Wnt and the cancer niche: paracrine interactions with gastrointestinal cancer cells undergoing asymmetric cell division

Abstract

Objective: Stem-like cancer cells contribute to cancer initiation and maintenance. Stem cells can self-renew by asymmetric cell division (ACD). ACD with non-random chromosomal cosegregation (ACD-NRCC) is one possible self-renewal mechanism. There is a paucity of evidence supporting ACD-NRCC in human cancer. Our aim was to investigate ACD-NRCC and its potential interactions with the cancer niche (microenvironment) in gastrointestinal cancers.

Design: We used DNA double and single labeling approaches with FACS to isolate live cells undergoing ACD-NRCC.

Results: Gastrointestinal cancers contain rare subpopulations of cells capable of ACD-NRCC. ACD-NRCC was detected preferentially in subpopulations of cells previously suggested to be stem-like/tumor-initiating cancer cells. ACD-NRCC was independent of cell-to-cell contact, and was regulated by the cancer niche in a heat-sensitive paracrine fashion. Wnt pathway genes and proteins are differentially expressed in cells undergoing ACD-NRCC vs. symmetric cell division. Blocking the Wnt pathway with IWP2 (WNT antagonist) or siRNA-TCF4 resulted in suppression of ACD-NRCC. However, using a Wnt-agonist did not increase the relative proportion of cells undergoing ACD-NRCC.

Conclusion: Gastrointestinal cancers contain subpopulations of cells capable of ACD-NRCC. Here we show for the first time that ACD-NRCC can be regulated by the Wnt pathway, and by the cancer niche in a paracrine fashion. However, whether ACD-NRCC is exclusively associated with stem-like cancer cells remains to be determined. Further study of these findings might generate novel insights into stem cell and cancer biology. Targeting the mechanism of ACD-NRCC might engender novel approaches for cancer therapy.

Keywords: Asymmetric Cell Division; Cancer Stem Cells; Microenvironment.; Non-Random Chromosomal Cosegregation.

Fig 1

Asymmetric cell division via non-random chromosomal cosegregation (ACD-NRCC). (A) ACD-NRCC is proposed as one potential mechanism by which stem cells self-renew. It is hypothesized that stem cells contain template DNA strands that are conserved during asymmetric cell divisions (orange). By segregating the “template DNA strands” into daughter cells destined to become stem cells, stem cells could avoid propagation of DNA replication errors. This is a potential mechanism by which mutations are preferentially segregated into daughter cells destined to differentiate and are eventually eliminated. (B) Double labeling procedure for the detection of ACD-NRCC (Additional file 1: figure S1 and Materials and Methods; IdU (Green): Iodo-deoxyuridine; CldU (Red): Chloro-deoxyuridine).

Fig 2

ACD-NRCC and SCD is shown in human liver cancer cells. (A) Fluorescent microscopy images showing ACD-NRCC (white arrow) and SCD with random chromosomal segregation (yellow arrow) in human liver cancers. The top four rows shows ACD-NRCC: Green fluorescent IdU is seen only in one nucleus after two cell cycles, while red fluorescent CldU is seen in both nuclei within the same cell arrested in cytokinesis. The bottom two rows show SCD, where both nuclei within one cell arrested in cytokinesis incorporated both nucleotides (Additional file 1: figure S2). (B) Three dimensional confocal microscopy images showing human liver cancer cells and lung cancer (iv) undergoing ACD-NRCC. In (i), (ii) and (iii), we show two nuclei in the same cytoplasmic space without intervening cytoplasmic membrane during ACD-NRCC (white arrow; Additional file 1: figure S2 and Additional file 2: Movie S1). In (v) we show ACD-NRCC after reverse labeling. In (vi) we show SCD for comparison.

Fig 3

ACD is detected preferentially in the Side Population of liver cancer cells. (A-B) FLOW CYTOMETRY sorting images of side population (SP) and non-side population cells (NSP). The side population is based on the ability of the ABCG2 transporter to efflux Hoechst 33342 (Ho). (B) In order to identify the population of cells that efflux Ho specifically by the ABCG2 transporter, we used Verapamil to block the activity of the ABCG2 transporter. (A) Here we show that the SP comprises 0.28% of the total cell population of Huh-7 liver cancer cells. (C) Side-population (SP) and non-SP hepatocellular carcinoma cells were plated initially at low concentrations, then allowed to proliferate, followed for 5 weeks, and tested for the presence of ACD-NRCC. At one week, SP cells did not exhibit ACD-NRCC, but when left to proliferate and differentiate while generating non-SP cells, demonstrated increasing levels of ACD-NRCC (p=0.0034); the non-SP cells neither generated SP cells nor demonstrated ACD-NRCC (p=0.024). While ACD-NRCC was never detected in NSP or CD133-negative cells (Vide Infra, figure 4), the maximal detection rate in total cells, in SP cells or in CD133+ cells seem to be constant suggesting that per a given condition the rate of ACD-NRCC is constant (steady state rate).

