The expression and significance of insulin-like growth factor-1 receptor and its pathway on breast cancer stem/progenitors

Introduction

Dysregulation of the insulin-like growth factor-1 receptor (IGF-1R)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway was shown to correlate with breast cancer disease progression. Cancer stem cells are a subpopulation within cancer cells that participate in tumor initiation, radio/chemoresistance and metastasis. In breast cancer, breast cancer stem cells (BCSCs) were identified as CD24^-CD44⁺ cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH⁺). Elucidation of the role of IGF-1R in BCSCs is crucial to the design of breast cancer therapies targeting BCSCs.

Methods

IGF-1R expression in BCSCs and noncancer stem cells sorted from xenografts of human primary breast cancers was examined by fluorescence-activated cell sorting (FACS), western blot analysis and immunoprecipitation. The role of IGF-1R in BCSCs was assessed by IGF-1R blockade with chemical inhibitor and gene silencing. Involvement of PI3K/Akt/mammalian target of rapamycin (mTOR) as the downstream pathway was studied by their phosphorylation status upon IGF-1R inhibition and the effects of chemical inhibitors of these signaling molecules on BCSCs. We also studied 16 clinical specimens of breast cancer for the expression of phosphor-Akt in the BCSCs by FACS.

Results

Expression of phosphorylated IGF-1R was greater in BCSCs than in non-BCSCs from xenografts of human breast cancer, which were supported by western blot and immunoprecipitation experiments. The sorted IGF-1R-expressing cells displayed features of cancer stem/progenitors such as mammosphere formation in vitro and tumorigenicity in vivo, both of which were suppressed by knockdown of IGF-1R. A specific inhibitor of the IGF-1R, picropodophyllin suppressed phospho-Akt^Ser473 and preferentially decreased ALDH⁺ BCSC populations of human breast cancer cells. Furthermore, picropodophyllin inhibited the capacity of CD24^-CD44⁺ BCSCs to undergo the epithelial-mesenchymal transition process with downregulation of mesenchymal markers. Inhibitors of signal molecules downstream of IGF-1R including PI3K/Akt/mTOR also reduced the ALDH⁺ population of breast cancer cells. Furthermore, the mTOR inhibitor, rapamycin, suppressed BCSCs in vitro and in vivo.

Conclusion

Our data support the notion that IGF-1R is a marker of stemness, and IGF-1R and its downstream PI3K/Akt/mTOR pathway are attractive targets for therapy directed against breast cancer stem/progenitors.     

Microarray-based analysis of microRNA expression in breast cancer stem cells

Background

This study aimed to determine the miRNA profile in breast cancer stem cells (BCSCs) and to explore the functions of characteristic BCSC miRNAs.

Methods

We isolated ESA⁺CD44⁺CD24^-/low BCSCs from MCF-7 cells using fluorescence-activated cell sorting (FACS). A human breast cancer xenograft assay was performed to validate the stem cell properties of the isolated cells, and microarray analysis was performed to screen for BCSC-related miRNAs. These BCSC-related miRNAs were selected for bioinformatic analysis and target prediction using online software programs.

Results

The ESA⁺CD44⁺CD24^-/low cells had up to 100- to 1000-fold greater tumor-initiating capability than the MCF-7 cells. Tumors initiated from the ESA⁺CD44⁺CD24^-/low cells were included of luminal epithelial and myoepithelial cells, indicating stem cell properties. We also obtained miRNA profiles of ESA⁺CD44⁺CD24^-/low BCSCs. Most of the possible targets of potential tumorigenesis-related miRNAs were oncogenes, anti-oncogenes or regulatory genes.

Conclusions

We identified a subset of miRNAs that were differentially expressed in BCSCs, providing a starting point to explore the functions of these miRNAs. Evaluating characteristic BCSC miRNAs represents a new method for studying breast cancer-initiating cells and developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation of cells in breast cancer.     

Can CD44+/CD24- Tumor Cells Be Used to Determine the Extent of Breast Cancer Invasion Following Neoadjuvant Chemotherapy?

Purpose

To investigate the distribution of CD44⁺/CD24^- cells in breast cancers in relation to tumor size before and after the administration of neoadjuvant chemotherapy.

