ASPM Is a Prognostic Biomarker and Correlates With Immune Infiltration in Kidney Renal Clear Cell Carcinoma and Liver Hepatocellular Carcinoma

Abstract

Background: Abnormal spindle microtubule assembly (ASPM) is a centrosomal protein and that is related to a poor clinical prognosis and recurrence. However, the relationship between ASPM expression, tumor immunity, and the prognosis of different cancers remains unclear.

Methods: ASPM expression and its influence on tumor prognosis were analyzed using the Tumor Immune Estimation Resource (TIMER), UALCAN, OncoLnc, and Gene Expression Profiling Interactive Analysis (GEPIA) databases. The relationship between ASPM expression and tumor immunity was analyzed using the TIMER and GEPIA databases, and the results were further verified using qPCR, western blot, and multiplex quantitative immuno fluorescence.

Results: The results showed that ASPM expression was significantly higher in most cancer tissues than in corresponding normal tissues, including kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), and breast invasive carcinoma (BRCA). ASPM expression was significantly higher in late-stage cancers than in early-stages cancers (e.g., KIRC, KIRP, LIHC, LUAD, and BRCA; p < 0.05), demonstrating a possible role of ASPM in cancer progression and invasion. Moreover, our data showed that high ASPM expression was associated with poor overall survival, and disease-specific survival in KIRC and LIHC (p < 0.05). Besides, Cox hazard regression analysis results showed that ASPM may be an independent prognostic factor for KIRC and LIHC. ASPM expression showed a strong correlation with tumor-infiltrating B cells, CD8⁺ T cells, and M2 macrophages in KIRC and LIHC.

Conclusions: These findings demonstrate that the high expression of ASPM indicates poor prognosis as well as increased levels of immune cell infiltration in KIRC and LIHC. ASPM expression may serve as a novel prognostic biomarker for both the clinical outcome and immune cell infiltration in KIRC and LIHC.

Keywords: ASPM; biomarker; gene expression; prognosis; tumor-infiltrating.

Figure 1

ASPM mRNA expression levels in various types of cancer. (A) The expression levels of ASPM in various types of cancer tissues and their corresponding normal tissues were analyzed using the Tumor Immune Estimation Resource (TIMER) database. It includes 10,897 samples for 32 cancer types from The Cancer Genome Atlas (TCGA) database to estimate the role of immune infiltration. The color intensity (red or blue) is directly proportional to the significance level of upregulation or downregulation, respectively. The expression of ASPM was upregulated in BLCA (bladder urothelial carcinoma), BRCA (breast invasive carcinoma), CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA (esophageal carcinoma), HNSC (head and neck squamous cell carcinoma), KICH (kidney chromophobe), KIRC (kidney renal clear cell carcinoma) and KIRP (kidney renal papillary cell carcinoma), LIHC (Liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), LUSC (lung squamous cell carcinoma), PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma), SKCM (skin cutaneous melanoma), STAD (stomach adenocarcinoma), THCA (thyroid carcinoma), and UCEC (uterine corpus endometrial carcinoma). P-value Significant Codes: 0 ≤ ***< 0.001 ≤ **< 0.01 ≤ *< 0.05 ≤. < 0.1. (B) The expression levels of ASPM in different tumor types using the UALCAN database. KIRC: primary tumor (n = 533), normal (n = 72); KIRP: primary tumor (n = 290), normal (n = 32); LIHC: primary tumor (n = 371), normal (n = 50); LUAD: primary tumor (n = 515), normal (n = 59); PAAD: primary tumor (n = 178), normal (n = 4); BRCA: primary tumor (n = 1097), normal (n = 114). a: The expression of ASPM in KIRC; b: The expression of ASPM in KIRP; c: The expression of ASPM in LIHC; d: The expression of ASPM in LUAD; e: The expression of ASPM in PAAD; f: The expression of ASPM in BRCA. n: Number of samples.

Figure 2

UALCAN analysis for the correlation between ASPM mRNA expression level based on cancer stage. KIRC: Normal (n = 72), stage 1 (n = 267), stage 2 (n = 57), stage 3 (n = 123), stage 4 (n = 84); KIRP: Normal (n = 50), stage 1 (n = 168), stage 2 (n = 84), stage 3 (n = 82), stage 4 (n = 6); LUAD: Normal (n = 59), stage 1 (n = 277), stage 2 (n = 125), stage 3 (n = 85), stage 4 (n = 28); PAAD: Normal (n = 4), stage 1 (n = 6), stage 2 (n = 146), stage 3 (n = 4), stage 4 (n = 4); BRCA: Normal (n = 114), stage 1(n = 183), stage 2(n = 615), stage 3(n = 247), stage 4 (n = 20). (A) Expression of ASPM in KIRC based on individual cancer stages. (B) Expression of ASPM in KIRP based on individual cancer stages. (C) Expression of ASPM in LIHC based on individual cancer stages. (D) Expression of ASPM in LUAD based on individual cancer stages. (E) Expression of ASPM in PAAD based on individual cancer stages. (F) Expression of ASPM in BRCA based on individual cancer stages. n: Number of samples.

