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Supplementary Table S1 from Aldehyde Dehydrogenase Discriminates the CD133 Liver Cancer Stem Cell Populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133+ HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133+ and CD133− subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133+ subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH+ to be CD133+, yet not all CD133+ HCC cells were ALDH+. Subsequent studies on purified subpopulations found CD133+ALDH+ cells to be significantly more tumorigenic than their CD133−ALDH+ or CD133−ALDH− counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133+ALDH+ > CD133+ALDH− > CD133−ALDH−. ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population. (Mol Cancer Res 2008;6(7):1146–53)

Representative immunohistochemical staining of high, medium and low FSTL1 in non-neoplastic squamous epithelium (non-tumor) and poorly differentiated ESCC tissue from 6 different patients.

FSTL1(+) cells in ESCC clinical samples that received (with treatment, n = 22) or did not receive (without treatment, n = 15) chemotherapy treatment prior to resection, as detected by IHC analyses.

Quantification of number of cells that migrated through a membrane, invaded through a Matrigel-coated membrane, formed spheroids or formed colonies in (A) KYSE150-Luc cells co-cultured with conditioned medium collected from EV or FSTL1 OE cells; or (B) KYSE150-Luc cells treated with control or recombinant FSTL1, in the presence or absence of IMD-0354.

Quantification of number of cells that migrated through a membrane, invaded through a Matrigel-coated membrane, formed spheroids or formed colonies in (A) KYSE150-Luc cells co-cultured with conditioned medium collected from EV or FSTL1 OE cells; or (B) KYSE150-Luc cells treated with control or recombinant FSTL1, in the presence or absence of C34.

Purpose: To investigate the autocrine/endocrine role of Id1-induced insulin-like growth factor-II (IGF-II) in esophageal cancer, and evaluate the potential of IGF-II- and IGF-type I receptor (IGF-IR)-targeted therapies.Experimental Design: Antibody array-based screening was used to identify differentially secreted growth factors from Id1-overexpressing esophageal cancer cells. In vitro and in vivo assays were performed to confirm the induction of IGF-II by Id1, and to study the autocrine and endocrine effects of IGF-II in promoting esophageal cancer progression. Human esophageal cancer tissue microarray was analyzed for overexpression of IGF-II and its correlation with that of Id1 and phosphorylated AKT (p-AKT). The efficacy of intratumorally injected IGF-II antibody and intraperitoneally injected cixutumumab (fully human monoclonal IGF-IR antibody) was evaluated using in vivo tumor xenograft and experimental metastasis models.Results: Id1 overexpression induced IGF-II secretion, which promoted cancer cell proliferation, survival, and invasion by activating AKT in an autocrine manner. Overexpression of IGF-II was found in 21 of 35 (60%) esophageal cancer tissues and was associated with upregulation of Id1 and p-AKT. IGF-II secreted by Id1-overexpressing esophageal cancer xenograft could instigate the growth of distant esophageal tumors, as well as promote metastasis of circulating cancer cells. Targeting IGF-II and IGF-IR had significant suppressive effects on tumor growth and metastasis in mice. Cixutumumab treatment enhanced the chemosensitivity of tumor xenografts to fluorouracil and cisplatin.Conclusions: The Id1–IGF-II–IGF-IR–AKT signaling cascade plays an important role in esophageal cancer progression. Blockade of IGF-II/IGF-IR signaling has therapeutic potential in the management of esophageal cancer. Clin Cancer Res; 20(10); 2651–62. ©2014 AACR.

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Representative images and quantification of number of cells that migrated or invaded in EC109 cells with or without FSTL1 suppressed and KYSE150-Luc cells with or without FSTL1 overexpressed, in the presence of mitomycin C.

Purpose: Previously, FTY720 was found to possess potent anticancer effects on various types of cancer. In the present study, we aimed to first verify the role of Runx2 in prostate cancer progression and metastasis, and, subsequently, assessed if FTY720 could modulate Runx2 expression, thus interfering downstream events regulated by this protein.Experimental Design: First, the association between Runx2 and prostate cancer progression was assessed using localized prostate cancer specimens and mechanistic investigation of Runx2-induced cancer aggressiveness was then carried out. Subsequently, the effect of FTY720 on Runx2 expression and transcriptional activity was investigated using PC-3 cells, which highly expressed Runx2 protein. Last, the involvement of Runx2 in FTY720-induced anticancer effects was evaluated by modulating Runx2 expression in various prostate cancer cell lines.Results: Runx2 nuclear expression was found to be up-regulated in prostate cancer and its expression could be used as a predictor of metastasis in prostate cancer. Further mechanistic studies indicated that Runx2 accelerated prostate cancer aggressiveness through promotion of cadherin switching, invasion toward collagen I, and Akt activation. Subsequently, we found that FTY720 treatment down-regulated Runx2 expression and its transcriptional activity, as well as inhibited its regulated downstream events. More importantly, silencing Runx2 in PC-3 enhanced FTY720-induced anticancer effects as well as cell viability inhibition, whereas overexpressing Runx2 in 22Rv1 that expressed very low endogenous Runx2 protein conferred resistance in the same events.Conclusion: This study provided a novel mechanism for the anticancer effect of FTY720 on advanced prostate cancer, thus highlighting the therapeutic potential of this drug in treating this disease.

Esophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, and molecular markers to improve early detection and predict outcomes are greatly needed. Here, we report that the BMP-binding follistatin-like protein FSTL1 is overexpressed in ESCCs, where it correlates with poor overall survival. Genetic amplification of FSTL1 or chromosome 3q, where it is located, occurred frequently in ESCC, where FSTL1 copy number correlated positively with higher FSTL1 protein expression. Elevating FSTL1 levels by various means was sufficient to drive ESCC cell proliferation, clonogenicity, migration, invasion, self-renewal, and cisplatin resistance in vitro and tumorigenicity and distant metastasis in vivo. Conversely, FSTL1 attenuation by shRNA or neutralizing antibody elicited the opposite effects in ESCC cells. mRNA profiling analyses suggested that FSTL1 drives ESCC oncogenesis and metastasis through various pathways, with deregulation of NFκB and BMP signaling figuring prominently. Cross-talk between the NFκB and BMP pathways was evidenced by functional rescue experiments using inhibitors of NFκB and TLR4. Our results establish the significance of FSTL1 in driving oncogenesis and metastasis in ESCC by coordinating NFκB and BMP pathway control, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in this disease setting. Cancer Res; 77(21); 5886–99. ©2017 AACR.

Supplementary Data from Suppression of Androgen-Independent Prostate Cancer Cell Aggressiveness by FTY720: Validating Runx2 as a Potential Antimetastatic Drug Screening Platform

Suppl Figure Legends 1-5 and Tables showing clinico-pathological correlation of endogenous and secretory FSTL1 as well as pathway analysis of ESCC cells with or without FSTL1 overexpressed.

PDF file - 1419KB Supplementary Fig S1. Human growth factor antibody array analysis of the conditioned media from HKESC-3-CON and HKESC-3-Id1 cells. Supplementary Fig S2. Effect of knockdown Id1 expression on IGF2 level using a second RNAi sequence. Supplementary Fig S3. Effect of ectopic Id1 expression on IGF2 level in multiple cancer types. Supplementary Fig S4. Effect of Id1 manipulation on IGF2 mRNA expression in ESCC cells. Supplementary Fig S5. IGF2, Id1, and p-AKT expressions in human esophageal cancer. Supplementary Fig S6. Western blot analysis of Id1 and IGF2 expressions in ESCC cell lines. Supplementary Fig S7. Effects of Id1 knockdown on autocrine stimulation of esophageal cancer cell proliferation, migration, and survival. Supplementary Fig S8. Ki-67 and TUNEL staining of tumor xenografts from three groups of mice inoculated with KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1- shIGF2 cells, respectively. Supplementary Fig S9. Ki-67 and TUNEL staining in "responder" tumors of different groups of mice bearing KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1- shIGF2 "instigating" tumors, respectively. Supplementary Fig S10. Anti-tumor effects of IGF2-neutralizing antibody in vivo. Supplementary Fig S11. Immunohistochemical analysis of tumor xenografts treated with cixutumumab or the isotype IgG. Supplementary Fig S12. TUNEL staining of tumor xenografts from two groups of mice inoculated with KYSE150 (A) and KYSE270 (B) respectively. Supplementary Fig S13. Evaluation of toxic effects of cixutumumab in nude mice. A, comparison of body weight of cixutumumab-treated mice and isotype IgG-treated control mice. B, histological examination of lung, liver and kidney specimens (H&E stained). Supplementary Fig S14. Increased thymidylate synthase expression in FR cells was inhibited by cixutumumab.

PDF file - 8KB, Immunohistochemical analysis of expression levels of Id1, IGF2, and p-AKT in human ESCC specimens.

PDF file - 490K, Supplemental Figure 1. A significantly deregulated network of genes in CD90+ esophageal T-ICs identified by genome-wide expression profiling and Ingenuity Pathway Analysis (IPA).

Supplementary Table 2 from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC. Cancer Res; 70(13); 5567–76. ©2010 AACR.

