Possess Cancer Stem Cell Properties Research Articles

Abstract PO1-24-02: Enhancement of TGF-β Receptor Inhibitor Efficacy through CD44 Suppression in Claudin-low Breast Cancer

Abstract Introduction: Claudin-low breast cancer is mostly classified as triple-negative breast cancer and is known to possess cancer stem cell properties with high expression of epithelial-to-mesenchymal transition (EMT) markers. However, specific therapeutic targets for this subtype have not yet been identified. While reports suggest that inhibition of EMT using EMT inhibitors can suppress cell and tumor proliferation, monotherapy may not be sufficient. The expression of CD44, a surface marker of cancer stem cells, has been reported to be involved in multiple signaling pathways, promoting cancer growth and invasion. In this study, we focus on CD44 and the TGF-β receptor, which is involved in EMT, to investigate the effect of inhibiting TGF-β receptor through CD44 inhibition in Claudin-low breast cancer and validate its potential as an effective treatment. Methods: SUM159 and MDA-MB-231 cell lines were used as Claudin-low breast cancer cell models. CD44 knockdown cells were established to examine the effects of TGF-β receptor inhibition and downstream signaling. Results: CD44 knockdown did not affect the expression of TGF-β receptor. Both SUM159 and MDA-MB-231 cells showed comparable cell growth inhibition through CD44 knockdown and TGF-β receptor inhibition, with enhanced effects observed in CD44 knockdown cells in response to TGF-β receptor inhibition. Furthermore, CD44 knockdown combined with TGF-β receptor inhibition significantly reduced the expression of Smad2/3. Conclusion: In Claudin-low breast cancer cells, CD44 knockdown enhances the effectiveness of TGF-β receptor inhibition and significantly influences the expression level of Smad2/3. Citation Format: Ryoichi Matsunuma, Sae Imada, Shoko Sato, Ryosuke Hayami, Michiko Tsuneizumi. Enhancement of TGF-β Receptor Inhibitor Efficacy through CD44 Suppression in Claudin-low Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-02.

Drug Design Targeting T-Cell Factor-Driven Epithelial-Mesenchymal Transition as a Therapeutic Strategy for Colorectal Cancer.

Metastasis is the cause of 90% of mortality in cancer patients. For metastatic colorectal cancer (mCRC), the standard-of-care drug therapies only palliate the symptoms but are ineffective, evidenced by a low survival rate of ∼11%. T-cell factor (TCF) transcription is a major driving force in CRC, and we have characterized it to be a master regulator of epithelial-mesenchymal transition (EMT). EMT transforms relatively benign epithelial tumor cells into quasi-mesenchymal or mesenchymal cells that possess cancer stem cell properties, promoting multidrug resistance and metastasis. We have identified topoisomerase IIα (TOP2A) as a DNA-binding factor required for TCF-transcription. Herein, we describe the design, synthesis, biological evaluation, and in vitro and in vivo pharmacokinetic analysis of TOP2A ATP-competitive inhibitors that prevent TCF-transcription and modulate or reverse EMT in mCRC. Unlike TOP2A poisons, ATP-competitive inhibitors do not damage DNA, potentially limiting adverse effects. This work demonstrates a new therapeutic strategy targeting TOP2A for the treatment of mCRC and potentially other types of cancers.

Autophagy-dependent generation of Axin2+ cancer stem-like cells promotes hepatocarcinogenesis in liver cirrhosis

