The level of metabolic tumor volume was correlated with changes in gene expression and biological functions in pancreatic cancer

Pancreatic cancer patient tissues were divided into MTV-low and MTV-high groups, each containing 7 cases, based on PET images presented in Fig. 1A. We followed a five-step process outlined in Additional file 1: Fig. S1 to identify potential markers and therapeutic targets. Initially, we conducted RNA sequencing (RNA-Seq) on both normal and cancerous sections of pancreatic cancer patient tissues. This allowed us to calculate the fold change in gene expression between the cancerous and normal counterparts. The clustered heatmap revealed that, compared to the MTV-low group, the MTV-high group exhibited general upregulation of 211 genes and downregulation of 166 genes (Fig. 1B). Among the 211 upregulated genes, 44 displayed a fold difference of more than 2 and statistical significance. Similarly, among the 166 downregulated genes, 56 exhibited a fold difference of more than 2 and statistical significance. The fold change distribution was centered around twofold for both upregulated and downregulated genes, although some downregulated genes demonstrated a fold difference of 10 or more (Fig. 1C). Functional annotation analysis revealed that the upregulated genes were enriched in processes related to mitosis (KW0498), cell cycle (KW-0131), and cell division (KW-0132), while the downregulated genes were enriched in ATP binding (GO:0005524) and the adenylate cyclase (AC)-activating G-protein coupled receptor(GPCR) signaling pathway (GO:0007189) (Fig. 1D). Notably, among the genes enriched in functional annotations, Kinesin Family Member 11 (KIF11), ATP Binding Cassette Subfamily A Member 13 (ABCA13), and Dicer 1, Ribonuclease III (DICER1) exhibited very significant and dramatic differences in expression between the MTV-low and MTV-high groups (Fig. 1E). Four genes (KIF11, Kinetochore Associated 1/KNTC1, Receptor Accessory Protein 4/REEP4, and Regulator Of Chromosome Condensation 1/RCC1) were commonly associated with cell division, mitosis, and cell cycle, while two genes (Adenylate Cyclase 1/ADCY1 and Adenylate Cyclase 5/ADCY5) were involved in ATP binding and adenylate cyclase(AC)-activating G-protein coupled receptor signaling pathway (Fig. 1F).

Fig. 1

The transcriptome-wide analysis of metabolic tumor volume-associated gene expression in pancreatic cancer. A PET images of pancreatic cancer patients with low MTV (a) and high MTV (b). B Heatmap analysis of gene expression in MTV-low and -high pancreatic cancer patient tissues. 377 genes with below 0.05 p-value between MTV-low and -high groups were presented as cancer-to-normal ratio's binary logarithm. C Bar graphical presentation of DEG fold-change in the MTV-high group compared to the MTV-low group. The genes showing more than twofold change and a statistical significance were presented. D Functional annotation analysis of upregulated and downregulated genes in the MTV-high group compared to the MTV-low group. The biological processes associated with DEGs were analyzed using DAVID and presented as an enrichment bubble. E Balloon plot presentation of annotation-related genes in individual patients. F Venn diagram presentation of annotation-related genes

MTV-associated gene expression was correlated with pancreatic cancer patient survival