Methods

CD44⁺/CD24^- tumor cells obtained from breast cancer specimens were characterized in vivo and in vitro using tumor formation assays and mammosphere generation assays, respectively. The distribution of CD44+/CD24- tumor cells in 78 breast cancer specimens following administration of neoadjuvant chemotherapy was also evaluated using immunofluorescence assays, and this distribution was compared with the extent of tumor invasion predicted by Response Evaluation Criteria in Solid Tumours (RECIST).

Results

In 27/78 cases, complete remission (CR) was identified using RECIST. However, 18 of these CR cases were associated with a scattered distribution of tumor stem cells in the outline of the original tumor prior to neoadjuvant chemotherapy. After neoadjuvant chemotherapy, 24 cases involved cancer cells that were confined to the tumor outline, and 21 cases had tumor cells or tumor stem cells overlapping the tumor outline. In addition, there were 6 patients who were insensitive to chemotherapy, and in these cases, both cancer cells and stem cells were detected outside the contours of the tumor volume imaged prior to chemotherapy.

Conclusion

CD44+/CD24- tumor cells may be an additional parameter to evaluate when determining the extent of breast cancer invasion.     

The Presence of EpCAM-/CD49f+ Cells in Breast Cancer Is Associated with a Poor Clinical Outcome

Purpose

It is well established that breast cancer stem cells (BCSCs) play an essential role in tumor invasion for both local and distant metastasis. The aim of this study was to establish whether BCSCs could act as a prognostic and clinical marker.

Methods

We analyzed tumor tissues from 161 breast cancer patients. Dual immunohistochemistry and immunofluorescence were performed on all the slides, and we analyzed the relationship between EpCAM^-/CD49f⁺ tumor cells and key clinical and prognostic factors.

Results

Univariate survival analysis using the Kaplan-Meier method showed that the presence of EpCAM^-/CD49f⁺ tumor cells in breast cancer was significantly associated with shorter disease-free survival (DFS) and overall survival (OS). Multivariate analysis showed that the presence of EpCAM^-/CD49f⁺ cells was associated with shorter DFS (p=0.010; hazard ratio [HR], 2.070) and OS (p=0.002; HR, 3.235). Tumors containing EpCAM^-/CD49f⁺ cells were also more likely to metastasize after initial surgery (p=0.048).

Conclusion

Our study suggests that breast tumors containing EpCAM^-/CD49f⁺ cells are more likely to undergo distant metastasis after initial surgery and are associated with a shorter DFS and OS.     

Tumor-Endothelial Interaction Links the CD44+/CD24- Phenotype with Poor Prognosis in Early-Stage Breast Cancer

Materials and Methods

The genomic effects of tumor-endothelial interactions in cancer are not yet well characterized. To study this interaction in breast cancer, we set up an ex vivo coculture model with human benign and malignant breast epithelial cells with endothelial cells to determine the associated gene expression changes using DNA microarrays.

Results

The most prominent response to coculture was the induction of the M-phase cell cycle genes in a subset of breast cancer cocultures that were absent in cocultures with normal breast epithelial cells. In monoculture, tumor cells that contained the stem cell-like CD44⁺/CD24^- signature had a lower expression of the M-phase cell cycle genes than the CD44^-/CD24⁺ cells, and in the CD44⁺/CD24^- cocultures, these genes were induced. Pretreatment gene expression profiles of early-stage breast cancers allowed evaluating in vitro effects in vivo. The expression of the gene set derived from the coculture provided a basis for the segregation of the tumors into two groups. In a univariate analysis, early-stage tumors with high expression levels (n = 137) of the M-phase cell cycle genes had a significantly lower metastasis-free survival rate (P = 1.8e - 5, 50% at 10 years) and overall survival rate (P = 5e - 9, 52% at 10 years) than tumors with low expression (n = 158; metastasis-free survival, 73%; overall survival, 84%).

Conclusions

Our results suggest that the interaction of endothelial cells with tumor cells that express the CD44⁺/CD24^- signature, which indicates a low proliferative potential, might explain the unexpected and paradoxical association of the CD44⁺/CD24^- signature with highly proliferative tumors that have an unfavorable prognosis.     