Autophagy is a pathophysiological phenomenon in liver cirrhosis that can further progress into hepatocarcinoma. Liver cancer stem cells (CSCs) are believed to initiate hepatocarcinogenesis. To investigate the precise mechanism related to the origin of CSCs in liver cirrhosis and hepatocarcinogenesis, we labeled Axin2+ hepatic cells with EGFP in Axin2Cre;Rosa26EGFP transgenic rats, and then stratified clinical and rat liver cirrhosis samples by autophagy flux. Clinical follow-up and lineage tracing in transgenic rat liver cirrhosis revealed that while Axin2/EGFP+ hepatic cells were present in normal livers and cirrhotic livers without aberrant autophagy, hepatic Axin2/EGFP+CD90+ cells were generated exclusively in cirrhotic livers with aberrant autophagy and promoted hepatocarcinogenesis. Aberrant autophagy in liver cirrhosis resulted in hepatocyte growth factor (HGF) expression, leading to activation of Met/JNK and Met/STAT3 signaling in sorted hepatic Axin2/EGFP+ cells and their transition into Axin2/EGFP+CD90+ cells that possess CSC properties. In a transgenic rat liver cirrhosis model, induction or inhibition of autophagy in cirrhotic livers by systemic administration of rapamycin or chloroquine or transfection with Atg3- and Atg7-shRNAs significantly induced or suppressed HGF expression, which in turn increased or reduced generation of EGFP+CD90+ hepatic cells by activating or inactivating Met/JNK and Met/STAT3 signaling, thereby promoting or preventing hepatocarcinogenesis. Systemic treatment with HGF-shRNA, SP600125 or stattic also reduced generation of EGFP(Axin2)+ hepatic cell-originated CD90+ CSCs in aberrant autophagic cirrhotic livers by inactivating HGF/Met/JNK or HGF/Met/STAT3 signaling, further preventing hepatocarcinogenesis. These data suggest that activation of Met/JNK and Met/STAT3 signaling in Axin2+ hepatic cells via autophagy-dependent HGF expression and the resultant generation of Axin2+CD90+ CSCs is a major mechanism of hepatocarcinogenesis in cirrhotic livers.

Abstract 2880: GANT61-mediated constraint of Fibulin-5/prorenin receptor/BMI1 signaling impacts nasopharyngeal carcinoma stemness and metastasis

Abstract Background: Cancer stem cells (CSCs) of NPC exhibit higher tumorigenesis and metastatic potential. Novel therapies based on molecular targets of CSCs have become the focus to cure NPC. GANT61, a GLI1 inhibitor, has been reported to exhibit potent anticancer effect on many cancers; here, we evaluated whether GANT61 has inhibitory effects on NPC-CSCs and further explored the possible mechanism. Methods: Condition medium was used to enrich NPC stem-like cells. The NPC stem-like cells characteristics were examined by Q-RT-PCR, Western blotting, MTT, BudU, colony formation, sphere formation, and subcutaneous and metastatic xenograft models. Genetic, pharmacological and biochemical approaches were applied in NPC stem-like cells to investigate molecular signals, to assess the efficacies under the inhibitors treatment, and to examine the correlation among the molecules in vitro, in vivo and NPC specimens. Results: In the present study, we had demonstrated that NPC tumorsphere cells (NPC-TCs) possess CSC properties. The proliferation, colony formation, spheroid formation, migration, and invasion of NPC-TCs were dramatic prevented in the presence of GANT61 in vitro and in vivo. ChIP reveals that Gli1 preferentially binds to the Fibulin-5 promoter, and Fibulin-5 transcriptional/post-transcriptional levels in NPC-TCs were regulated upon GANT61 treatment. However, ectopic expression of Fibulin-5 in NPC-TCs not only enhanced self-renewal and ALDH1 expression, but also reduced the effects of GANT61-eilicted anti-tumor in vitro and in vivo. We further identified that a novel signaling, prorenin receptor/BMI1 pathway was modulated by Fibulin-5 and involved in Fibulin-5-raised self-renewal capacity of NPC-TCs in vitro and in vivo. Notably, GANT61 inhibited NPC-TCs phenotypes was associated with suppression of Fibulin-5/prorenin receptor/BMI1 signaling. Finally, a significant correlation was observed among GLI1, Fibulin-5, AT1R and BMI1 in the specimens of NPC. Conclusions: Our data highlight that Fibulin-5/prorenin receptor/BMI1 signaling has critical role in maintaining stem-like properties of NPC-CSCs and can be efficiently targeted by GANT61 representing a preclinical therapeutic strategy to repress NPC-CSCs. Note: This abstract was not presented at the meeting. Citation Format: Hsin-Ting Tasi, Chang-Han Chen. GANT61-mediated constraint of Fibulin-5/prorenin receptor/BMI1 signaling impacts nasopharyngeal carcinoma stemness and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2880. doi:10.1158/1538-7445.AM2017-2880

