Cancer Stem Cell Activity Research Articles

Spheroid-Derived Cells From Renal Adenocarcinoma Have Low Telomerase Activity and High Stem-Like and Invasive Characteristics.

Cancer stem cells (CSCs) are a theorized small subpopulation of cells within tumors thought to be responsible for metastasis, tumor development, disease progression, treatment-resistance, and recurrence. The identification, isolation, and biological characterization of CSCs may therefore facilitate the development of efficient therapeutic strategies targeting CSCs. This study aims to compare the biology and telomerase activity of CSCs to parental cells (PCs) in renal cancer. Renal CSCs were enriched from the ACHN cell line using a sphere culture system. Spheroid-derived cells (SDCs) and their adherent counterparts were compared with respect to their colony and sphere formation, expression of putative CSC markers, tumorigenicity in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and invasiveness. The expression of genes associated with CSCs, stemness, EMT, apoptosis, and ABC transporters was also compared between the two populations using quantitative real-time PCR (qRT-PCR). Finally, telomerase activity, hTERT expression, and sensitivity to MST-312, a telomerase inhibitor, was investigated between the two populations. We demonstrated that a subpopulation of ACHN cells was capable of growing as spheroids with many properties similar to CSCs, including higher clonogenicity, superior colony- and sphere-forming ability, and stronger tumorigenicity and invasiveness. In addition, SDCs demonstrated a higher expression of markers for CSCs, stemness, EMT, apoptosis, and ABC transporter genes compared to PCs. The expression of hTERT and telomerase activity in SDCs was significantly lower than PCs; however, the SDC population was more sensitive to MST-312 compared to PCs. These findings indicate that the SDC population exhibits stem-like potential and invasive characteristics. Moreover, the reduced expression of hTERT and telomerase activity in SDCs demonstrated that the expressions of hTERT and telomerase activity are not always higher in CSCs. Our results also showed that MST-312 treatment inhibited SDCs more strongly than PCs and may therefore be useful as a complementary targeted therapy against renal CSCs in the future.

Capsaicin suppressed activity of prostate cancer stem cells by inhibition of Wnt/β-catenin pathway.

Cancer stem cells (CSCs) are considered as the origin and driving cells of cancer, and play a key role in the progress of cancer. Studies have shown that capsaicin exerted inhibitory effect on prostate cancer cells, however, the effects of capsaicin on prostate CSCs remain undefined. In the present study, we showed that capsaicin could downregulate prostate CSCs markers and inhibit the growth of PC-3 and DU145 prostate cancer stem cells. Further, we found capsaicin suppressed the expression of Wnt-2, p-GSK3β and β-catenin, along with downregulation of Wnt/β-catenin pathway target genes c-myc and cyclinD1. Using LiCl, a activator of Wnt/β-catenin pathway, we found activation of Wnt/β-catenin pathway could ameliorate the downregulation of prostate CSCs markers and the growth inhibition induced by capsaicin in prostate cancer stem cells. Those data suggested that the inhibition effect of capsaicin on prostate cancer stem cells and the anti-cancer effect of capsaicin on prostate cancer stem cell may be mediated by Wnt/β-catenin pathway. Findings from this study reveals for the first time the potential role and mechanisms of capsaicin on prostate cancer stem cells.

Comparative molecular and genetic particularities in human and animals mammary cancers

During normal mammary gland development, s-SHIP promoter expression marks a distinct type of mammary stem cells, at two different stages, puberty and early mid-pregnancy. To determine whether s-SHIP is a marker of mammary cancer stem cells (CSCs), we generated bitransgenic mice by crossing the C3(1)-SV40 T-antigen transgenic mouse model of breast cancer, and a transgenic mouse (11.5kb-GFP) expressing green fluorescent protein from the s-SHIP promoter. Here we show that in mammary tumors originating in these bitransgenic mice, s-SHIP promoter expression enriches a rare cell population with CSC activity as demonstrated by sphere-forming assays in vitro and limiting dilution transplantation in vivo. These s-SHIP-positive CSCs are characterized by lower expression of Delta-like non-canonical Notch ligand 1 (DLK1), a negative regulator of the Notch pathway. Inactivation of Dlk1 in s-SHIP-negative tumor cells increases their tumorigenic potential, suggesting a role for DLK1 in mammary cancer stemness.