A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype

Introduction

Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro, they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties.

Methods

We describe a sub-population expressing the stem-like marker CD44⁺/CD24^-/low in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44⁺/CD24^low. Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments.

Results

Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo.

Conclusion

In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A.     

Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells

Introduction

Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors.

Methods

Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation.

Results

Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH⁺ cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH^- cells. GD2⁺ cells showed a 3.9-fold greater capacity than GD2^- cells. ALDH⁺/GD2⁺cells displayed 12.8-fold greater mammosphere forming ability than ALDH^-/GD2^- cells. In vivo, the tumor-initiating frequency of ALDH⁺/GD2⁺ cells were up to 33-fold higher than that of ALDH⁺ cells, with as few as 50 ALDH⁺/GD2⁺ cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH⁺/GD2⁺ cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH⁺ or ALDH⁺/GD2⁺ cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes.

Conclusions

Our findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH⁺ and ALDH⁺/GD2⁺ subpopulations.     

Cancer-associated fibroblasts induce epithelial–mesenchymal transition of breast cancer cells through paracrine TGF-β signalling

Background:

Cancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised.

Methods:

Stromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-β (TGF-β) signalling derived from CAF-CM, which effect on breast cancer cells.

Results:

The stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of α-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell–extracellular matrix adhesion, migration and invasion, and promoted epithelial–mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-β1 than TGF-β2 and TGF-β3, and activated the TGF-β/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-β1 signalling.

Conclusion:

Cancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-β1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.     

CD44+/CD24- phenotype contributes to malignant relapse following surgical resection and chemotherapy in patients with invasive ductal carcinoma

Background

Invasive ductal carcinoma is the most common type of breast malignancy, with varying molecular features and resistance to treatment. Although CD44+/CD24- cells are believed to act as breast cancer stem cells and to be linked to poor prognosis in some patients, the association between these cells and tumor recurrence or metastasis in all or some types of invasive ductal carcinoma is unclear.

Methods

A total of 147 randomly selected primary and secondary invasive ductal carcinoma samples were assayed for expression of CD44, CD24, ER, PR, and Her2. The association between the proportions of CD44+/CD24- tumor cells and the clinico-pathological features of these patients was evaluated.

Results

CD44+/CD24- tumor cells were detected in 70.1% of the tumors, with a median proportion of 5.8%. The proportion of CD44+/CD24- tumor cells was significantly associated with lymph node involvement (P = 0.026) and PR status (P = 0.038), and was correlated with strong PR status in patients with recurrent or metastatic tumors (P = 0.046) and with basal-like features (p = 0.05). The median disease-free survival (DFS) of patients with and without CD44⁺/CD24^-/low tumor cells were 22.9 ± 2.2 months and 35.9 ± 3.8 months, and the median overall survival (OS) of patients with and without CD44⁺/CD24^-/low tumor cells were 39.3 ± 2.6 months and 54.0 ± 3.5 months, respectively, and with both univariate and multivariate analyses showing that the proportion of CD44⁺/CD24^-/low tumor cells was strongly correlated with DFS and OS.

Conclusion

The prevalence of CD44+/CD24- tumor cells varied greatly in invasive ductal carcinomas, with the occurrence of this phenotype high in primary tumors with high PR status and in secondary tumors. Moreover, this phenotype was significantly associated with shorter cumulative DFS and OS. Thus, the CD44⁺/CD24^- phenotype may be an important factor for malignant relapse following surgical resection and chemotherapy in patients with invasive ductal carcinoma.     

CD24+ cells fuel rapid tumor growth and display high metastatic capacity

Introduction

Breast tumors are comprised of distinct cancer cell populations which differ in their tumorigenic and metastatic capacity. Characterization of cell surface markers enables investigators to distinguish between cancer stem cells and their counterparts. CD24 is a well-known cell surface marker for mammary epithelial cells isolation, recently it was suggested as a potential prognostic marker in a wide variety of malignancies. Here, we demonstrate that CD24⁺ cells create intra-tumor heterogeneity, and display highly metastatic properties.