Bone marrow-derived mesenchymal stem cells increase drug resistance in CD133-expressing gastric cancer cells by regulating the PI3K/AKT pathway.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are recruited to primary tumours to compose the tumour microenvironment. In various cancers, CD133-positive cells have been shown to possess cancer stem cell properties that confer chemoresistance. This study aimed to investigate the role of BM-MSCs in the anti-tumour drug resistance of CD133-expressing gastric cancer cells and explore the underlying mechanisms that governing this role. We found that CD133+ gastric cancer cells displayed more resistance to chemotherapeutics than CD133- cells. In addition, BM-MSCs increased the anti-apoptotic abilities and chemoresistance of CD133+ cells via upregulation of Bcl-2 and downregulation of BAX. Mechanistically, BM-MSCs triggered activation of the PI3K/Akt signalling cascade in CD133+ cells. Blocking the PI3K/Akt pathway inhibited the promotion of chemoresistance. Furthermore, BM-MSCs enhanced the drug resistance of CD133-overexpressing cells in vitro and in vivo, but not that of CD133-knockdown cells, which demonstrated the contribution of CD133 to this process. In conclusion, we demonstrated that BM-MSCs increased the anti-apoptotic abilities and drug resistance of CD133-expressing cells via activation of the PI3K/Akt pathway following Bcl-2 upregulation and BAX downregulation, in which CD133 played a significant role. Targeting this route may help improve the efficacy of chemotherapy in gastric cancer.

Abstract 1525: FAK and PI3K/mTOR inhibitors target cancer stem cells: Implications for SCLC treatment strategies

Abstract Small cell lung cancer (SCLC) is an extremely aggressive cancer with poor outcome. In most patients with SCLC tumors initially respond to first line chemotherapy, but subsequently experience aggressive recurrence that may be attributed to the presence of a cancer stem cell (CSC) population with tumor-initiating and metastatic potential. Inhibitors of FAK and PI3K/mTOR have been shown to preferentially target CSCs in preclinical models of other cancers, where the evidence indicates that CSC populations are increased following chemotherapy. FAK and PI3K/mTOR inhibitors may be particularly valuable in a SCLC setting, where CSCs may be strong mediators of recurrence. Tumors of patients with SCLC are generally not resectable, such that tumor biopsies are not available for CSC evaluation. Therefore, it will be important to be able to monitor CSC markers by blood-based testing. A drug-resistant population of SCLC CSCs (side population, SP) can be monitored by enhanced ability to exclude Hoechst 33342 dye. Additionally, SCLC CD133+ cells have been demonstrated to possess CSC properties and presence of CD133+ cells in SCLC has been linked to poor prognosis. Our data indicate that cisplatin and etoposide, first line chemotherapy for SCLC, enrich for SP and CD133+ cells, suggesting that chemotherapy is not effective against CSCs and should be combined with anti-CSC agents for more durable response. Here we investigate the antitumor activity of FAK and PI3K/mTOR inhibitors in SCLC xenograft models in vivo. Both a FAK inhibitor (VS-4718) and PI3K/mTOR inhibitor (VS-5584) were found to enhance the antitumor activity of chemotherapy in an NCI-H69 SCLC xenograft model. Additionally, oral administration of VS-5584 reduced the proportion of CSCs in vivo in an NCI-H841 SCLC tumor model as evidenced by a decrease in the percentage of SP cells and approximately 70-fold reduction in in vivo tumor-initiating capability. The combination of chemotherapy and FAK or PI3K/mTOR inhibitors demonstrate an extended time to relapse after first line therapy. VS-5584 significantly delayed tumor regrowth following cessation of cisplatin treatment in H69 xenograft tumors and a SCLC patient-derived xenograft (PDX) model. Patients with relapsed SCLC have elevated circulating tumor cells, consistent with the poor prognosis of their disease status. Blood samples from these patients are under evaluation for CSC markers in the circulation. The preferential targeting of CSCs in preclinical SCLC models provides an important rationale for clinical development of FAK and PI3K inhibitors, where combination with chemotherapeutic agents or single agent activity post chemotherapy may potentially lengthen the time to relapse and improve outcome for patients with small cell lung cancer. Citation Format: Vihren N. Kolev, Qunli Xu, Jonathan A. Pachter, David T. Weaver. FAK and PI3K/mTOR inhibitors target cancer stem cells: Implications for SCLC treatment strategies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1525. doi:10.1158/1538-7445.AM2015-1525

