The class I HDAC inhibitor MGCD0103 induces cell cycle arrest and apoptosis in colon cancer initiating cells by upregulating Dickkopf-1 and non-canonical Wnt signaling

Abstract

Colorectal cancer metastatic recurrence and chemoresistance are major causes of morbidity and mortality. Colon cancer initiating cells (CCIC) are thought to contribute to both these processes. To identify drugs with anti-CCIC activity we screened a number of FDA approved and investigational compounds. We found that the class I selective histone deacetylase inhibitor (HDACi) MGCD0103 has significant activity against CCIC, and also significantly inhibits non-CCIC CRC cell xenograft formation. Both MGCD0103 and the pan-HDAC inhibitor Trichostatin impairs CCIC clonogenicity and cause cell cycle arrest and cell death. Gene expression profiling revealed that the canonical WNT ligand DKK-1 is a highly upregulated target of HDAC inhibitors. Despite the presence of APC mutations and constitutive WNT signaling in CCIC, both transfected and recombinant DKK-1 dramatically inhibit CCIC proliferation and clonogenicity. Overall, these data show that inhibition of class I HDACs is a promising novel approach to target both CCIC and non-CCIC CRC cells. Our studies also provide novel insights into roles for DKK1 in addition to canonical WNT signaling and the mechanism of CCIC tumor formation.

Figure 1. MCCD0103 inhibits xenograft growth of multiple commonly used CRC cell lines

NOD/SCID mice were injected in the lateral flank with one million cells of the indicated cell line and mice were treated with 40mg/kg MGCD0103 i.p. daily injections for three weeks. The decrease in xenograft tumor volume is indicated. Inhibition is the mean of two independent experiments for each cell line.

Figure 2. MGCD0103 impairs viability of CCIC

Cells were cultured in stem cell media in suspension and treated with MGCD0103 and/or the combination of 5-FU/Oxaliplatin (FOLFOX). MTT assays were performed to assess cell viability after drug treatment. Error bars are S.E.M.

Figure 3. MGCD0103 inhibits CCIC D culture tumor formation

A. Representative pictures of xenograft tumors (left) and 3D culture glandular crypt-like tumors (right) from CCIC cell lines. B. Light microscopy view of 10cm plate of CCIC after MGCD0103 treatment in 3D matrigel culture at low magnification (left), 10X (middle) and 40X (right).

Figure 4. HDAC inhibitors induce CCIC apoptosis

FACS cell cycle analysis plot of CCIC treated with vehicle only (mock), 200nM TSA or 800nM TSA for 24 hours.

Figure 5. Gene expression profiling reveals CCIC targets of MGCD0103 and TSA upregulation

A. Venn diagram of differentially expressed genes (DEG) common for 2 CCIC cell lines treated with TSA or MGCD0103. B. Treatment with HDAC inhibitors upregulates expression of DKK-1 and DKK-3. Fold upregulation and p-value from quadruplicate data points for each of 2 CCIC lines from Affymetrix array data is shown. C. Q-PCR confirmation of DKK-1 upregulation by increasing concentrations of TSA.

Figure 6. DKK1 inhibits CCIC tumor formation through non-canonical WNT signaling pathways

Light microscopy of CCIC 3D culture clonogenicity in cells transfected with DKK-1 expression vector (A) or addition of recombinant DKK-1 (C). B. Reduction in CCIC colony number with DKK-1 transfection. Error bars are S.E.M. D. Western blot showing loss of wild-type APC in CCIC-1 and CCIC-2 lines. Hela WT APC is shown as a positive control. E. Immunohistochemistry of CCIC xenograft tumors stained for beta-catenin.