To facilitate convenience, the differentially expressed genes (DEGs) between the low and high MTV groups were designated as MTV-associated genes (MAGs), which were further divided into MTV-upregulated genes (MUGs) and MTV-downregulated genes (MDGs). To assess their potential as markers for pancreatic cancer, the expression levels of MUGs and MDGs were compared between the groups of living and deceased patients in the TCGA-PAAD dataset, a large dataset lacking MTV information. As shown in Fig. 2A, out of the 43 MUGs (excluding BST2 Interferon Stimulated Positive Regulator/BISPR, a gene not present in the dataset), 12 genes exhibited a significant positive correlation with patient death, 4 genes displayed a significant inverse correlation, and 27 genes showed no correlation. Among the MDGs, 17 genes demonstrated a significant negative correlation with patient death, 7 genes exhibited a significant inverse correlation, 27 genes showed no correlation, and 2 genes were not expressed. When comparing the living and deceased groups of pancreatic cancer patients, the deceased group displayed a significant upregulation of the MUGs (Cadherin EGF LAG Seven-Pass G-Type Receptor 1/CELSR1, CCR4-NOT Transcription Complex Subunit 1/CNOT1, DNA Cross-Link Repair 1B/DCLRE1B, Integrin Subunit Alpha 3/ITGA3, KIAA1217, KIF11, Membrane Bound O-Acyltransferase Domain Containing 2/MBOAT2, Regulator Of Chromosome Condensation 1/RCC1, Solute Carrier Family 44 Member 1/SLC44A1, SON DNA And RNA Binding Protein/SON, MTOR Associated Protein, Eak-7 Homolog /TLDC1/MEAK7, and ZFP69 Zinc Finger Protein /ZFP69) (Fig. 2B), while showing a significant downregulation of the MDGs (ADCY1, ADP Ribosylation Factor Like GTPase 6 Interacting Protein 4/ARL6IP4, ATPase Phospholipid Transporting 8A1/ATP8A1, Cytochrome C Oxidase Assembly Factor COX14/COX14, Erythropoietin Receptor/EPOR, Family With Sequence Similarity 110 Member D/FAM110D, Growth Arrest And DNA Damage Inducible Gamma/GADD45G, Hematopoietically Expressed Homeobox/HHEX, Inositol Polyphosphate-5-Phosphatase B/INPP5B, Potassium Inwardly Rectifying Channel Subfamily J Member 8/KCNJ8, Lin-7 Homolog B, Crumbs Cell Polarity Complex Component/LIN7B, Musashi RNA Binding Protein 1/MSI1, P21 (RAC1) Activated Kinase 3/PAK3, Retinol Binding Protein 5/RBP5, Sidekick Cell Adhesion Molecule 1/SDK1, Small Nucleolar RNA Host Gene 7/SNHG7, and Synaptogyrin 1/SYNGR1) (Fig. 2C). Additional file 2: Fig. S2 provided information on genes with no or inverse correlation. Groups with high expression of 10 MUGs exhibited significantly poorer patient survival than groups with low expression of MUGs (Fig. 2D). Similarly, groups with low expression of 16 MDGs showed significantly poorer patient survival than groups with high expression of MDGs (Fig. 2E). Furthermore, 10 MUGs and 16 MDGs displayed a significant correlation both in gene expression and patient survival (Fig. 2F). Among the 29 MAGs, 6 genes (ADCY1, ATP8A1, KCNJ8, KIF11, PAK3, and RCC1) were associated with functional annotations (Fig. 2G). Annotation-related genes other than the 6 genes did not show a significant correlation between expression and patient survival (Additional file 3: Fig. S3). In addition to differential gene expression, genetic alteration can affect the survival of patients with pancreatic cancer. Therefore, the genetic alterations of MAGs were analyzed. Of 175 patients in the TCGA-PAAD dataset, genetic alterations were found in 39 (22.3%) but not seen in 136 (77.7%) (Additional file 4: Fig. S4A). Except for MBOAT2, FAM110D, GADD45G, and ARL6IP4, amplification, deletion, and mutation (missense, splice, and truncating) occurred in MUGs and MDGs, but their frequencies were 3% or less (Additional file 4: Fig. S4B). Most of the patients with genetic alteration had less than 3 genetic alterations. However, a patient (TCGA-2J-AAB8) had 16 alterations (Additional file 4: Fig. S4C). Genetic alterations in MAGs were not associated with patient survival. There was no significant difference in patient survival between the group with alteration and non-alteration (Additional file 4: Fig. S4D).