Transforming growth factor-beta1 promotes the migration and invasion of sphere-forming stem-like cell subpopulations in esophageal cancer

Esophageal cancer is one of the most lethal solid malignancies. Mounting evidence demonstrates that cancer stem cells (CSCs) are able to cause tumor initiation, metastasis and responsible for chemotherapy and radiotherapy failures. As CSCs are thought to be the main reason of therapeutic failure, these cells must be effectively targeted to elicit long-lasting therapeutic responses. We aimed to enrich and identify the esophageal cancer cell subpopulation with stem-like properties and help to develop new target therapy strategies for CSCs. Here, we found esophageal cancer cells KYSE70 and TE1 could form spheres in ultra low attachment surface culture and be serially passaged. Sphere-forming cells could redifferentiate and acquire morphology comparable to parental cells, when return to adherent culture. The sphere-forming cells possessed the key criteria that define CSCs: persistent self-renewal, overexpression of stemness genes (SOX2, ALDH1A1 and KLF4), reduced expression of differentiation marker CK4, chemoresistance, strong invasion and enhanced tumorigenic potential. SB525334, transforming growth factor-beta 1(TGF-β1) inhibitor, significantly inhibited migration and invasion of sphere-forming stem-like cells and had no effect on sphere-forming ability. In conclusion, esophageal cancer sphere-forming cells from KYSE70 and TE1 cultured in ultra low attachment surface possess cancer stem cell properties, providing a model for CSCs targeted therapy. TGF-β1 promotes the migration and invasion of sphere-forming stem-like cells, which may guide future studies on therapeutic strategies targeting these cells.

Abstract 3016: Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived exosomes/microvesicles

Abstract It is widely accepted that the microenvironment/niche exert genetic and/or epigenetic effects on stem cells. Current studies have demonstrated that an aberrant microenvironment may contribute to the genesis of cancer stem cells (CSCs). In the case of cancer, growing evidence indicate a cancerous niche can play an active role in the regulation of tumor cell maintenance and progression through exosomes/microvesicles-based intercellular communication. It has not been reported, however, whether this vesicle-mediated communication induces the malignant transformation of normal stem cells/differentiating progenitors, result in CSC generation. Recentry, we have reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into CSCs. Here, we investigated the contribution of tumor-derived exosomes/microvesicles (TEMVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. The differentiating miPSCs were exposed to TEMVs derived from LLC cells for 4 weeks. We used Nanog-GFP miPS, in which GFP expression is under control of Nanog promoter, indicative of undifferentiation state. During this period, we observed the reemergence of the GFP-positive colonies, whereas most of cells were differentiated and GFP-negative without TEMV-treatment. The resultant TEMVs treated cells (miPS-LLCemv) expressed Nanog and Oct3/4 proteins comparable to miPSCs assessed by Western blotting. The sphere formation of the miPS-LLCemv cells in suspension culture indicated the selfrenewal capacity of the miPS-LLCemv cells. When the miPSLLCemv cells were subcutaneously allografted into immunodeficient mice, malignant tumors with extensive angiogenesis developed. Phathological features such as vementin, Ki67, PPAR gamma expression, and accumulation of lipid droplets in tumor revealed these tumors were liposarcomas. Tumor cells established from liposarcomas showed the capacity of spheroid formation in suspension culture and the capacity of differntiation into adipocytes and endothelial cells. These results indicate that miPS-LLCemv cells and established tumor cells possess CSC properties. Interestingly, these cells expressed Sox2 and Klf4 genes significantly higher than parental miPS cells, might be related to selfrenewal and addipogenesis. Thus, we concluded that TEMVs should have the potential to induce CSC properties in normal stem cells. Citation Format: Yan Ting, Junko Masuda, Akifumi Mizutani, Ling Chen, Tsukasa Shigehiro, Shuichi Matsuda, Tomonari Kasai, Takayuki Kudoh, Hiroshi Murakami, Mary J.C. Hendrix, Luigi Strizzi, David S. Salomon, Li Fu, Masaharu Seno. Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived exosomes/microvesicles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2014-3016