HER2 Regulates Cancer Stem Cell Activities via the Wnt Signaling Pathway in Gastric Cancer Cells

Introduction: Human epidermal growth factor 2 (HER2) gene overexpression in breast carcinoma cell lines has been shown to drive mammary carcinogenesis and tumor growth and invasion through its effects on mammary stem cells. Objective: Therefore, we investigated the mechanism by which HER2 regulates cancer stem cell (CSC) activity in gastric cancer cells. Methods: HER2 was transfected into MKN28 gastric cancer cells, and its role in regulating CSC activity was determined by characterizing the HER2-overexpressing cells. Results: The sphere formation assay revealed that the sphere sizes and frequency of sphere formation were significantly greater for the HER2-overexpressing cells than for the MKN28 control cells. The CSC markers Oct-4 and BMI1 were more highly expressed in the HER2-overexpressing cells, as were the EMT markers. This was accompanied by a significant enhancement in cellular invasion of the Matrigel and migration. The E-cadherin level was significantly downregulated, and the mesenchymal marker Snail upregulated, in the HER2-transfected cells. HER2 overexpression activated the well-characterized CSC-associated Wnt/β-catenin signaling pathway, as shown by the luciferase assay. After treatment of these cells with the Wnt signal inhibitor PRI-724, the BMI1 and Oct-4 levels were decreased for 24 h and Snail was also downregulated. Immunofluorescence staining revealed the significant restoration of E-cadherin levels in the HER2-transfected cells after PRI-724 treatment. Conclusions: These results established a role for HER2 in regulating gastric CSC activity, with Wnt/β-catenin signaling being mediated via a HER2-dependent pathway. In summary, HER2-overexpressing gastric cancer cells exhibited increased stemness and invasiveness and were regulated by Wnt/β-catenin signaling.

P11.46 Discovery of a new HDAC6 inhibitor for the treatment of glioblastoma

Abstract BACKGROUND Glioblastoma’s origin and development is not only associated to genetic alterations, but also to epigenetic changes. Indeed, an altered expression or activity of epigenetic enzymes such as histone deacetylases (HDAC) has been associated to cancer stem cell activity, which has been widely described as a major feature for therapy resistance and tumor recurrence. In particular, inhibition of HDAC6 is an increasingly attractive pharmacological strategy, due to its association with low toxicity. Thus, the aim of the present study was to determine the impact of a new HDAC6-selective-inhibitor in glioblastoma and glioma stem cells. MATERIAL AND METHODS To test the effect of QTX compound in glioblastoma and glioma stem cell lines, cell viability after 72h of treatment was studied by MTT assay. After evaluation of IC50, QTX in vitro activity was analyzed, focusing on proliferation, apoptosis and stemness of U87-MG cell line and confirmed in a patient-derived glioma stem cell line. In vivo antitumor effect was evaluated using U87-MG cells xenografted in immunocompromised mice; after tumor formation, 5 mice were randomly selected as control group and another 5 for QTX treatment (intraperitoneal administration of 50 mg/kg; 5 days of dosing / 2 days off for 2 weeks). Mice weight was measured daily and tumor volume every two days. RESULTS We demonstrated that QTX reduces viability of all tested glioblastoma cells, even more greatly than normal astrocytes. Indeed, QTX diminishes proliferation and induces apoptosis in both conventional and patient-derived glioma cell lines. In particular, this effect was accompanied by a reduction of self-renewal properties of glioma stem cells. Interestingly, QTX in vitro activity was more effective comparing to the pan-inhibitor SAHA or the HDAC6-selective inhibitor Tubastatin A. Furthermore, QTX delayed tumor initiation and progression in vivo, without presenting significant side effects. CONCLUSION QTX compound presents a promising anti-tumor effect both in vitro and in vivo in glioblastoma, at least in part, inhibiting glioma stem cell activity.

