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The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

ML264 treatment decreases EGR1 expression levels in DLD-1-derived xenografts.

Supplementary Data from Sox7 Is an Independent Checkpoint for β-Catenin Function in Prostate and Colon Epithelial Cells

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The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was done in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC50 < 10 μmol/L for KLF5 inhibition and EC50 > 10 μmol/L for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized on the basis of computational, Western blot, and cell viability analyses. Both of these compounds, and two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results show the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5. Mol Cancer Ther; 10(11); 2043–51. ©2011 AACR.

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Statistical analysis of apoptosis assay.

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Supplementary Figure Legends

Klf4ΔIS/ApcMin/+ mice have stronger beta-catenin, Ki67 and cyclin D1 staining in the small intestine than Klf4fl/fl/ApcMin/+ mice.

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Beta-catenin, Ki67 and cyclin D1 staining in colonic adenomas of Klf4fl/fl/ApcMin/+ and Klf4ΔIS/ApcMin/+ mice.

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The list of antibodies used during this study.

ML264 treatment decreases KLF5 expression levels in DLD-1-derived xenografts.

Klf4ΔIS/ApcMin/+ mice develop significantly more small intestinal adenomas than Klf4fl/fl/ApcMin/+ mice.

Krüppel-like factor 5 (KLF5), a member of the SP/KLF family of zinc finger transcription factors, is overexpressed in human colorectal cancer specimens, and this overabundance is associated with aggressive cancer development and progression. We demonstrated that mice haploinsufficient for Klf5 had reduced intestinal tumor burden in the background of germline mutation in Apc, a gatekeeper of intestinal tumorigenesis. Based on a high-throughput screening strategy, we developed ML264, a small-molecule compound that inhibits KLF5, and showed that it inhibits growth of colorectal cancer in vitro and in vivo. Through optimization efforts based on the structure of ML264, we have now identified a new lead compound, SR18662. We find that treatment with SR18662 significantly reduces growth and proliferation of colorectal cancer cells as compared with treatment with vehicle control, ML264, or SR15006 (a less optimized analogue from SAR efforts leading to SR18662). SR18662 showed improved efficacy in reducing the viability of multiple colorectal cancer cell lines. Flow cytometry analysis following SR18662 treatment showed an increase in cells captured in either S or G2–M phases of the cell cycle and a significant increase in the number of apoptotic cells, the latter a unique property compared with ML264 or SR15006. SR18662 treatment also reduces the expression of cyclins and components of the MAPK and WNT signaling pathways. Importantly, we observed a significant dose-dependent inhibition of xenograft growth in mice following SR18662 treatment that exceeded the effect of ML264 at equivalent doses. These findings support further development of SR18662 and its analogues for colorectal cancer therapy.

The presence of somatic β-catenin mutations in some prostate cancers implies that aberrant WNT signaling is involved in the cancer development. Although β-catenin stability is regulated by a multicomponent destruction complex, mutational alterations of β-catenin or other components of the destruction complexes are rare in prostate tumors. Therefore, β-catenin may be regulated by another protein in the prostate. In fact, recent linkage and somatic deletion analyses in prostate cancers reveal a 1.4-Mb candidate tumor suppressor locus on 8p23.1, which includes the Sox7 gene. Here we show that Sox7 protein expression was indeed down-regulated in 47% (15 of 32) of prostate adenocarcinomas. In addition, Sox7 mRNA was down-regulated in 60% of snap-frozen tumors. This down-regulation was found to be due to tumor-specific promoter hypermethylation, which was present in 48% (10 of 21) of primary prostate tumors and 44% (11 of 25) of prostate cancer cell lines/xenografts. We discovered that Sox7 protein physically interacts with β-catenin and suppresses β-catenin–mediated transcription by depleting active β-catenin. Furthermore, in HCT116 colorectal cancer cell lines with Sox7 inactivation, ectopic Sox7 expression suppressed cell proliferation and inhibited transcription that was activated by an endogenous mutant β-catenin. Although nearly all colorectal cancers contain mutations in β-catenin or adenomatous polyposis coli/axin, epigenetic silencing of Sox7 was still observed. These data suggest that Sox7 is a tumor suppressor that functions as an independent checkpoint for β-catenin transcriptional activity. Inactivation of Sox7 could promote the development of a majority of colorectal tumors and approximately half of prostate tumors. (Mol Cancer Res 2008;6(9):1421–10)

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Supplementary Figures Legends

Supplementary Figure S1. Synthesis steps of SR15006 and SR18662. Supplementary Figure S2. SR18662 changes cell cycle profiles of colorectal cancer cell lines. Supplementary Figure S3. SR18662 increases apoptosis of colorectal cancer cell lines. Supplementary Figures S4 - S6. SR18662 inhibits activity of MAPK, WNT/b-catenin signaling pathways and decreases the levels of cyclins. Supplementary Figure S7. The effect of SR18662 on the mice weight during xenograft studies.