Enrichment and functional characterization of cancer stem cells from squamous cell carcinoma of head and neck by suspension culture

Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinoma (HNSCC) have been related to the cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH-1) activity. In this study, we try to prove that suspension culture can enrich ALDH-1 high expression cells within HNSCC cell lines and the enriched cells possess cancer stem cell properties. Cells from five HNSCC cell lines were cultured in ultra-low attachment plates in serum-free Quantum 263 medium supplemented with 10 ng/ml EGF and 10 ng/ml bFGF, and ALDH-1 expression level was evaluated by ALDEFLUOR assay. ALDH-1 high expression cells were separated by FACS sorting, and their phenotypical and functional properties were characterized. Spheroids can be formed from all five HNSCC cell lines (UD-SCC1, UT-SCC22, UM-SCC11B, UT-SCC9 and UT-SCC24A) under anchorage independent culture condition. The proportion of ALDH1 high expression cells were highly increased in speroids derived cells (SDCs) compared with their monolayers (P < 0.05). The clones formed by ALDH1 high expression cells on average contained 197 (197 ± 47) cells compared with 33 (33 ± 16) cells in clones generated from ALDH1 low expression cells (P < 0.01). Single ALDH1 high expression cell could significantly better regenerate a spheroid (UT-SCC9: 17.1%, UD-SCC1:19.3%), whereas under the same conditions single ALDH1 low expression cells regenerated only in one case a spheroid (P < 0.01). SDCs from all five tested cell lines also showed a significantly increased invasion capacity (P < 0.05). We also found that the mRNA levels of Oct-4, Sox2, and Nanog were all significantly increased in the SDC. The reactive oxygen species (ROS) levels in SDCs from UD-SCC1 and UT-SCC24A were significantly increased compared with their monolayer counterpart [(26.3 ± 4.9)% vs (8.6 ± 1.7)% and (72.1 ± 6.1)% vs (23.7 ± 7.5)%, P < 0.05)]. Cancer stem cells can be enriched by suspension culture, which may be of importance in investigation of their contribution to therapy resistance, tumor recurrence and metastasis.

Characterization of cancer stem-like cells derived from mouse induced pluripotent stem cells transformed by tumor-derived extracellular vesicles.