Fig. 2

Analysis of metabolic tumor volume-associated genes in TCGA-PAAD dataset. A Correlation analysis between MAGs and TCGA-PAAD dataset. Expression of (B) MUGs and (C) MDGs between the live and dead groups of patients. Survival plot analysis of patient groups with low and high expression of (D) MUGs and (E) MDGs. F Pie graph presentation of the MUGs and MDGs showing statistically significant gene expression and patient survival. G Annotation-related gene analysis of TCGA-PAAD dataset

MTV-associated genes significantly correlated with TCGA-suggested pancreatic and blood test cancer markers

To assess the potential of MAGs as markers for pancreatic cancer, we examined their correlation with TCGA-suggested markers, the top 20 genes associated with unfavorable and favorable prognoses based on TCGA-PAAD data analysis (The human protein atlas) [45]. As shown in Fig. 3A, most MUGs exhibited a positive correlation with unfavorable genes and a negative correlation with favorable genes. Conversely, most MDGs showed a negative correlation with unfavorable genes and a positive correlation with favorable genes. Additionally, we investigated the correlation between MAGs and CEA Cell Adhesion Molecule (CEACAMs), which are blood test markers for pancreatic cancer [46, 47]. Except for CELSR1 and SON, most MAGs correlated with CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM20, or CEACAM21. CEACAM1, 5, and 6 notably exhibited a significant correlation with many MAGs (Fig. 3B, Additional file 5: Fig. S5A, B). Combinations of tumor markers can potentially improve cancer diagnosis and prognosis prediction compared to individual markers [48]. Therefore, we performed a correlation matrix analysis using MAGs from the MTV RNA-Seq and TCGA-PAAD datasets (deceased patient group) to identify promising combinations. In the MTV RNA-Seq, there was a clear positive correlation between MUGs or MDGs and a negative correlation between MUGs and MDGs. Similarly, the general correlation pattern in the TCGA-PAAD dataset resembled that of the MTV RNA-Seq, although there were some variations in individual correlations (Additional file 6: Fig. S6A). Significant correlations observed in the MTV RNA-Seq, TCGA-PAAD, or between the MTV RNA-Seq and TCGA-PAAD datasets were arranged as a half correlation matrix and further categorized into MTV RNA-Seq only, TCGA-PAAD only, and MTV ∩ PAAD (Additional file 6: Fig. S6B).

Fig. 3

Correlation analysis between MAGs, TCGA-suggested markers, and blood test markers. A Correlation matrix between MAGs and the unfavorable and favorable genes of TCGA-PAAD dataset. The number of significant correlations was further presented as pie graphs. B Correlation analysis between MAGs and CEACAMs. CEACAM1, 5, and 6 were further presented using a correlation dot

Higher tumor grade and unfavorable clinical outcomes correlated with MTV-associated gene expression

The presence of MAGs was observed to be correlated with patient death, as shown in Fig. 2. Therefore, we assumed that the expression of MAGs could potentially be linked to both tumor grade and clinical outcomes. In Fig. 4A, the patient group exhibiting high expression of MUGs demonstrated a greater prevalence of tumor grade (G) 2 and 3 than the group with low expression. Conversely, the patient group with low expression of MDGs exhibited a higher distribution of tumor grade 2 and 3 than the group with high expression. Tumors with higher grades displayed more upregulated MUGs and downregulated MDGs than those with lower grades. The overexpression of KIF11 and RCC1 and the underexpression of ADCY1 and SDK1 were detected in ~ 60% of tumor grade 2 pancreatic cancer patients. Treatment response was analyzed by being categorized into four groups: CR (complete regression), PR (partial regression), PD (partial disease), and SD (stable disease). In Fig. 4B, the expression of MUGs was mainly associated with CR and PD. The patient group with low MUG expression exhibited a higher distribution of CR than the group with high MUG expression, while the patient group with high MUG expression showed a higher distribution of PD than the group with low MUG expression. The expression of MDGs was also mainly associated with CR and PD. The patient group with high MDG expression displayed a higher distribution of CR than the group with low MDG expression, whereas the patient group with low MDG expression exhibited a higher distribution of PD than the group with high MDG expression. Favorable treatment responses (CR and PR) displayed more downregulated MUGs and upregulated MDGs. In contrast, unfavorable treatment responses (PD and SD) exhibited more upregulated MUGs and downregulated MDGs. Post-treatment outcomes were analyzed in DF (disease-free) and RP (recurred or progressed). As shown in Fig. 4C, the patient group with high MUG expression showed a higher distribution of RP and a lower distribution of DF, whereas the group with low MDG expression showed a higher distribution of RP and a lower distribution of DF. Among the MUGs, high expression of KIF11 and RCC1 was commonly correlated with high tumor grades (G2 and G3) and unfavorable clinical outcomes (treatment response-PD, SD, and post-treatment outcome-RP). Conversely, among the MDGs, low expression of ATP8A1 was commonly associated with tumor grades (G2 and G3) and unfavorable clinical outcomes (treatment response-PD, SD, and post-treatment outcome-RP) (Fig. 4D). To assess whether MUGs and MDGs can be used as early detection markers for pancreatic cancer, the mortality rates of stages I and II patients from the TCGA-PAAD dataset were analyzed. In Fig. 5A, ~ 94% of patients in the dataset were in stages I and II. The patients with high MUG expression (marked with a white asterisk) showed ~ 40–60% mortality (Fig. 5B). On the other hand, those with low MDG expression (marked with a white asterisk) showed around 50 to 88 percent mortality (Fig. 5C).

