Heterochromatin Protein 1 Binding Protein 3 Expression as a Candidate Marker of Intrinsic 5-Fluorouracil Resistance

Abstract

Background: Despite receiving post-operative 5-fluorouracil (5-FU)-based chemotherapy, approximately 50% of patients with stage IIIC colon cancer experience recurrence. Currently, no molecular signature can predict response to 5-FU.

Materials and methods: Mouse models of colon cancer have been developed and characterized. Individual tumors in these mice can be longitudinally monitored and assessed to identify differences between those that are responsive and those that are resistant to therapy. Gene expression was analyzed in serial biopsies that were collected before and after treatment with 5-FU. Colon tumors had heterogeneous responses to treatment with 5-FU. Microarray analysis of pre-treatment biopsies revealed that Hp1bp3, a gene encoding heterochromatin protein 1 binding protein 3, was differentially expressed between sensitive and resistant tumors.

Conclusion: Using mouse models of human colorectal cancer, Hp1bp3 was identified as a candidate marker of intrinsic 5-FU resistance and may represent a potential biomarker for patient stratification or a target of clinical importance.

Keywords: 5-fluorouracil; Colorectal cancer; chemotherapy; heterochromatin protein 1 binding protein 3; intrinsic resistance; mouse models.

Figure 1

Categorization of F1.Min tumor response to 5-FU. F1.Min tumors exhibit both resistance and sensitivity to 5-FU as evidenced by tumor size changes. Panel A shows the parameters used to categorize tumor size changes into response. Panel B shows tumors that are sensitive, unclassifiable, and resistant, before and following treatment. Panel C shows median and quartiles for the sensitive and resistant tumors.

Figure 2

Overexpression of Hp1bp3 in tumors is a marker of tumor resistance. The level of Hp1bp3 in sensitive and resistant tumors before and after treatment was determined by quantitative PCR. Tumors that were classified as sensitive had lower levels of Hp1bp3 expression (0.86±0.18-fold change over normal colon epithelium; n=9) than tumors that were classified as resistant (1.28±0.18-fold change over normal colon epithelium; n=9). This difference was statistically significant (p<0.001, two-sided Wilcoxon rank sum test). All sensitive tumors had tissue that persisted throughout 5-FU treatment as none fully responded. These tissue samples, along with post-treatment samples of resistant tumors, were analyzed for Hp1bp3 expression. Expression levels were not significantly different when comparing tissue that persisted after treatment in sensitive tumors and tissue from resistant tumors (p=0.44).

Figure 3

Hp1bp3 expression varies between tumors. Hp1bp3 expression was analyzed in 26 tumors; some were later treated with 5-FU and determined to be sensitive or resistant, while others were not treated. Tumors ranged from 0.56–2.30-fold change with a median of 1.16 compared to normal intestinal epithelium. The median is denoted by the horizontal dotted line. All tumors that were sensitive to 5-FU are below the median; 8/9 resistant tumors were above the median (Fisher’s exact test, p<0.001).

Figure 4

c-Myc expression is low in F1.Min tumors and does not predict resistance. F1.Min tumors had low c-Myc expression both before and after treatment compared to normal colon epithelium. There was no significant difference (Wilcoxon rank sum test, p=0.47) in c-Myc levels between sensitive (0.80±0.23-fold change) and resistant tumors (0.92±0.32 fold change) prior to treatment. Following treatment, c-Myc levels also were not distinguishable between sensitive and resistant tumors (Wilcoxon rank sum test, p=0.42).