Several studies have shown that cancer niche can perform an active role in the regulation of tumor cell maintenance and progression through extracellular vesicles-based intercellular communication. However, it has not been reported whether this vesicle-mediated communication affects the malignant transformation of normal stem cells/progenitors. We have previously reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into cancer stem cells (CSCs), indicating that normal stem cells when placed in an aberrant microenvironment can give rise to functionally active CSCs. Here, we focused on the contribution of tumor-derived extracellular vesicles (tEVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. We isolated tEVs from the conditioned medium of LLC cells, and then the differentiating miPSCs were exposed to tEVs for 4 weeks. The resultant tEV treated cells (miPS-LLCev) expressed Nanog and Oct3/4 proteins comparable to miPSCs. The frequency of sphere formation of the miPS-LLCev cells in suspension culture indicated that the self-renewal capacity of the miPS-LLCev cells was significant. When the miPS-LLCev cells were subcutaneously transplanted into Balb/c nude mice, malignant liposarcomas with extensive angiogenesis developed. miPS-LLCevPT and miPS-LLCevDT, the cells established from primary site and disseminated liposarcomas, respectively, showed their capacities to self-renew and differentiate into adipocytes and endothelial cells. Moreover, we confirmed the secondary liposarcoma development when these cells were transplanted. Taken together, these results indicate that miPS-LLCev cells possess CSC properties. Thus, our current study provides the first evidence that tEVs have the potential to induce CSC properties in normal tissue stem cells/progenitors.

RETRACTED ARTICLE: Characterization of cancer stem-like cells in the side population cells of human gastric cancer cell line MKN-45

Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many kinds of cell lines and tissues have demonstrated the presence of SP cells, including several gastric cancer cell lines. This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45. We used fluorescence activated cell sorting (FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45 (cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells. This study found that the SP cells had higher clone formation efficiency than major population (MP) cells. Five stemness-related gene expression profiles, including OCT-4, SOX-2, NANOG, CD44, and adenosine triphosphate (ATP)-binding cassette transporters gene ABCG2, were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Western blot was used to show the difference of protein expression between SP and MP cells. Both results show that there was significantly higher protein expression in SP cells than in MP cells. When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, SP cells show higher tumorigenesis tendency than MP cells. These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.

Spheroid body-forming cells in the human gastric cancer cell line MKN-45 possess cancer stem cell properties

The cancer stem cell theory hypothesizes that cancer stem cells (CSCs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor formation, recurrence and metastasis. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. In this study, we enriched gastric cancer stem cells through spheroid body formation by cultivating the human gastric cancer cell line MKN-45 in defined serum-free medium. The stemness characteristics of spheroid body-forming cells, including self-renewal, proliferation, chemoresistance, tumorigenicity of the MKN-45 spheroid body-forming cells were evaluated, and the expression levels of stemness genes and related proteins in the MKN-45 spheroid body-forming cells were assessed. Furthermore, immunofluorescence staining for the stem cell markers on spheroid body-forming cells was examined to evaluate the association between stemness factors (Oct4, Sox2, Nanog) and the proposed CSC marker CD44. Our data demonstrated that non-adherent spheroid body-forming cells from the gastric cancer cell line MKN-45 cultured in stem cell-conditioned medium possessed gastric CSC properties, such as persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and overexpression of CSC-related genes and proteins (Oct4, Sox2, Nanog and CD44), compared with the parental cells. More importantly, CD44-positive cells co-expressing the pluripotency genes Oct4, Sox2 and Nanog may represent gastric CSCs. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify CSCs.

Chk1 knockdown confers radiosensitization in prostate cancer stem cells

Radioresistance is responsible for treatment failure after radiotherapy in localized prostate cancer, while prostate cancer stem cells promote radioresistance by preferential activation of the DNA damage response. Chk1 inhibition has been shown to sensitize many tumor cells to radiation. However, whether Chk1 inhibition can potentiate the cytotoxic effects of radiation on prostate cancer stem cells remains to be elucidated. In this study, CD133+CD44+ cells were isolated using microbeads and were found to possess cancer stem cell properties. Using shRNA, Chk1 was knocked down in the sorted CD133+CD44+ cells. Our results demonstrated that Chk1 knockdown abrogated the radiation-induced G2/M arrest, inhibited DNA damage repair and promoted premature mitosis, leading to increased apoptosis in the radiated sorted CD133+CD44+ cells. Moreover, these effects were accompanied by caspase-2 activation and the inactivation of phosphorylated Cdc25C and Cdc2. Our results suggest that Chk1 knockdown increases the radiosensitivity of CD133+CD44+ prostate cancer stem cells. Chk1 knockdown in prostate cancer stem cells may be an effective therapeutic strategy against prostate cancer.