The Milk Protein Alpha-Casein Suppresses Triple Negative Breast Cancer Stem Cell Activity Via STAT and HIF-1alpha Signalling Pathways in Breast Cancer Cells and Fibroblasts

Triple negative breast cancer (TNBC) is the most lethal breast cancer subtype. Extended periods of lactation protect against breast cancer development, but the mechanisms underlying this protection are unknown. We examined the effects of the milk protein alpha-casein over expression in the triple negative MDA-MB-231 breast cancer cell line. The effects of recombinant alpha-casein added exogenously to MDA-MB-231 breast cancer cells, and immortalised human fibroblasts were also investigated. We used transcriptional reporters to understand the signalling pathways downstream of alpha-casein in breast cancer cells and these fibroblasts that were activated by breast cancer cells. To extend our findings to the clinical setting, we analysed public gene expression datasets to further understand the relevance of these signalling pathways in triple negative breast cancer cells and patient samples. Finally, we used small molecular inhibitors to target relevant pathways and highlight these as potential candidates for the treatment of TN breast cancer. High levels of alpha-casein gene expression were predictive of good prognosis across 263 TNBC patient tumour samples. Alpha-casein over expression or exogenous addition reduces cancer stem cell (CSC) activity. HIF-1alpha was identified to be a key downstream target of alpha-casein, in both breast cancer cells and activated fibroblasts, and STAT transcription factors to be upstream of HIF-1alpha. Interestingly, HIF-1alpha is regulated by STAT3 in breast cancer cells, but STAT1 is the regulator of HIF-1alpha in activated fibroblasts. In analysis of 573 TNBC patient samples, alpha-casein expression, inversely correlated to HIF-1alpha, STAT3 and STAT1. STAT1 and STAT3 inhibitors target HIF-1alpha signalling in activated fibroblasts and MDA-MB-231 breast cancer cells respectively, and also abrogate CSC activities. Our findings provide an explanation for the protective effects of lactation in TNBC. Clinical data correlates high alpha-casein expression with increased recurrence-free survival in TNBC patients. Mechanistically, alpha-casein reduces breast cancer stem cell activity in vitro, and STAT3 and STAT1 were identified as regulators of pro-tumorigenic HIF-1alpha signalling in breast cancer cells and fibroblasts respectively.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

Role of the Microenvironment in Regulating Normal and Cancer Stem Cell Activity: Implications for Breast Cancer Progression and Therapy Response.

The epithelial cells in an adult woman’s breast tissue are continuously replaced throughout their reproductive life during pregnancy and estrus cycles. Such extensive epithelial cell turnover is governed by the primitive mammary stem cells (MaSCs) that proliferate and differentiate into bipotential and lineage-restricted progenitors that ultimately generate the mature breast epithelial cells. These cellular processes are orchestrated by tightly-regulated paracrine signals and crosstalk between breast epithelial cells and their tissue microenvironment. However, current evidence suggests that alterations to the communication between MaSCs, epithelial progenitors and their microenvironment plays an important role in breast carcinogenesis. In this article, we review the current knowledge regarding the role of the breast tissue microenvironment in regulating the special functions of normal and cancer stem cells. Understanding the crosstalk between MaSCs and their microenvironment will provide new insights into how an altered breast tissue microenvironment could contribute to breast cancer development, progression and therapy response and the implications of this for the development of novel therapeutic strategies to target cancer stem cells.

4-Methylumbelliferone Inhibits Cancer Stem Cell Activation and Overcomes Chemoresistance in Ovarian Cancer.