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Klf4ΔIS mice develop micro adenomas at an earlier time point (8 weeks) following AOM treatment.

The zinc finger-containing transcription factor, Krüppel-like factor 4 (KLF4), inhibits cell proliferation. An in vivo tumor-suppressive role for KLF4 is shown by the recent finding that Klf4 haploinsufficiency in ApcMin/+ mice promotes intestinal tumorigenesis. Studies also show that KLF4 is required for the terminal differentiation of goblet cells in the mouse intestine. The Notch signaling pathway suppresses goblet cell formation and is up-regulated in intestinal tumors. Here, we investigated the relationship between Notch signaling and KLF4 expression in intestinal epithelial cells. The rate of proliferation of HT29 human colon cancer cells was reduced when treated with the γ-secretase inhibitor dibenzazepine to inhibit Notch signaling or small interfering RNA directed against Notch. KLF4 levels were increased in dibenzazepine-treated or Notch small interfering RNA-treated cells. Conversely, overexpression of Notch in HT29 cells reduced KLF4 levels, suppressed KLF4 promoter activity, and increased proliferation rate. Treatment of ApcMin/+ mice with dibenzazepine resulted in a 50% reduction in the number of intestinal adenomas compared with the vehicle-treated group (P < 0.001). Both the normal-appearing intestinal mucosa and adenomas obtained from dibenzazepine-treated ApcMin/+ mice had increased goblet cell numbers and Klf4 staining accompanied by reduced cyclin D1 and Ki-67 staining when compared with those from vehicle-treated mice. Results of these studies indicate that Notch signaling suppresses KLF4 expression in intestinal tumors and colorectal cancer cells. Inhibition of Notch signaling increases KLF4 expression and goblet cell differentiation and reduces proliferation and tumor formation. KLF4 is therefore a potential mediator for the antitumor effect of Notch inhibitors such as dibenzazepine. (Mol Cancer Res 2008;6(12):1920–7)

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Supplementary Figures S1-S8 from Notch Inhibits Expression of the Krüppel-Like Factor 4 Tumor Suppressor in the Intestinal Epithelium

Colorectal cancer is one of the leading causes of cancer mortality and morbidity worldwide. Previous studies indicate that the zinc finger-containing transcription factor Krüppel-like factor 5 (KLF5) positively regulates proliferation of intestinal epithelial cells and colorectal cancer cells. Importantly, inhibition of KLF5 expression in intestinal epithelial cells and colorectal cancer cells by pharmacologic or genetic means reduces their rate of proliferation. To identify additional and novel small molecules that inhibit KLF5 expression and thus colorectal cancer proliferation, we developed a reporter assay using colorectal cancer cell line (DLD-1) that stably expressed a luciferase reporter gene directed by 1,959 bp of the human KLF5 promoter upstream of the ATG start codon and performed a cell-based high-throughput screen with the Library of Pharmacologically Active Compounds that contains 1,280 biologically active compounds. The screen identified 8 potential inhibitors and 6 potential activators of the KLF5 promoter. Three potential inhibitors, wortmannin, AG17, and AG879, were further evaluated by secondary analyses. All three significantly reduced both KLF5 promoter-luciferase activity and protein level in DLD-1 cells in a dose- and time-dependent manner when compared with controls. They also significantly reduced the rate of proliferation of DLD-1 and two other colorectal cancer cell lines, HCT116 and HT29. Our results show the principle of using high-throughput screening to identify small-molecule compounds that modulate KLF5 activity and consequently inhibit colorectal cancer proliferation. [Mol Cancer Ther 2009;8(3):563–70]

Supplementary Fig. from Identification of novel small-molecule compounds that inhibit the proproliferative Krüppel-like factor 5 in colorectal cancer cells by high-throughput screening