Methods

The mammary carcinoma Mvt1 cells were sorted into CD24⁻ and CD24⁺ cells. Both subsets were morphologically and phenotypically characterized, and tumorigenic capacity was assessed via orthotopic inoculation of each subset into the mammary fat pad of wild-type and MKR mice. The metastatic capacity of each subset was determined with the tail vein metastasis assay. The role of CD24 in tumorigenesis was further examined with shRNA technology. GFP-labeled cells were monitored in vivo for differentiation. The genetic profile of each subset was analyzed using RNA sequencing.

Results

CD24⁺ cells displayed a more spindle-like cytoplasm. The cells formed mammospheres in high efficiency and CD24⁺ tumors displayed rapid growth in both WT and MKR mice, and were more metastatic than CD24- cells. Interestingly, CD24-KD in CD24+ cells had no effect both in vitro and in vivo on the various parameters studied. Moreover, CD24⁺ cells gave rise in vivo to the CD24⁻ that comprised the bulk of the tumor. RNA-seq analysis revealed enrichment of genes and pathways of the extracellular matrix in the CD24⁺ cells.

Conclusion

CD24⁺ cells account for heterogeneity in mammary tumors. CD24 expression at early stages of the cancer process is an indication of a highly invasive tumor. However, CD24 is not a suitable therapeutic target; instead we suggest here new potential targets accounting for early differentiated cancer cells tumorigenic capacity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13058-015-0589-9) contains supplementary material, which is available to authorized users.     

Silencing NOTCH signaling causes growth arrest in both breast cancer stem cells and breast cancer cells

Background:

Breast cancer stem cells (BCSCs) are characterized by high aldehyde dehydrogenase (ALDH) enzyme activity and are refractory to current treatment modalities, show a higher risk for metastasis, and influence the epithelial to mesenchymal transition (EMT), leading to a shorter time to recurrence and death. In this study, we focused on examination of the mechanism of action of a small herbal molecule, psoralidin (Pso) that has been shown to effectively suppress the growth of BSCSs and breast cancer cells (BCCs), in breast cancer (BC) models.

Methods:

ALDH⁻ and ALDH⁺ BCCs were isolated from MDA-MB-231 cells, and the anticancer effects of Pso were measured using cell viability, apoptosis, colony formation, invasion, migration, mammosphere formation, immunofluorescence, and western blot analysis.

Results:

Psoralidin significantly downregulated NOTCH1 signaling, and this downregulation resulted in growth inhibition and induction of apoptosis in both ALDH⁻ and ALDH⁺ cells. Molecularly, Pso inhibited NOTCH1 signaling, which facilitated inhibition of EMT markers (β-catenin and vimentin) and upregulated E-cadherin expression, resulting in reduced migration and invasion of both ALDH⁻ and ALDH⁺ cells.

Conclusion:

Together, our results suggest that inhibition of NOTCH1 by Pso resulted in growth arrest and inhibition of EMT in BCSCs and BCCs. Psoralidin appears to be a novel agent that targets both BCSCs and BCCs.     

Oestrogen increases the activity of oestrogen receptor negative breast cancer stem cells through paracrine EGFR and Notch signalling

Introduction

Although oestrogen is essential for the development of the normal breast, adult mammary stem cells are known to be oestrogen receptor alpha (ER) negative and rely on paracrine signals in the mammary epithelium for mediation of developmental cues. However, little is known about how systemic oestrogen regulates breast cancer stem cell (CSC) activity.

Methods

Here, we tested the effects of oestrogen on CSC activity in vitro and in vivo and investigated which paracrine signalling pathways locally mediate oestrogen effects.

Results

CSC-enriched populations (ESA⁺CD44⁺CD24^low) sorted from ER positive patient derived and established cell lines have low or absent ER expression. However, oestrogen stimulated CSC activity demonstrated by increased mammosphere and holoclone formation in vitro and tumour formation in vivo. This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA. These data suggest that the oestrogen response is mediated through paracrine signalling from non-CSCs to CSCs. We have, therefore, investigated both epidermal growth factor (EGF) and Notch receptor signals downstream of oestrogen. We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency.

Conclusions

These data establish that EGF and Notch receptor signalling pathways operate downstream of oestrogen in the regulation of ER negative CSCs.     