Not All Side Population Cells Contain Cancer Stem-Like Cells in Human Gastric Cancer Cell Lines

Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many kinds of cell lines and tissue have demonstrated presence of SP cells including different gastric cancer cell lines. However, is that true all SP cells contain cancer stem-like cells in gastric cancer cell lines? MKN-45 and BGC-823 cells labeled with Hoechst 33342 were chosen to obtain SP cells, then characterized the cancer stem-like properties of SP cells both in vitro and in vivo. Five stemness-related genes expression profiles, including OCT-4, SOX-2, NANOG, CD44 and ATP-binding cassette transporters gene ABCG-2, were tested in SP and MP cells using quantitative real-time RT-PCR. Western blot was chosen to show the difference of protein expression between SP and MP cells. When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, SP cells from MKN-45 showed higher tumorigenesis tendency than MP cells, but SP cells from BGC-823 showed same tumorgenesis tendency as MP cells. SP cells from MKN-45 possess cancer stem cell properties and proved that they were gastric cancer stem-like cells. SP cells from BGC-823 didn't possess cancer stem cell properties and proved that not all SP cells contain cancer stem-like cells in gastric cancer cell lines.

Abstract 1294: Suppression of uPA in chemo-sensitization and p53 regulation in pancreatic cancer cells

Abstract Despite new knowledge of the molecular profile of pancreatic cancer and its precursor lesions, survival rates have changed very little over the last 40 years. The ability to predict which patients would benefit most from surgical intervention and/or chemotherapy would be a great clinical asset. Researchers have demonstrated that uPA is frequently present in a higher concentration in the serum of pancreaticobiliary cancer patients. It is known that uPA is involved in ECM degradation and has been correlated with malignant transformation of cancer cells. Emerging data have suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor called cancer stem cells (CSCs) or cancer initiating cells. Under in vitro cell culture conditions, these cells possess sphere-forming ability, which is known to be one of the properties of CSCs. Interestingly, the significance of this sphere formation ability still remains unclear. Recent studies have demonstrated that these sphere-forming cells acquired chemoresistance to cisplatin. In the present study, as observed by FACS analysis, we show that a sub-population of MIA PaCa-2 and PANC-1 pancreatic cancer cells possess cancer stem cell properties. RNAi-mediated suppression of uPA in these cells retarded their tumor forming ability in a nude mouse model. We also observed that uPA-suppressed MIA PaCa-2 and PANC-1 cells, after supplementation with uPA protein regained their cancer stem cell phenotype and showed increased expression of Lhx-2. To determine whether these sub-population cells possess any chemoresistance, we treated these cells with varying concentrations of gemcitabine (0-1000 nM) and observed that these cells were resistant to gemcitabine even at the 1000 nM concentration. Further suppression of uPA using siRNA in these cells sensitized these cells to gemcitabine. In addition, to determine whether uPA has any transcriptional role, we examined whether uPA interacted with any known transcription factors we used a TF-protein array. We observed that uPA interacted with Hoxa-5 very strongly. To further determine whether the interaction of uPA with Hoxa-5 invokes any regulatory response, we used a p53 promoter known to possess Hoxa-5 binding regions to drive a Luciferace gene, both with and without the presence of uPA. We observed that uPA retarded p53 promoter activity. Finally, nude mice implanted with MIA PaCa-2 cancer stem cells were susceptible to gemcitabine treatment after uPA downregulation. Taken together, these results indicate that targeting uPA can sensitize chemoresistant pancreatic cells to gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1294. doi:1538-7445.AM2012-1294

Genistein inhibits the stemness properties of prostate cancer cells through targeting Hedgehog–Gli1 pathway