Purpose: Near-IR fluorescence imaging has great potential for noninvasive in vivo imaging of tumors. In this study, we show the preferential uptake and retention of two hepatamethine cyanine dyes, IR-783 and MHI-148, in tumor cells and tissues.Experimental Design: IR-783 and MHI-148 were investigated for their ability to accumulate in human cancer cells, tumor xenografts, and spontaneous mouse tumors in transgenic animals. Time- and concentration-dependent dye uptake and retention in normal and cancer cells and tissues were compared, and subcellular localization of the dyes and mechanisms of the dye uptake and retention in tumor cells were evaluated using organelle-specific tracking dyes and bromosulfophthalein, a competitive inhibitor of organic anion transporting peptides. These dyes were used to detect human cancer metastases in a mouse model and differentiate cancer cells from normal cells in blood.Results: These near-IR hepatamethine cyanine dyes were retained in cancer cells but not normal cells, in tumor xenografts, and in spontaneous tumors in transgenic mice. They can be used to detect cancer metastasis and cancer cells in blood with a high degree of sensitivity. The dyes were found to concentrate in the mitochondria and lysosomes of cancer cells, probably through organic anion transporting peptides, because the dye uptake and retention in cancer cells can be blocked completely by bromosulfophthalein. These dyes, when injected to mice, did not cause systemic toxicity.Conclusions: These two heptamethine cyanine dyes are promising imaging agents for human cancers and can be further exploited to improve cancer detection, prognosis, and treatment. Clin Cancer Res; 16(10); 2833–44. ©2010 AACR.

Supplementary Data from Near IR Heptamethine Cyanine Dye–Mediated Cancer Imaging

Supplementary Figure Legends 1-3 from Haploinsufficiency of Krüppel-Like Factor 4 Promotes Adenomatous Polyposis Coli–Dependent Intestinal Tumorigenesis

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Supplementary Figure 3 from Haploinsufficiency of Krüppel-Like Factor 4 Promotes Adenomatous Polyposis Coli–Dependent Intestinal Tumorigenesis

Supplementary Methods and Materials from Haploinsufficiency of Krüppel-Like Factor 5 Rescues the Tumor-Initiating Effect of the ApcMin Mutation in the Intestine

Cells engage sophisticated programs of DNA damage response (DDR) and repair to guard against genetic mutations. Although there is significant knowledge concerning DDR in interphase cells, much less is known about these processes in mitosis. Direct interaction between MDC1, a master DDR organizer, and a marker of DNA damage, histone γH2AX, is required to trigger robust repair. Here we show that the DNA damage-induced interaction between MDC1 and γH2AX is attenuated in mitosis. Furthermore, inhibition in the activity of the core mitotic regulator CDK1, either by pharmacologic inhibition or siRNA attenuation, enhances MDC1–γH2AX colocalization in mitosis. Our findings offer key new insights into how DDR is controlled during mitosis. Cancer Res; 72(21); 5448–53. ©2012 AACR.

Supplementary Figure 3 from Haploinsufficiency of Krüppel-Like Factor 5 Rescues the Tumor-Initiating Effect of the ApcMin Mutation in the Intestine

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The mitotic checkpoint is a mechanism that arrests the progression to anaphase until all chromosomes have achieved proper attachment to mitotic spindles. In cancer cells, satisfaction of this checkpoint is frequently delayed or prevented by various defects, some of which have been causally implicated in tumorigenesis. At the same time, deliberate induction of mitotic arrest has proved clinically useful, as antimitotic drugs that interfere with proper chromosome-spindle interactions are effective anticancer agents. However, how mitotic arrest contributes to tumorigenesis or antimitotic drug toxicity is not well defined. Here, we report that mitotic chromosomes can acquire DNA breaks during both pharmacologic and genetic induction of mitotic arrest in human cancer cells. These breaks activate a DNA damage response, occur independently of cell death, and subsequently manifest as karyotype alterations. Such breaks can also occur spontaneously, particularly in cancer cells containing mitotic spindle abnormalities. Moreover, we observed evidence of some breakage in primary human cells. Our findings thus describe a novel source of DNA damage in human cells. They also suggest that mitotic arrest may promote tumorigenesis and antimitotic toxicity by provoking DNA damage. [Cancer Res 2007;67(24):11487–92]

Supplementary Figure 1 from Haploinsufficiency of Krüppel-Like Factor 4 Promotes Adenomatous Polyposis Coli–Dependent Intestinal Tumorigenesis

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