Pharmacological inhibition of AKT sensitizes MCF-7 human breast cancer-initiating cells to radiation

Background

Caner-initiating cells (CICs or cancer stem cells) have been shown both experimentally and clinically to be resistant to radiation. The mechanism underlying radioresistance remains unclear.

Methods

In the present study, we screened 51 genes which are potentially important in mediating radioresistance of breast CICs.

Results

The expression of AKT1 and AKT2 at protein and mRNA levels was dramatically increased among the screened genes by 8 Gy radiation treatment in MCF-7 mammosphere cells (predominantly CD24^–/low/CD44⁺ CICs), but not in the bulk population of MCF-7 cells (predominantly CD24⁺/CD44⁺). Using apoptosis and clonogenic survival assays, we found pharmacological inhibition of AKT with selective inhibitors of AKT sensitized MCF-7 mammosphere cells, but not MCF-7 monolayer cells to radiation.

Conclusion

The present findings suggest that treatment with AKT inhibitors prior to ionizing radiation treatment may be a potential benefit to patients with breast cancer, in particular to eradiate breast CICs.     

CD44 targets Na+/H+ exchanger 1 to mediate MDA-MB-231 cells' metastasis via the regulation of ERK1/2

Background:

CD44, a transmembrane glycoprotein expressed in a variety of cells and tissues, has been implicated in tumour metastasis. But the molecular mechanisms of CD44-mediated tumour cell metastasis remain to be elucidated.

Methods:

The downregulation of CD44 was determined by immunofluorescence. Moreover, the motility of breast cancer cells was detected by wound-healing and transwell experiments. Then the spontaneous metastasis of CD44-silenced MDA-MB-231 cells was tested by histology with BALB/c nude mice.

Results:

A positive correlation between CD44 and Na⁺/H⁺ exchanger isoform 1 (NHE1) was found in two breast cancer cells. CD44 downregulation could inhibit the metastasis of MDA-MB-231 cells and the expressions of Na⁺/H⁺ exchanger 1. Moreover, CD44 overexpression upregulated the metastasis of MCF-7 cells, but the elevated metastatic ability was then inhibited by Cariporide. Interestingly, during these processes only the p-ERK1/2 was suppressed by CD44 downregulation and the expression of matrix metalloproteinases and metastatic capacity of MDA-MB-231 cells were greatly inhibited by the MEK1 inhibitor PD98059, which even had a synergistic effect with Cariporide. Furthermore, CD44 downregulation inhibits breast tumour outgrowth and spontaneous lung metastasis.

Conclusions:

Taken together, this work indicates that CD44 regulates the metastasis of breast cancer cells through regulating NHE1 expression, which could be used as a novel strategy for breast cancer therapy.     

Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells

Introduction

Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44^high/CD24^-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.

Methods

A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44^high subpopulation and sorted CD44^high/CD24^-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44^high/CD24^-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.

Results

Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER⁺/HER2⁺, a metastatic variant), and HR5 (ER⁺/HER2⁺, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44^high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44^high/CD24^-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44^high/CD24^-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse.

Conclusion

Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44^high/CD24^-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.     

CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma

Background:

Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC).

Methods:

Circulating CD44⁺CD90⁺ cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro.

Results:

CD44⁺CD90⁺ cells in the blood acquired epithelial–mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44^high population showed higher anoikis resistance and sphere-forming ability than did the CD44^low population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44^high population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown.

Conclusions:

CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.     

The prognostic value of peripheral CD4+CD25+ T lymphocytes among early stage and triple negative breast cancer patients receiving dendritic cells-cytokine induced killer cells infusion

Objective

This study aimed to assess the prognostic value of CD4⁺CD25⁺ T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy.

Methods

217 patients participated in the follow-up study. CD4⁺CD25⁺ proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD4⁺CD25⁺ proportion more than 5% and less than or equal to 5% in peripheral T cells.