We have recently shown that the extracellular matrix molecule hyaluronan (HA) plays a role in the development of ovarian cancer chemoresistance. This present study determined if HA production is increased in chemotherapy-resistant ovarian cancers and if the HA inhibitor 4-methylubelliferone (4-MU) can overcome chemoresistance to the chemotherapeutic drug carboplatin (CBP) and inhibit spheroid formation and the expression of cancer stem cell (CSC) markers. We additionally assessed whether 4-MU could inhibit in vivo invasion of chemoresistant primary ovarian cancer cells in the chicken embryo chorioallantoic membrane (CAM) assay. The expression of the HA synthases HAS2 and HAS3 was significantly increased in chemoresistant compared to chemosensitive primary ovarian cancer cells isolated from patient ascites. 4-MU significantly inhibited HA production, cell survival, and spheroid formation of chemoresistant serous ovarian cancer cells. In combination with CBP, 4-MU treatment significantly decreased ovarian cancer cell survival and increased apoptosis of chemoresistant primary cells compared to CBP alone. 4-MU significantly reduced spheroid formation, expression of CSC markers ALDH1A1 and ABCG2 in primary cell spheroid cultures, and ALDH1 immunostaining in patient-derived tissue explant assays following treatment with CBP. Furthermore, 4-MU was very effective at inhibiting in vivo invasion of chemoresistant primary cells in CAM assays. Inhibition of HA is therefore a promising new strategy to overcome chemoresistance and to improve ovarian cancer survival.

WDR76 degrades RAS and suppresses cancer stem cell activation in colorectal cancer

BackgroundStabilization of RAS is a key event for the hyper-activation of Wnt/β-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). WD Repeat protein 76 (WDR76) mediates the polyubiquitination-dependent degradation of RAS in hepatocellular carcinoma (HCC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC.MethodsWe generated mice with deletion of Wdr76 (Wdr76−/−) and crosses of Wdr76−/− with ApcMin/+ (Wdr76−/−; ApcMin/+) and compared them with wildtype mice (Wdr76+/+) and ApcMin/+ mice (Wdr76+/+; ApcMin/+), respectively. Intestinal crypt lengthening, tumorigenesis and CSC activation were analyzed by histology, immunohistochemistry, and immunoblotting. CRC cell line was engineered to stably express or knockdown WDR76 or control vector and was analyzed after spheroid culture.ResultsWdr76−/− mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76−/−; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and β-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. In human CRC, lower level of WDR76 was associated with poor patient survival.ConclusionsIn analyses of mice with deletion of Wdr76 and CRC spheroids, we found that RAS stability plays important roles in tumorigenesis by affecting proliferation and CSC activation. Our results suggest that destabilization of RAS by WDR76 is a potential strategy for targeting malignant CRC involving CSC activation.Graphic abstract

Sonic hedgehog pathway mediates genistein inhibition of renal cancer stem cells

Cancer stem cells (CSCs) have been implicated in the genesis, progression and recurrence of renal cancer. The sonic hedgehog (Shh) pathway serves a critical role in maintaining the stemness of CSCs. Genistein, a major isoflavone component extracted from soybeans and soy products, has been demonstrated to possess anticancer activity. However, the effects of genistein on renal CSCs and its underlying mechanisms remain to be fully elucidated. The aim of the present study was to investigate the role of the Shh pathway in genistein inhibition of renal CSCs. The results of the present study demonstrated that expression levels of renal CSC markers were markedly upregulated in the sphere-forming cells, which were isolated and enriched from 786-O and ACHN cells in a tumor sphere formation assay, and more cells were arrested at the G0/G1 phase instead of the S1 phase compared with the adherent cells. Furthermore, the present study demonstrated that genistein could effectively diminish the activity of renal CSCs by suppressing tumor sphere formation, decreasing renal CSCs markers, inhibiting proliferation and inducing apoptosis. Additionally, the downregulation of Shh pathway activity could inhibit renal CSCs. Genistein exhibited an inhibitory effect on renal CSCs by attenuating the activation of the Shh pathway. In conclusion, the results illustrated the role of the Shh pathway in regulating renal CSC traits and the intervention of renal CSCs by genistein, which could provide novel insights into the molecular mechanisms of renal CSC intervention.

Integrin α6 (CD49f), The Microenvironment and Cancer Stem Cells.

Cancer is a highly prevalent and potentially terminal disease that affects millions of individuals worldwide. Here, we review the literature exploring the intricacies of stem cells bearing tumorigenic characteristics and collect evidence demonstrating the importance of integrin α6 (ITGA6, also known as CD49f) in cancer stem cell (CSC) activity. ITGA6 is commonly used to identify CSC populations in various tissues and plays an important role sustaining the self-renewal of CSCs by interconnecting them with the tumorigenic microenvironment.