Expression of microRNA genes is profoundly altered in cancer but their role in the development of androgen-independent prostate cancer has received limited attention as yet. In this study, we report a functional impact in prostate cancer cells for overexpression of the microRNA miR-616, which occurred consistently in cells that were androgen-independent (AI) versus androgen-dependent (AD). miR-616 overexpression was confirmed in malignant prostate tissues as opposed to benign prostate specimens. Stable miR-616 overexpression in LNCaP cells by a lentiviral-based approach stimulated AI prostate cancer cell proliferation in vitro whereas concomitantly reducing androgen-induced cell growth. More importantly, miR-616 overexpressing LNCaP cells overcame castration resistance as shown by an enhanced ability to proliferate in vivo after bilateral orchiectomy. Conversely, antagonizing miR-616 in AI prostate cancer cells yielded opposite effects. Microarray profiling and bioinformatics analysis identified the tissue factor pathway inhibitor TFPI-2 mRNA as a candidate downstream target of miR-616. In support of this candidacy, we documented interactions between miR-616 and the 3′UTR of TFPI-2 and determined TFPI-2 expression to be inversely correlated to miR-616 in a series of prostate cell lines and clinical specimens. Notably, reexpression of TFPI-2 in LNCaP cells with stable miR-616 overexpression rescued the AD phenotype, as shown by a restoration of androgen dependence and cell growth inhibition. Taken together, our findings define a functional involvement for miR-616 and TFPI-2 in the development and maintenance of androgen-independent prostate cancer. Cancer Res; 71(2); 583–92. ©2011 AACR.

Supplementary Table 1 from Extracellular Protease ADAMTS9 Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis

PDF file, 720K, Detection of DNA copy number change of Rab25 by dual-color fluorescent in situ hybridization (FISH) in ESCC cell lines KYSE520, EC109 and formalin-fixed paraffin-embedded ESCC clinical tissue specimens (#1 and #2) with no or low Rab25 expression. BAC probe to Rab25 and the control reference probe to the centromere of chromosome 1 are represented by red and green signals, respectively. Magnification at 1000x.

PDF file, 153K, Supplementary Materials and Methods, Tables, Figure Legends, References

DOC file - 322K, Supplemental Table 1. Primer sequences for real-time qPCR analyses. Supplemental Table 2. RPKM values of other reported CSC markers in the original 3 pairs of non-tumor (N) and ESCC (T) clinical samples (Patients #16, #18 and #19) used for RNA-Seq profiling. Supplemental Table 3. Cancer stem cell marker expression (CD90, CD44 and p75NTR / CD271) in a panel of ESCC cell lines. Supplemental Table 4. Clinicopathological correlation of CD44 and p75NTR / CD271 expression in ESCC. Supplemental Table 5. List of deregulated genes in CD90+ vs. CD90- cells in ESCC cells KYSE140 (cut off ≥ 3 folds).

PDF file - 1414K, Supplemental Figure 2. CD90, CD44 and p75NTR / CD271 expression in matched non-tumor (N) and primary ESCC (T) (n = 33) as detected by real-time qPCR.

Supplementary Figure Legend from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is a devastating disease characterized by distinctly high incidences and mortality rates. However, there remains limited understanding of molecular events leading to development and progression of the disease, which are of paramount importance to defining biomarkers for diagnosis, prognosis, and personalized treatment. By high-throughout transcriptome sequence profiling of nontumor and ESCC clinical samples, we identified a subset of significantly differentially expressed genes involved in integrin signaling. The Rab25 gene implicated in endocytic recycling of integrins was the only gene in this group significantly downregulated, and its downregulation was confirmed as a frequent event in a second larger cohort of ESCC tumor specimens by quantitative real-time PCR and immunohistochemical analyses. Reduced expression of Rab25 correlated with decreased overall survival and was also documented in ESCC cell lines compared with pooled normal tissues. Demethylation treatment and bisulfite genomic sequencing analyses revealed that downregulation of Rab25 expression in both ESCC cell lines and clinical samples was associated with promoter hypermethylation. Functional studies using lentiviral-based overexpression and suppression systems lent direct support of Rab25 to function as an important tumor suppressor with both anti-invasive and -angiogenic abilities, through a deregulated FAK–Raf–MEK1/2–ERK signaling pathway. Further characterization of Rab25 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target in ESCC treatment. Cancer Res; 72(22); 6024–35. ©2012 AACR.

PDF file, 2272K, Expression tiling of Rab25 in 6 unpaired ESCC and non-tumor clinical samples.

Supplementary Figure 1 from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2

Supplementary References from MicroRNA-616 Induces Androgen-Independent Growth of Prostate Cancer Cells by Suppressing Expression of Tissue Factor Pathway Inhibitor TFPI-2