Results

Peripheral CD4⁺CD25⁺ T lymphocytes had not a relationship with progression-free survival. It was featured that above 5% peripheral CD4⁺CD25⁺ proportion of T cells was related with the median overall survival by a shorten of 51 months (p < 0.05) with the HR 1.65 (95%CI 1.04, 2.62). Above 5% CD4⁺CD25⁺proportion of T cells produced the HR to be 1.76 (95%CI 1.07, 2.87) In stage 0-II patients, and 3.59 (95%CI 1.05, 12.29) in triple negative breast cancer patients.

Conclusion

Cellular immunity restoration recovered by adoptive T cell infusions which resulted in less proportion of peripheral CD4⁺CD25⁺T lymphocytes could be a potential prognostic indicator among early stage and triple negative patients.     

TGM2 inhibition attenuates ID1 expression in CD44-high glioma-initiating cells

Background

CD44 is a molecular marker associated with cancer stem cell populations and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting glioma cells high in CD44.

Methods

Glioma-initiating cell lines were derived from fresh surgical glioblastoma samples. Expression of tissue transglutaminase 2 (TGM2) was attenuated through lentivirus-mediated short hairpin RNA knockdown. MTT assay [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used to evaluate the growth inhibition induced by TGM2 inhibitor. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling was used to evaluate cell apoptosis following TGM2 inhibition. CD44⁺ glioma stem cells were sorted by flow cytometry. A nude mice orthotopic xenograft model was used to evaluate the in vivo effect of TGM2 inhibitor.

Results

TGM2 was highly expressed in CD44-high glioblastoma tissues and tumor-derived glioma-initiating cell lines. TGM2 knockdown impaired cell proliferation and induced apoptosis in CD44-high glioma-initiating cell lines. Further studies indicated that expression of inhibitor of DNA binding 1 protein (ID1) is regulated by TGM2 and might be an important mediator for TGM2-regulated cell proliferation in CD44-high glioma-initiating cell lines. TGM2 inhibitor reduces ID1 expression, suppresses cell proliferation, and induces apoptosis in CD44-high glioma-initiating cell lines. Furthermore, TGM2 is highly expressed in CD44⁺ glioma stem cells, while pharmacological inhibition of TGM2 activity preferentially eliminates CD44⁺ glioma stem cells. Consistently, TGM2 inhibitor treatment reduced ID1 expression and induced apoptosis in our orthotopic mice xenograft model, which can be translated into prolonged median survival in tumor-bearing mice.

Conclusions

TGM2 regulates ID1 expression in glioma-initiating cell lines high in CD44. Targeting TGM2 could be an effective strategy to treat gliomas with high CD44 expression.     

Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics

Introduction

Whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response is not known.

Methods

We generated and characterized 16 cell lines from five distinct Brca1deficient mouse mammary tumors with respect to their cancer stem cell characteristics.

Results

All cell lines derived from one tumor included increased numbers of CD44⁺/CD24^- cells, which were previously identified as human breast cancer stem cells. All cell lines derived from another mammary tumor exhibited low levels of CD44⁺/CD24^- cells, but they harbored 2% to 5.9% CD133⁺ cells, which were previously associated with cancer stem cells in other human and murine tumors. When plated in the absence of attachment without presorting, only those cell lines that were enriched in either stem cell marker formed spheroids, which were further enriched in cells expressing the respective cancer stem cell marker. In contrast, cells sorted for CD44⁺/CD24^- or CD133⁺ markers lost their stem cell phenotype when cultured in monolayers. As few as 50 to 100 CD44⁺/CD24^- or CD133⁺ sorted cells rapidly formed tumors in nonobese diabetic/severe combined immunodeficient mice, whereas 50-fold to 100-fold higher numbers of parental or stem cell depleted cells were required to form few, slow-growing tumors. Expression of stem cell associated genes, including Oct4, Notch1, Aldh1, Fgfr1, and Sox1, was increased in CD44⁺/CD24^- and CD133⁺ cells. In addition, cells sorted for cancer stem cell markers and spheroid-forming cells were significantly more resistant to DNA-damaging drugs than were parental or stem cell depleted populations, and they were sensitized to the drugs by the heat shock protein-90 inhibitor 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride).

Conclusion

Brca1-deficient mouse mammary tumors harbor heterogeneous cancer stem cell populations, and CD44⁺/CD24^- cells represent a population that correlates with human breast cancer stem cells.