Cancer stem cells (CSCs) are involved in tumorigenesis and progression of prostate cancer (PCa). Conventional anticancer therapeutics failed to eradicate CSCs, which may eventually lead to the disease relapse and metastasis. Therefore, targeting prostate CSCs may be an ideal strategy to cure PCa. Genistein is a major isoflavone constituent of soybeans and soy products, which has been shown to exhibit potent anticancer effect on many cancers. We have previously reported that genistein can inhibit PCa cell invasion by reversing epithelial to mesenchymal transition, suggesting that genistein may be effective against metastatic PCa. In addition, we have recently demonstrated that PCa tumorsphere cells (TCs) possess CSC properties. Here, we found that tumorsphere formation and colony formation of Pca cells were noticeably suppressed in the presence of genistein. Pretreatment of PCa TCs with genistein also suppressed tumorigenicity in vivo. Additionally, genistein treatment inhibited tumor growth of PCa TCs. Further studies showed that genistein treatment not only led to the down-regulation of PCa CSC markers CD44 in vitro and in vivo, but also inhibited Hedgehog–Gli1 pathway, which may contribute to the anti-CSC effect of genistein in PCa TCs. Therefore, our findings demonstrated that genistein may be a dietary phytochemical with potential to target prostate CSCs.

Human ovarian cancer stem-like cells can be efficiently killed by γδ T lymphocytes.

Ovarian cancer comprises a small population of cancer stem cells (CSCs) that are responsible for tumor maintenance and resistant to cancer therapies, it would be desirable to develop a therapy that could selectively target ovarian CSCs. Recently, cellular immune-based therapies have improved the prognosis of cancer patients clinically. In this study, we isolated a subset of ovarian cancer sphere cells that possess CSC properties and explored the cell cytotoxicity of γδ T cells to ovarian cancer sphere cells using a transwell cocultured cell system. The proliferation rate of the cancer sphere cells decreased to 40% after cocultured with γδ T cells. The γδ T cells increased the sensitivity of SK-OV-3 sphere cells to chemotherapeutic drugs. After the treatment of γδ T cells, the expression of stem cell marker genes decreased in sphere cells, while the expression of HLA-DR antigen on tumor cells was increased in a time-dependent manner. Further, γδ T cells induced G2/M phase cell cycle arrest and subsequent apoptosis in SK-OV-3 sphere cells. Xenograft mouse models demonstrated that γδ T cells dramatically reduced the tumor burden. Notably, the level of IL-17 production significantly increased after cocultured with γδ T cells. We conclude that γδ T cells may efficiently kill ovarian CSCs through IL-17 production and represent a promising immunotherapy for ovarian cancer.

Cancer stem cell traits in squamospheres derived from primary head and neck squamous cell carcinomas

A subpopulation of cancer stem cells (CSCs), but not the majority of non-tumorigenic cancer cells, in a variety of human malignancies plays a critical role in cancer cell proliferation, invasion, metastasis, and tumor recurrence post-therapies. We report the isolation of sphere-forming cells (squamospheres) from primary head and neck squamous cell carcinomas (HNSCCs), and characterization of their CSC properties. Squamospheres appeared within 2 weeks after seeding as single-dissociated cells obtained from primary HNSCC specimens in serum-free culture conditions. Real-time RT-PCR and immunocytochemistry assays revealed that a number of stem cell markers, including CK5, OCT4, SOX2, and nestin, were up-regulated in HNSCC-driven squamospheres. Fluorescence-activated cell sorting (FACS) analysis showed that squamospheres contain enriched side population cells compared to serum-induced differentiated squamosphere cells. Furthermore, HNSCC-driven squamospheres appeared to be chemoresistant to cisplatin, 5-fluorouracil (FU), paclitaxel and doxetaxel, and showed increased levels of ABCG2, one of the ATP-binding cassette (ABC) transporters. Of particular interest, in sharp contrast to subcutaneous injection of 1×10(6) differentiated squamosphere cells, as few as 100 squamosphere cells were able to give rise to tumors in nude mice. Altogether, we assert that primary HNSCC-driven squamospheres possess CSC properties, and its functional analysis may provide a novel tool for investigating the tumorigenic process of HNSCC.