpression in LUAD cells. Furthermore, MIMP, MTG, and ATP assays were utilized to quantify changes in mitochondria metabolism. In vitro functional assays were performed to evaluate the influence of FAM83B overexpression on the malignant progression and resistance mechanisms to chemotherapy in LUAD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eIn the context of this study, it was determined that LUAD patients with increased FAM83B expression had shorter survival times, and tissue samples with FAM83B overexpression were more prone to metastasis compared to primary samples. As a result, FAM83B is identified as an adverse prognostic marker. The mechanistic analysis demonstrated that FAM83B impedes the translocation of calbindin 2 (CALB2) from the cytoplasm to the mitochondria, resulting in the inhibition of apoptosis and the promotion of mitochondrial activity. Consequently, this ultimately confers resistance to chemotherapy in LUAD. Furthermore, the administration of metformin, which blocks mitochondrial oxidative phosphorylation (OXPHOS), can restore sensitivity to drug resistance in LUAD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Taken together, these findings provide substantial evidence supporting the notion that FAM83B enhances chemotherapy resistance in LUAD through the upregulation of mitochondrial metabolism and the inhibition of apoptosis.\u003c/p\u003e","manuscriptTitle":"Title: FAM83B Regulates Mitochondrial Metabolism and Anti-Apoptotic Activity in pulmonary adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-09 19:21:04","doi":"10.21203/rs.3.rs-3837359/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-29T08:11:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-01-25T14:29:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6d20950f-197d-4ecd-a413-86d96541f4f1","date":"2024-01-17T11:24:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-07T14:08:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-06T08:05:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-06T08:05:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Apoptosis","date":"2024-01-05T13:25:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"apoptosis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"appt","sideBox":"Learn more about [Apoptosis](http://link.springer.com/journal/10495)","snPcode":"10495","submissionUrl":"https://submission.nature.com/new-submission/10495/3","title":"Apoptosis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ff37d57e-7364-4f48-b2cd-b52eee3644b2","owner":[],"postedDate":"January 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-16T20:06:10+00:00","versionOfRecord":{"articleIdentity":"rs-3837359","link":"https://doi.org/10.1007/s10495-024-01944-7","journal":{"identity":"apoptosis","isVorOnly":false,"title":"Apoptosis"},"publishedOn":"2024-03-13 20:06:10","publishedOnDateReadable":"March 13th, 2024"},"versionCreatedAt":"2024-01-09 19:21:04","video":"","vorDoi":"10.1007/s10495-024-01944-7","vorDoiUrl":"https://doi.org/10.1007/s10495-024-01944-7","workflowStages":[]},"version":"v1","identity":"rs-3837359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3837359","identity":"rs-3837359","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}