Fig. 4

Correlation analysis of MAGs with tumor grades and clinical outcomes. Correlation of MAGs with (A) tumor grades, (B) treatment response, and (C) post-treatment outcomes. The correlation of MAG expression with tumor grades and clinical outcomes was presented as patient distribution (stacked bar) and distribution comparison between MAG-low and MAG-high groups (symbols with connecting lines). The number of MAGs showing a higher patient distribution for tumor grades, treatment response, and post-treatment outcomes was displayed as a pie graph. D Venn diagram presentation of the MAGs related to clinical outcomes

Fig. 5

MAG expression-dependent mortality rates of stages I and II patients in the TCGA-PAAD dataset. A Distribution of tumor stages in the TCGA-PAAD dataset. Mortality rates of (B) the group with high expression of MUGs and (C) the group with low expression of MDGs in stages I and II pancreatic cancer patients. 166 patients in stages I or II were classified into the groups with low and high MAGs using the Human Protein Atlas cut-off values. The mortality percentages of patient groups with high MUG expression or low MDG expression were marked with a white asterisk

Potential therapeutic options for pancreatic cancer may include miRNAs that show a reverse correlation with the MAGs

Potential therapeutic options for pancreatic cancer could be found in the MUG-binding miRNAs or inhibitors against the MDG-binding miRNAs (Fig. 6A). Therefore, we first identified the miRNAs that potentially bind to the MAGs using a common group of three databases (miRWalk, TargetScan, and miRDB). 25 MAGs were commonly analyzed in all the databases, but 4 MAGs (LIN7B, SNHG7, SON, and TLDC1) were not (Additional file 7: Fig. S7). Subsequently, we investigated the miRNAs showing a reverse expression correlation with the 25 MAGs in the TCGA-PAAD dataset. Of the 10 MUGs, KIAA1217, MBOAT2, and SLC44A1 showed significant and inverse correlations with 3, 11, and 7 miRNAs, respectively (Fig. 6B-a). In contrast, among the 15 MDGs, INPP5B, PAK3, SDK1, and SYNGR1 exhibited significant and inverse correlations with 2, 5, 3, and 2 miRNAs, respectively (Fig. 6B-b). In Fig. 6C, the patient population with high expression of KIAA1217 demonstrated significant downregulation of miRNA(MIR)130B, MIR301A, and MIR301B compared to those with low expression. The patient population with high expression of MBOAT2 showed significant downregulation of MIR27B, MIR29B-2, MIR30B, MIR32, MIR33B, MIR125A, MIR148B, MIR181D, MIR429, MIR642A, and MIR676 compared to those with low expression. The patient population with high expression of SLC44A1 displayed significant downregulation of MIR7-1, MIR7-2, MIR32, MIR301A, MIR1179, MIR1185-1, and MIR1301 compared to those with low expression. In Fig. 6D, the patient population with low expression of INPP5B demonstrated significant upregulation of MIR654 and MIR1185-1 compared to those with high expression. The patient population with low expression of PAK3 exhibited significant upregulation of MIR18A, MIR23A, MIR138-1, MIR193B, and MIR378A compared to those with high expression. The patient population with low expression of SDK1 showed significant upregulation of MIR7-2, MIR299, and MIR495 compared to those with high expression. The patient population with low expression of SYNGR1 displayed significant upregulation of MIR485 and MIR654 compared to those with high expression. To assess the risk of the therapeutic manipulation of corresponding miRNAs, we analyzed patient survival based on the low and high expression of the miRNAs. Patient survival was classified into three groups: MUG (16 miRNAs), MUG ∩ MDG (2 miRNAs), and MDG (9 miRNAs). As shown in Fig. 6E, the patient group with high expression of miRNAs targeting the MUGs did not show a significantly poorer survival than those with low expression. The expression of miRNAs commonly involved in MUG and MDG did not significantly affect patient survival. Except for one miRNA, MIR193B, the patient group with low expression of miRNAs targeting the MDG did not show a significantly poorer survival than those with high expression. The Patients with high expression of MIR193B displayed better survival than those with low expression. Therefore, the inhibition of MIR193B may not be helpful for patient survival.