Abstract 2820: Macrophages recruited to and function to promote activation of cancer stem cells in response to chemotherapy

Abstract Failure of chemoradiotherapy for treating colorectal cancer (CRC) is often caused by the relapse of residual tumor cells that enrich cancer stem cells (CSCs). However, how CSCs are regulated by tumor microenvironment (TME), particularly myeloid derived cells (MDCs), remains unclear. Here, we used the APCMin/+ adenoma model to study the dynamic interaction between MDCs and CSCs in vivo following chemoradiotherapy. Using 3D scanning electronic microscopy, we observed that while proliferating tumor cells were dying following chemoradiotherapy, MDCs or its derivative tumor associated macrophages (TAMs) were recruited to the site where CSCs paired with necroptotic cells. TAMs functioned to promote activation of slow-cycling CSCs. Dissected with single cell RNA-seq, we observed that while proliferating tumor cells were declining following chemotherapy, an increase in MDCs was accompanied with a reduction of immune cells as well as an activation and short-term expansion of initially quiescent CSCs. Depletion of TAMs using a drugs or genetic means resulting in reduced CSCs and attenuated tumorigenesis. Mechanistically, we uncovered that TAM with secreted PGE2 activated Akt and Wnt signaling to promote activation of CSCs. Hence, targeting TAMs will benefit clinical treatment of CRC by reducing drug-resistant CSCs. Note: This abstract was not presented at the meeting. Citation Format: Xi C. He, Linheng Li. Macrophages recruited to and function to promote activation of cancer stem cells in response to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2820.

Abstract 3673: The BMI1/Sp1/IGF1R pathway mediates pemetrexed resistance in non-small cell lung cancer cells

Abstract Background: Lung cancer is the leading cause of cancer death in Taiwan and worldwide and non-small cell lung cancers (NSCLCs) account for more than 85% of lung cancers. Pemetrexed is approved for several steps of nonsquamous NSCLC therapy but drug resistance often occurs in long-term treatment. Cancer stem cells (CSCs) are considered as the rare subpopulation of cancer cells with the features of tumor initiation, drug resistance, and metastatic capability. The signaling pathway and potential targets contribute to the maintenance of pemetrexed-resistant cancer stem cell remain unknown. Methods: To investigate the mechanism of pemetrexed resistance in lung cancers, the pemetrexed-resistant A549 cells were established (A400). The sphere-formation assay, western blot analysis and real-time qPCR were employed to identify cancer stem cells and study their properties. Result: We first discovered that the pemetrexed-resistant A549 cells (called A400) contained more CSC activity than the parental A549 cells including the increased number of tumorspheres and the upregulation of BMI1, c-Myc, Sox2 and p-IGF1Rtyr1165/1166 expression. The treatment of PTC-209, a BMI1 inhibitor, displayed a greater inhibitory effect in cell proliferation in A400 than A549 cells and it suppressed tumorsphere formation capability of A400 cells. In addition, PTC-209 treatment downregulated the expression of IGF1R, p-IGF1Rtyr1165/1166, and Sp1, a positive regulatory transcriptional factor in IGF1R expression, in A400 cells. We also found that Sp1 was increased in A400 when compared to parental A549 cells. With the treatment of Sp1 inhibitor in A400 cells, the expression of IGF1R was suppressed without changing BMI1 level. Finally, we examined the therapeutic effect of BMI1 inhibition in vivo and resulted found that intraperitoneal administration of PTC-209 enhanced the pemetrexed efficacy in NOD/SCID mice harboring A400 xenograft tumors. Conclusion: Our data reveal that BMI1/Sp1/IGF1R pathway is upregulated in pemetrexed resistant NSCLCs and targeting BMI1 could be a therapeutic strategy in NSCLCs when pemetrexed resistance occurs. Citation Format: Wen-Ling Wang, Peng-Ju Chien, Wen-Wei Chang, Bing-Yen Wang. The BMI1/Sp1/IGF1R pathway mediates pemetrexed resistance in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3673.

