p53 and p63 Proteoforms Derived from Alternative Splicing Possess Differential Seroreactivity in Colorectal Cancer with Distinct Diagnostic Ability from the Canonical Proteins

Abstract

Colorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer-related death worldwide. The detection in plasma samples of autoantibodies against specific tumor-associated antigens has been demonstrated to be useful for the early diagnosis of CRC by liquid biopsy. However, new studies related to the humoral immune response in cancer are needed to enable blood-based diagnosis of the disease. Here, our aim was to characterize the humoral immune response associated with the different p53 and p63 proteoforms derived from alternative splicing and previously described as aberrantly expressed in CRC. Thus, here we investigated the diagnostic ability of the twelve p53 proteoforms and the eight p63 proteoforms described to date, and their specific N-terminal and C-terminal end peptides, by means of luminescence HaloTag beads immunoassays. Full-length proteoforms or specific peptides were cloned as HaloTag fusion proteins and their seroreactivity analyzed using plasma from CRC patients at stages I-IV (n = 31), individuals with premalignant lesions (n = 31), and healthy individuals (n = 48). p53γ, Δ40p53β, Δ40p53γ, Δ133p53γ, Δ160p53γ, TAp63α, TAp63δ, ΔNp63α, and ΔNp63δ, together with the specific C-terminal end α and δ p63 peptides, were found to be more seroreactive against plasma from CRC patients and/or individuals with premalignant lesions than from healthy individuals. In addition, ROC (receiver operating characteristic) curves revealed a high diagnostic ability of those p53 and p63 proteoforms to detect CRC and premalignant individuals (AUC higher than 85%). Finally, electrochemical biosensing platforms were employed in POC-like devices to investigate their usefulness for CRC detection using selected p53 and p63 proteoforms. Our results demonstrate not only the potential of these biosensors for the simultaneous analysis of proteoforms' seroreactivity, but also their convenience and versatility for the clinical detection of CRC by liquid biopsy. In conclusion, we here show that p53 and p63 proteoforms possess differential seroreactivity in CRC patients in comparison to controls, distinctive from canonical proteins, which should improve the diagnostic panels for obtaining a blood-based biomarker signature for CRC detection.

Keywords: POC-like device; alternative splicing; autoantibodies; biosensor; colorectal cancer; diagnosis; humoral immune response; immunomics; p53 and p63; p53 family; proteoform.

Figure 1

Schematic representation of the domains of described human p53 and p63 proteoforms derived from the alternative splicing of TP53 and TP63 genes. Alternative promoters (P1 and P2) are indicated. Alternative splicing events at the C-terminal generate variants α, β, and γ. Exon skipping or premature transcription termination produces variant δ. Truncated transactivating domains of p53 produce Δ40, Δ133, and Δ163 p53 proteoforms, whereas truncation of transactivation domains in p63 produce ΔN proteoforms. Transactivation domains (TAD), DNA-binding domain (DBD), proline-rich domain (PRD), sterile alpha motif domain (SAM), C-terminal inhibitory domain (TID), oligomerization domain (OD), and hinge domain (HD) are depicted.

Figure 2

Prediction of the 3D structures of p53 and p63 proteoforms. (A) Prediction of the 3D structures of the p53 and p63 proteoforms. (B) Prediction of the electrostatic potential of the p53 and p63 proteoforms. Electropositive and electronegative charged regions are colored in blue and red, respectively. Neutral regions are colored in white.

Figure 3

Cloning, and in vitro protein expression of the p53 and p63 proteoforms used as HaloTag fusion proteins in subsequent experiments. (A) Confirmation of the in vitro protein expression of full-length p53 and p63 proteoforms derived from alternative splicing as HaloTag fusion proteins was assessed by WB using an anti-HaloTag mAb. (B) Confirmation of the in vitro protein expression of the indicated specific p53 and p63 peptides derived from alternative splicing as HaloTag fusion proteins was assessed by WB using an anti-HaloTag mAb. (C) Immunostaining analysis using an anti-p53 mAb against the N-terminal end of unmodified p53 proteoforms confirmed their correct expression in vitro. (D) Immunostaining analysis using an anti-p63 mAb against a N-terminal peptide present in all p63 proteoforms confirmed their correct expression in vitro. The original western blot figures could be found in Figure S2.

Figure 4

Evaluation of the seroreactivity levels of the p53 and p63 proteoforms and indicated specific cryptic peptides derived from the alternative splicing of TP53 and TP63 genes. (A) Significant CRC autoantibody levels against the indicated p53 proteoforms comparing healthy individuals, premalignant colorectal individuals, and CRC patients. (B) Significant CRC autoantibody levels against the indicated p63 proteoforms comparing healthy individuals, premalignant colorectal individuals, and CRC patients. (C) Autoantibody levels against the specific cryptic peptides of p63α and p63δ.

Figure 5

Diagnostic potential of the p53 and p63 proteoforms’ autoantibodies. The diagnostic value of autoantibodies against seroreactive p53 (A) and p63 (B) proteoforms was evaluated by means of ROC curves to discriminate between CRC patients, colorectal premalignant individuals, pathological individuals (CRC patients and premalignant lesion patients), and healthy individuals. AUC, sensitivity, and specificity for indicated comparisons are depicted.

Figure 6

Evaluation of the diagnostic value of CRC and colorectal premalignant autoantibodies. The combination of the significant p53 and p63 proteoforms autoantibodies to discriminate (A) CRC and premalignant individuals from healthy individuals, (B) CRC from healthy individuals, and (C) premalignant individuals from healthy individuals showed AUCs higher than 85%, and sensitivity and specificity up to 96.8% and 87.5%, respectively.

Figure 7

Autoantibody measurement in plasma samples by the proteoform-multiplexed electrochemical biosensing platform. (A) Amperometric responses obtained for the indicated proteoforms were larger for the CRC patients in comparison with the healthy individuals. (B) ROC curve of autoantibody detection against all proteoforms using the electrochemical biosensing platform. CRC, colorectal cancer patients.