Fig. 6

MAG-targeting miRNA expression and its association with patient survival. A Schematic presentation of MAG-targeting miRNA therapeutic approaches. B Number of the miRNAs showing reverse expression correlation with putative target MAGs. The number of total miRNAs, the number of the miRNAs showing a significant reverse expression correlation with putative target MAGs, and the number of others were labeled with black, orange, and gray, respectively. Expression of miRNAs between the patient groups with low and high expression of (C) MUGs and (D) MDGs. E Survival plot analysis between patient groups with low and high expression of miRNA potentially targeting MAGs. The significant and nonsignificant correlations of miRNAs with MUG or MDG were presented as pie graphs

MAG proteins may be specific markers and therapeutic targets for pancreatic cancer

The MAGs validated in the TCGA-PAAD dataset were protein-coding genes, except for SHNG7 (long non-coding RNA). Therefore, their protein expression was analyzed between pancreatic normal and cancer tissues using the human protein atlas. In Fig. 7A, KIF11, RCC1, KIAA1217, SLC44A1, ITGA3, and SON were overexpressed in cancer tissues compared to normal ones. Among those MUG proteins, KIF11 and RCC1 were the most validated markers according to the number of analyzed subjects. In Fig. 7B, compared to normal tissues, ATP8A1, ADCY1, INPP5B, and SDK1 were underexpressed in cancer tissues. Among those MDG proteins, ATP8A1 and INPP5B were the most validated markers according to the number of analyzed subjects. Like the correlation matrix analysis with MAG transcripts, the possible combinations of MAG proteins were presented. ITGA3 was combinable with ADCY1, ATP8A1, and SDK1. KIAA1217 was combinable with SLC44A1 and SON. KIF11 were combinable with RCC1, ADCY1, and SDK1. SLC44A1 was combinable with SON and SDK1. ADCY1 was combinable with ATP8A1 and SDK1. ATP8A1 was combinable with SDK1 (Fig. 7C). To evaluate the potential of the MAG combinations in the early detection of pancreatic cancer, patient mortality rates were analyzed using stages I and stage II patients from the TCGA-PAAD dataset. In Additional file 8: Fig. S8, the MUG x MUG combination showed ~ 60–70% mortality, the MDG × MUG combination showed ~ 66–70% mortality, and the MDG × MDG combination showed ~ 63–66% mortality.

Fig. 7

Expression analysis and combinable sets of MAG proteins. Immunohistochemical staining of (A) MUGs and (B) MDGs in pancreatic normal and cancer tissues. C Transcriptome analysis-based combinable sets of MAG proteins for markers and therapeutic targets