Abstract 5250: Notch signaling in ER+ breast cancer in response to endocrine therapy agents

Abstract Breast cancer is the most frequent cancer in women in the world, and more than 70% of breast cancer cases are estrogen receptor α positive (ER+). Standard treatment for ER+ breast cancers is endocrine therapy that consists of selective estrogen receptor modulators (SERMs) such as tamoxifen in pre-menopausal patients; aromatase inhibitors (AIs); or selective estrogen receptor down-regulators (SERDs); fulvestrant, and treatment is dependent on the menopausal state of the patient. Many luminal tumors acquire resistance to therapy and recur, leading to disease progression and mortality. Resistance may result from the activation of quiescent signaling pathways that drive cancer stem cell (CSC) activity, such as the Notch signaling pathway. Previous studies demonstrate that activation of Notch via the NOTCH1 receptor recruits ERα in the absence of estrogen to facilitate transcription of estrogen responsive genes. However, NOTCH4 is often upregulated in resistant ER+ breast cancers, driving endocrine therapy resistance via breast CSC activity. We hypothesize that NOTCH4 promotes the transcription of estrogen-responsive genes in the absence of estrogen in a selective manner that is unlike NOTCH1. Understanding how NOTCH4 regulates the transcription of estrogen responsive genes upon treatment with endocrine therapy may suggest the need for combination therapy for ER+ breast cancer that includes a Notch targeted therapy. We have measured mRNA expression of classic estrogen responsive genes in ER+ breast cancer cell lines that either overexpress NOTCH1 or NOTCH4. Our data demonstrate differential expression of estrogen-responsive genes between NOTCH1 and NOTCH4 in the presence of tamoxifen. We have also measured Notch contribution to breast cancer stem cell activity through mammosphere formation assays using ER+ breast cancer cell lines in the presence of fulvestrant and Notch signaling inhibitors. The expression of classical estrogen responsive genes from mammospheres was altered in the presence of fulvestrant, Notch signaling inhibitors, or combinations of the two treatments. Further, combination treatment led to the disruption of mammosphere formation suggesting that combination therapy in the clinic may be suitable for the prevention of endocrine resistance by inhibition of BCSC activity through NOTCH4. Citation Format: Ciera S. Singleton, Lucio Miele, Judy S. Crabtree. Notch signaling in ER+ breast cancer in response to endocrine therapy agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5250.

Abstract 4672: WDR26 regulates an AKT-Gsk3-Wnt/b-catenin signaling cascade to maintain the breast cancer stem cell population and controls cancer metastasis

Abstract Cancer metastasis is the major cause of tumor mortality and has been attributed in part to the presence of a minority subpopulation of cancer stem cells (CSCs) in the bulk of tumor cells. We showed previously that WDR26, a scaffolding/adaptor protein that is highly upregulated in breast cancer, promotes breast cancer growth and metastasis. Here we show WDR26 was required for maintaining the CSC populations in breast cancer cells and the formation of lung metastases. Downregulation of WDR26 in breast cancer cells impaired the CSC-like activities and reduced the CSC population. Mammary gland-specific deletion of WDR26 in the MMTV-PyMT mouse model of breast cancer had a little effect on primary tumor formation but largely abolished spontaneous lung metastasis. WDR26 promoted β-catenin activation via AKT and GSK3, and the activity of AKT, GSK3 and β-catenin was required for maintaining the CSC population in breast cancer cells. Our results have identified a novel, WDR26-dependent pathway that links breast CSC activities to tumor metastatic potential. Citation Format: Dharmendra K. Bhargava, Wei Wang, Maddison Lensing, Songhai Chen. WDR26 regulates an AKT-Gsk3-Wnt/b-catenin signaling cascade to maintain the breast cancer stem cell population and controls cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4672.

Bone microenvironment signaling of cancer stem cells as a therapeutic target in metastatic prostate cancer.

Prostate cancer (PCa) is one of the most prevalent cancers and the second leading cause of cancer death among US males. When diagnosed in an early disease stage, primary tumors of PCa may be treated with surgical resection or radiation, sometimes combined with androgen deprivation therapy, with favorable outcomes. Unfortunately, the treatment efficacy of each approach decreases significantly in later stages of PCa that involve metastasis to soft tissues and bone. Metastatic PCa is a heterogeneous disease containing host cells, mature cancer cells, and subpopulation of cancer stem cells (CSC). CSCs are highly tumorigenic due to their self-renewing and differentiating potential, clinically resulting in recurrence and resistance to standard therapies. Therefore, there is a large unmet clinical need to develop therapies, which target CSC activity. In this review, we summarize the main signaling pathways that are implicated in the current pre-clinical and clinical studies of recurrent metastatic PCa within the bone microenvironment targeting CSCs and discuss the trajectory of therapeutics moving forward.

