Side Population Expression Research Articles

Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44+ tumorigenic cells in Akt/β-catenin-driven hepatocellular carcinoma

Hepatic resection and liver transplantation with adjuvant chemo- and radiotherapy are the mainstay of hepatocellular carcinoma (HCC) treatment, but the 5-year survival rate remains poor because of frequent recurrence and intrahepatic metastasis. Only sorafenib and lenvatinib are currently approved for the first-line treatment of advanced, unresected HCC, but they yield modest survival benefits. Thus, there is a need to identify new therapeutic targets to improve current HCC treatment modalities. The HCC tumor model was generated by hydrodynamic transfection of AKT1 and β-catenin (CTNNB1) oncogenes. Cancer cells with stemness properties were characterized following isolation using side population (SP) and CD44 surface markers by flow cytometry. The effect of Jak/Stat inhibitors was analyzed in vitro by using tumorsphere culture and in vivo using an allograft mouse model. Co-activation of both Wnt/β-catenin and Akt/mTOR pathways was found in 14.4% of our HCC patient cohort. More importantly, these patients showed poorer survival than those with either Wnt/β-catenin or Akt/mTOR pathway activation alone, demonstrating the clinical relevance of our study. In addition, we observed that Akt/β-catenin tumors contained a subpopulation of cells with stem/progenitor-like characteristics identified through SP analysis and expression of the cancer stem cell-like marker CD44, which may contribute to tumor self-renewal and drug resistance. Consequently, we identified small molecule inhibitors of the Jak/Stat pathway that demonstrated efficacy in mitigating tumor proliferation and formation in Akt/β-catenin-driven HCC. In conclusion, we have shown that Akt/β-catenin tumors contain a subpopulation of tumor-initiating cells with stem/progenitor-like characteristics which can be effectively targeted with inhibitors of the Jak/Stat pathway, demonstrating that inhibition of the Jak/Stat pathway could be an alternative method to overcome drug resistance and effectively treat Akt/β-catenin-driven HCC tumors. The prognosis for patients with hepatocellular carcinoma is poor, partly because of the lack of effective treatment options for those with more advanced disease. In this study, we identified a subpopulation of cancer cells with stem cell-like properties that were critical for tumor maintenance and growth in a mouse model of hepatocellular carcinoma. Through further experiments, we demonstrated that the Jak/Stat pathway is a promising therapeutic target in hepatocellular carcinoma.

Interleukin-8 is related to poor chemotherapeutic response and tumourigenicity in hepatocellular carcinoma

AimInterleukin-8 (IL-8) has been suggested as a prognostic biomarker for human hepatocellular carcinoma (HCC), but its roles in HCC progression and drug resistance have not been studied. This study investigates the role and underlying mechanism of IL-8 in the chemoresistance and progressive growth of HCC. MethodsThe change of chemosensitivity and proportion of side population in hepatoma cells was examined by cell growth and flow cytometric analyses after anti-cancer treatments or knockdown of IL-8. Expression of IL-8 and ATP-binding cassette (ABC) transporters in hepatoma cells, xenograft and clinical HCC tissues was determined by Western blot and immunohistochemical analyses. Tumourigenicity of hepatoma cells was evaluated in vivo after silencing IL-8 gene. ResultsTreatment of hepatoma cells with anti-cancer drugs increased the production of IL-8 and its receptor, as well as the proportion of side population (SP). Exogenous IL-8 increased the SP fraction and expression of multidrug resistance-1, decreasing the drug sensitivity. Silencing of IL-8 gene decreased the ratio of SP cells and drug resistance properties. Both IL-8 and ABC transporters were highly expressed in xenograft and clinical HCC tissues, and knockdown of IL-8 significantly reduced tumour size in vivo. ConclusionAnti-cancer drug-induced IL-8 secretion increased the expression of ABC transporters and SP cells, promoting the growth of HCC in vitro. Thus IL-8 may be a potential therapeutic target in the treatment of HCC.

Side population in LX2 cells decreased by transforming growth factor‐β

Side population (SP) cells are known to be enriched in stem/progenitor-like cells. Transforming growth factor (TGF)-β signaling is associated with extracellular matrix (ECM) production in hepatic stellate cells. We hypothesized that the SP fraction in LX2 cells is associated with ECM deposition, which is regulated through TGF-β signaling. We investigated the relationship between SP cells and TGF-β signaling in the hepatic stellate cell line LX2. The effects of TGF-β and SB431542 on the SP fraction and expression of collagen type I and phospho-Smad2 was determined. We identified 0.8-3% SP cells in LX2 cells. The growth rate of sorted SP and non-SP cells was similar to that of the original LX2 population, but population of the G0/G1 phase was increased in SP cells. Treatment of LX2 cells with TGF-β decreased the SP fraction in a dose-dependent manner and increased the production of collagen type I. Treatment of LX2 cells with SB431542 blocked the effect of TGF-β on the SP fraction and the expression of collagen type I. We cultured LX2 cells on collagen-coated dishes to observe the effect of ECM deposition on the SP fraction. The growth rate and cell cycle distribution was similar to that observed on normal tissue culture dishes, but the SP fraction was decreased when LX2 cells were cultured on collagen-coated plates. These results show that LX2 cells contain an SP fraction and that TGF-β signaling is involved in the induction of ECM deposition as well as the number of SP cells.

Side population in human non-muscle invasive bladder cancer enriches for cancer stem cells that are maintained by MAPK signalling.

Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. In vitro and in vivo phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2hi) relative to the non-SP (NSP) fraction (ABCG2low). ABCG2hi SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2low NSP cells. In vivo, ABCG2hi SP cells enriched for tumour growth compared with ABCG2low NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2hi SP cells and MEK inhibition also inhibited the ABCG2hi SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2hi SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.

Sorafenib inhibits cancer side population cells by targeting c‑Jun N‑terminal kinase signaling.

Sorafenib is a systemic chemotherapeutic agent for advanced hepatocellular carcinoma (HCC). The aim of the present study was to evaluate the anticancer effect of sorafenib in cancer stem cell‑like cells, such as side population (SP) cells, in HCC and to analyze the signaling pathway for drug‑resistance. To evaluate the anticancer effects of sorafenib, Huh7 and Huh‑BAT cells were treated with sorafenib, fluorouracil (5‑FU), and sorafenib plus 5‑FU. These cells were examined for growth rates, the SP fraction, sphere‑forming efficacy and expression of c‑Jun N‑terminal kinase (JNK) signaling molecules. Sorafenib and 5‑FU treatment decreased growth rates in Huh7 and Huh‑BAT cells; however, the treatments exerted different effects in SP cells and on the expression levels of JNK signaling molecules. Treatment with 5‑FU increased the SP cell number and upregulated the expression of JNK signaling molecules. By contrast, sorafenib decreased the SP cell number and downregulated the expression of JNK signaling molecules. No significant differences in sphere‑forming efficacy were observed subsequent to 5‑FU and sorafenib treatment in Huh7 and Huh‑BAT cells. These results indicate that sorafenib exerted anticancer effects in HCC and SP cells by targeting JNK signaling.

Abstract 503: Instability of ABCG2-associated side population expression in human A549 lung cancer cells and its contribution to drug resistance

Abstract Dynamic changes in tumour subpopulations may contribute to post-therapeutic repopulation and treatment failure. Human tumours and established cell lines can maintain ‘side’ populations (SPs) – identified by a reduced accumulation of Hoechst dye 33342 attributable to ABCG2-mediated efflux. Progenitors in SP fractions present a challenge for therapies that attempt to target active S phase if they also comprise agents subject to active cellular efflux. The current study addresses the impact of the SP phenotype on drug resistance characteristics with a focus on a predicted resistance to the S-phase targeting anticancer drug DNA topoisomerase I poison topotecan (TPT). The human lung cancer A549 cell line system was used to isolate subclones that initially presented a range of SP expression (< 0.5 – >30 %) and a positive correlation was found between the size of the SP fraction and reduced TPT accumulation associated with ABCG2 expression. Representative clones were established with an initially low SP fraction (<1% SP; A549LowSP) or an initially high SP fraction (>30% SP; A549HighSP) compared with the A549parent (∼7% SP). Using RT-PCR, cells were compared for the expression of drug resistance-associated ATP binding cassette transporters (ABCG2, ABCB1). Increased ABCG2, but not ABCB1, expression, correlated with the degree of SP expression. A database survey of 59 human tumor NCI60 panel cell lines suggested that expression of the cancer stem cell marker aldehyde dehydrogenase (ALDH1A1) may augment TPT resistance in cells with low-level expression of ABCG2. However, parental, A549LowSP and A549HighSP cells showed similar co-expression patterns for ALDH genes (ALDH1A1, ALDH2, and ALDH3A1). A549LowSP increased its SP fraction during continued culture while A549HighSP maintained the SP fraction. The extent of the SP fraction reflected the degree of TPT efflux that was sensitive to the iABCG2-inhibitor Fumitremorgin C. To address whether innate SP expression provided an initial advantage for recovery from TPT exposure we studied the dynamic changes in the SP under in vitro selection of A549parent cells. We found that human SPs have a selective advantage, with enrichment of >20-fold, in cell populations recovering from multiple rounds of TPT treatment. Clonal analysis of TPT recovering populations found no evidence of long-term enrichment of SP fraction suggesting innate instability in SP expression. The data suggest that A549 non-SP cell populations can re-create SP fractions indicating that they are likely to arise by variations that provide a proliferative advantage rather than a unique stem-like progenitor. Innate SP fractions provide the tumor population with a short term selective advantage following TPT exposure related to their enhanced ABCG2 expression, and become enriched under dose fractionation. (LC is funded by UK-EPSRC and KP, LHP by Yorkshire Cancer Research) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 503. doi:10.1158/1538-7445.AM2011-503

Berberine Diminishes the Side Population and ABCG2 Transporter Expression in MCF-7 Breast Cancer Cells

The plant alkaloid berberine has many biological activities including the ability to induce cell cycle arrest and apoptosis, making it a potentially useful agent for targeting cancer cells. We have analyzed the effects of berberine on MCF-7 breast cancer cells. Berberine was added to MCF-7 cells in culture, and proliferation, side population (SP) cells and expression of ABCG2 were examined. Berberine caused a dose-dependent reduction in proliferation. Hoechst 33,342 dye staining and FACS analysis revealed that berberine treatment caused a decrease in SP cells relative to untreated controls. In addition, berberine treatment was associated with a decrease in expression of ABCG2 relative to untreated controls. These results indicate that the growth inhibitory effects of berberine treatment on MCF-7 cells may be partly via effects on SP and ABCG2 expression. Further work is warranted to explore whether berberine may be a novel therapeutic drug useful for targeting breast cancer stem cells.

Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.