Figure 3

The functional protein association network generated by the STRING database. (A) The network of ASPM and its co-expression genes was set up visually. (B) The biological functions (GO enrichment) of the identified genes via the Enrichr online database. (a) biological process, (b) molecular function, and (c) cellular component. The biological processes of these proteins mainly involved the regulation of mitotic metaphase/anaphase transition (GO:0030071), mitotic spindle assembly checkpoint (GO:0007094), and mitotic spindle checkpoint (GO:0071174). The molecular function term was mainly enriched in microtubule motor activity (GO:0003777), histone kinase activity (GO:0035173), and cyclin-dependent protein kinase activity (GO:0097472). The cell component term was significantly enriched in the mitotic spindle (GO:0072686), spindle microtubule (GO:0005876), and kinetochore microtubule (GO:0005828).

Figure 4

Correlation between ASPM expression and survival outcomes using the OncoLnc online tool. The relationship between the expression level of ASPM and the survival rate of (A) KIRC (Low: n = 261; High: n = 261); (B) KIRP (Low: n = 255; High: n = 255); (C) LIHC (Low: n = 180; High: n = 180); (D) LUAD (Low: n = 246; High: n = 246); (E) PAAD(Low: n = 87; High: n = 87); and (F) BRCA (Low: n = 503; High: n = 503) patients.

Figure 5

The relationship between the transcription level of ASPM and cancer prognosis. Survival curves showing association of ASPM expression with prognosis: overall survival (OS). For the accurate interpretation of ASPM expression detection results, we set the cut-off value as 50%. (A) KIRC (HR = 1.9, p = 0.00044), (B) KIRP (HR = 3.8, p = 0.00016) (C) LIHC (HR = 1.8, p = 0.00071), (D) LUAD (HR = 1.7, p = 0.00044), (E) PAAD (HR = 1.9, p = 0.00035), and (F) BRCA (HR = 1.1, p = 0.76). HR: Hazard Ratio.

Figure 6

Relationship between ASPM expression and tumor-infiltrating immune cells using TIMER database. ASPM expression is related to immune cell infiltration (B cells, CD8⁺ T cells, CD4⁺ T cells macrophages, neutrophils, and DCs) in KIRC, KIRP, LIHC, LUAD, PAAD, and BRCA using the TIMER database. cor: Correlation.

Figure 7

Expression analysis of ASPM in KIRC and LIHC tissues. (A) Relative level of ASPM mRNA using quantitative RT-PCR; **p < 0.01. (B) The expression of ASPM was analyzed by Western-blot analysis using a compound samples. (C) Tumor infiltration of B cells, CD8⁺ T cells, and M2 macrophages in KIRC and LIHC using immunohistochemistry. 20 diagnosed cases of KIRC and 20 diagnosed cases of LIHC samples for immunohistochemistry. We validate the relationship between ASPM expression and B cells (marker: CD19), CD8⁺ T cells (marker: CD8A), and M2 macrophages (marker: CD163), we performed immunohistochemistry to assess ASPM, CD19, CD8A, and CD163. Muscle and lymph nodes as control samples. (a-d, f-i) Tumor infiltration of B cells, CD8⁺ T cells, and M2 macrophages in KIRC. (k-n, p-s) Tumor infiltration of B cells, CD8⁺ T cells, and M2 macrophages in LIHC. (a-d) High expression of ASPM (+++), CD8A (++), CD19 (++), and CD163 (++) in KIRC. (f-i) Low expression of ASPM (+), CD8A (+), CD19 (+), and CD163 (+) in KIRC. (k-n) High expression of ASPM (+++), CD8A (++), CD19 (++), and CD163 (++) in LIHC. (p-s) Low expression of ASPM (+), CD8A (+), CD19 (+), and CD163 (+) in LIHC. (e, o) lymph nodes was used as a positive control in KIRC (+++) and LIHC (+++). (j, t) muscle was used as a negative control in KIRC (-) and LIHC (-). The expression density of ASPM, CD8A, CD19, and CD163 in KIRC and LIHC tissues were quantitated by scoring staining intensity, including negative (–) and weak (+) staining, moderate (++) and strong (+ + +) staining, respectively.

Figure 8

Detection of tumor-infiltrating levels of CD8⁺ T cells, B cells, and M2 macrophages in KIRC and LIHC using multiplex quantitative immunofluorescence. 20 diagnosed cases of KIRC and 20 diagnosed cases of LIHC samples for ultiplex quantitative immunofluorescence. Representative fluorescence images showing the detection of immune cells samples by simultaneous staining of DAPI, ASPM (rose red channel), CD8A (red channel), CD19 (pink channel), and CD163 (green channel) in KIRC (A, B) and LIHC (C, D). CD8⁺ T cells marker: CD8A; B cells marker: CD19; M2 macrophages marker: CD163. (E, F): Immune cell infiltration level at high or low expression of ASPM in KIRC and LIHC. **p < 0.01.

Figure 9

Univariate and Multivariate Cox analysis of ASPM and pathological parameters in KIRC (A) and LIHC (B). 611 KIRC patients and 424 LIHC patients were used for analysis. Patients were divided into different subgroups according to, age, clinical stage, clinical TNM stage, and ASPM expression. For each subgroup, the prognostic performance of ASPM on overall survival was evaluated by Cox regression, and the results are presented as hazard ratio (HR). The bar represents the 95% confidence interval of HR.