ALDH1A3-regulated long non-coding RNA NRAD1 is a potential novel target for triple-negative breast tumors and cancer stem cells

To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1 A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.

S-SHIP Promoter Expression Identifies Mouse Mammary Cancer Stem Cells.

SummaryDuring normal mammary gland development, s-SHIP promoter expression marks a distinct type of mammary stem cells, at two different stages, puberty and early mid-pregnancy. To determine whether s-SHIP is a marker of mammary cancer stem cells (CSCs), we generated bitransgenic mice by crossing the C3(1)-SV40 T-antigen transgenic mouse model of breast cancer, and a transgenic mouse (11.5kb-GFP) expressing green fluorescent protein from the s-SHIP promoter. Here we show that in mammary tumors originating in these bitransgenic mice, s-SHIP promoter expression enriches a rare cell population with CSC activity as demonstrated by sphere-forming assays in vitro and limiting dilution transplantation in vivo. These s-SHIP-positive CSCs are characterized by lower expression of Delta-like non-canonical Notch ligand 1 (DLK1), a negative regulator of the Notch pathway. Inactivation of Dlk1 in s-SHIP-negative tumor cells increases their tumorigenic potential, suggesting a role for DLK1 in mammary cancer stemness.

PRP‑1 significantly decreases the ALDHhigh cancer stem cell population and regulates the aberrant Wnt/β‑catenin pathway in human chondrosarcoma JJ012cells.

Chondrosarcomas are malignant bone tumors refractory to chemotherapy and radiation treatment; thus, novel therapeutic strategies are required. Proline-rich polypeptide 1 (PRP-1) has previously demonstrated antitumor properties in chondrosarcoma. To further investigate the role of PRP-1 in chondrosarcoma cells, its effects on cancer stem cell (CSC) populations were determined by analyzing aldehyde dehydrogenase (ALDH) activity, an established marker of CSCs, in association with regulation of the Wnt/β-catenin signaling. A significant decrease in ALDHhigh CSCs was observed following treatment of chondrosarcoma JJ012 cells with PRP-1. For RT2 profiler PCR array analysis of Wnt/β-catenin signaling genes, cells were sorted into: i) Bulk JJ012 cells; ii) ALDHhigh cells sorted from untreated JJ012 cells (ALDHhigh-untreated); and iii) ALDHlow cells sorted from PRP-1-treated JJ012 cells (ALDHlow-PRP-1). The expression levels of Wnt/β-catenin signaling genes were determined to be downregulated in the ALDHhigh-untreated cells and upregulated in ALDHlow-PRP-1 cells when compared to the bulk JJ012 cells. Additionally, two important oncogenes involved in this pathway, MMP7 and CCND2, were found to be downregulated in the ALDHlow-PRP-1 cells. Immunocytochemistry demonstrated the localization of β-catenin in the nuclei of the PRP-1-treated cells. Western blotting indicated increased β-catenin expression in the ALDHlow-PRP-1 cells compared with the bulk JJ012 cells. Analysis of the cytoplasmic and nuclear fractions of cells treated with increasing concentrations of PRP-1 and β-catenin nuclear translocation inhibitor CGP57380, suggested the nuclear translocation of β-catenin following PRP-1 treatment. In addition, treatment of JJ012 cells with a specific ALDH inhibitor, diethylaminobenzaldehyde, and PRP-1 resulted in a significant decrease in cytoplasmic β-catenin protein expression. This indicated that ALDH inactivation may be associated with the nuclear translocation of β-catenin. Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway has been previously documented. The findings of the present study support the notion that Wnt/β-catenin activation may serve a differential role in sarcomas, limiting tumor progression in association with decreased CSC activity.