SMAD4 Loss in Colorectal Cancer Patients Correlates with Recurrence, Loss of Immune Infiltrate, and Chemoresistance

Abstract

Purpose: SMAD4 has shown promise in identifying patients with colorectal cancer at high risk of recurrence or death.Experimental Design: A discovery cohort and independent validation cohort were classified by SMAD4 status. SMAD4 status and immune infiltrate measurements were tested for association with recurrence-free survival (RFS). Patient-derived xenografts from SMAD4-deficient and SMAD4-retained tumors were used to examine chemoresistance.

Results: The discovery cohort consisted of 364 patients with stage I-IV colorectal cancer. Median age at diagnosis was 53 years. The cohort consisted of 61% left-sided tumors and 62% stage II/III patients. Median follow-up was 5.4 years (interquartile range, 2.3-8.2). SMAD4 loss, noted in 13% of tumors, was associated with higher tumor and nodal stage, adjuvant therapy use, fewer tumor-infiltrating lymphocytes (TIL), and lower peritumoral lymphocyte aggregate (PLA) scores (all P < 0.04). SMAD4 loss was associated with worse RFS (P = 0.02). When stratified by SMAD4 and immune infiltrate status, patients with SMAD4 loss and low TIL or PLA had worse RFS (P = 0.002 and P = 0.006, respectively). Among patients receiving 5-fluorouracil (5-FU)-based systemic chemotherapy, those with SMAD4 loss had a median RFS of 3.8 years compared with 13 years for patients with SMAD4 retained. In xenografted mice, the SMAD4-lost tumors displayed resistance to 5-FU. An independent cohort replicated our findings, in particular, the association of SMAD4 loss with decreased immune infiltrate, as well as worse disease-specific survival.

Conclusions: Our data show SMAD4 loss correlates with worse clinical outcome, resistance to chemotherapy, and decreased immune infiltrate, supporting its use as a prognostic marker in patients with colorectal cancer.

Figure 1:

Scoring of IHC results for SMAD4 and tumor infiltrating lymphocytes (TIL) in the discovery cohort. a) Representative IHC staining for each SMAD4 score. SMAD4 loss was defined as a score of 0 (left image), and SMAD4 retention was defined as a score of 1-3 (right three images). The magnification bars are 200 microns in the main images and 100 microns in the inset images. b) Representative H&E images for low and high TIL scores. Each H&E-stained image is shown next to the SMAD4 IHC staining from the same tumor core. Scale bars are 200 microns.

Figure 2:

Kaplan-Meier graphs with number of subjects at risk comparing recurrence-free survival (RFS) by SMAD4 IHC status. (a) RFS among all patients. (b) RFS among patients older than the median age (53 years). (c) RFS among patients who received systemic treatment. *The median age of the cohort is 53; RFS did not significantly differ by SMAD4 status among patients younger than 53.

Figure 3:

Kaplan-Meier graphs with number of subjects at risk comparing recurrence-free survival (RFS) by SMAD4 IHC status and (a) TIL status or (b) PLA status.

Figure 4:

SMAD4 and the 5-fluorouracil (5-FU) sensitivity of colorectal cancer xenografts, tumoroids, and cell lines. (a) Western blot analysis validating SMAD4 status of patient-derived tumor cells. The blots analyzed tumoroids grown from cells from a SMAD4-retained tumor (patient 1; SMAD4 wild-type) and a SMAD4-lost tumor (patient 2; SMAD4-mutant). (b) Weight of tumors grown in mice from cells from the patient 1 tumoroid (retained SMAD4) after treatment with 5-FU or vehicle. (c) Weight of tumors grown in mice from cells from the patient 2 tumoroid (SMAD4 loss) after treatment with 5-FU or vehicle. The central horizontal lines represent the mean, and the whiskers represent the SD. NS, not significant. (d) Western blot analysis of two additional patient-derived tumoroids (patients 3 and 4) transfected with a control sgRNA lentiviral vector or sgRNA lentiviral against SMAD4, showing that SMAD4 was knocked down by the SMAD4-targeting sgRNA. (e) Sensitivity of tumoroids with and without SMAD4-knockdown to 5-FU (left) and FOLFOX (right). Data shown are the averages of biological duplicate samples; error bars display SE. The differences in the control group and SMAD4-knockdown group were assessed using ANOVA. (f) Western blot confirming the expected lack of SMAD4 in SW480 colorectal cancer cells (which are SMAD4-mutant) and restoration of SMAD4 in SW480^SMAD4 cells (which were transduced with SMAD4). (g) Cell viability of SW480 and SW480^SMAD4 cells after treatment with increasing concentration (1x, 2x, and 4x) of 5-FU (left) or FOLFOX (right). The data represent mean ± SD; *P<0.05; **P< 0.01 vs. control).