Fig 4

ACD is detected preferentially in CD133+ cells, and is modulated by the cancer microenvironment in a paracrine nature. (A) Here we show that ACD-NRCC is detected in CD133+ cells of Huh-7 liver cancer cells; no ACD-NRCC was detected in CD133-negative cells under any-condition. Additionally, we show that in order for CD133+ cells to undergo ACD-NRCC they must be cultured together with CD133-negative cells. The effect of CD133-negative cells on CD133+ cells is paracrine in nature. Thus, the effect of CD133-negative cells on CD133+ cells undergoing ACD-NRCC is not dependent on cell-to-cell contact. CD133-negative or CD133+ cells growing separately alone do not undergo ACD-NRCC. All experiments were repeated three times in a prospective blinded fashion. (B) Here we show that the permissive effect of CD133-negative cells on CD133+ cells undergoing ACD-NRCC is heat sensitive, and can be abolished by heat denaturation. While ACD-NRCC was never detected in CD133-negative cells or NSP cells (figure 3), the maximal detection rate in total cells, in CD133+ cells or in SP cells seem to be constant suggesting that per a given condition the rate of ACD-NRCC is constant (steady state rate).

Fig 5

Wnt pathway genes are expressed asymmetrically in cells undergoing asymmetric cell division (ACD-NRCC). We studied representative Wnt associated proteins representing each of the cellular compartments (membrane, cytoplasm and nucleus). TCF4 was the only Wnt associated protein distributed asymmetrically during liver cancer cell divisions (3.1% ± 1.5%). (A) Using confocal microscopy with tridimensional rendering, we show asymmetric distribution of TCF4, and (Green=TCF4, Blue=DAPI and Red=CFSE), and (B) symmetric distribution of TCF4. (C) We isolated live cells undergoing ACD-NRCC or SCD; using qRT-PCR, we show that TCF4 is upregulated in cells undergoing ACD-NRCC when compared to cells undergoing SCD. (D) We performed Wnt SuperArray analysis on cells undergoing ACD-NRCC or SCD. Among 84 Wnt associated genes tested only 5 were found to be differentially expressed, TCF4, TCF7, CSBK2A1 and Sox17 were upregulated, and RB1 was down-regulated by cells undergoing ACD-NRCC.

Fig 6

Inhibition of Wnt results in significant suppression of ACD-NRCC. We detected ACD-NRCC or SCD before and after treatment with the Wnt-antagonist IWP2 or siRNA-TCF4. (A) IWP-2 didn't affect cell proliferation (0.29 ± 0.01 vs. 0.33 ± 0.0, p=0.45 for PLC/PRF/5; 0.27 ± 0.01% vs. 0.25 ± 0.03, p=0.59 for HuH-7). (B) To understand Wnt effects on ACD-NRCC in a quantitative manner, we tested for ACD-NRCC in live cells before and after treatment with the Wnt-antagonist IWP2. The suppression of ACD-NRCC after treatment with the Wnt-antagonist IWP2 was statistically significant. (C-D) To further validate these results and because TCF4 was differentially expressed in cells undergoing ACD-NRCC, we tested for ACD-NRCC by confocal microscopy in fixed cells before and after treatment with the Wnt-antagonist IWP2 or TCF4-siRNA. We detected ACD-NRCC in cells before treatment with IWP2 or TCF4-siRNA. After treatment with the Wnt-antagonist IWP2 or knock down of TCF4, we couldn't detect cells undergoing ACD-NRCC; only cells undergoing SCD were observed. (E) Treatment with the Wnt-antagonist IWP2 reversed or reduced the differential expression of TCF4, TCF7, SOX17 and CSNK2A1 in cells undergoing ACD-NRCC vs. SCD (figure 5 and S5). (F) Immunofluorescence staining for TCF4 before and after treatment with IWP2 or TCF4-siRNA showed that the Wnt antagonist IWP-2 or TCF4-siRNA reduced TCF4 levels by 64% (p=0.011) and 61% (p=0.0093), respectively (Additional file 1: figure S8).