Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy

Introduction

The phenotypic and functional differences between cells that initiate human breast tumors (cancer stem cells) and those that comprise the tumor bulk are difficult to study using only primary tumor tissue. We embarked on this study hypothesizing that breast cancer cell lines would contain analogous hierarchical differentiation programs to those found in primary breast tumors.

Methods

Eight human breast cell lines (human mammary epithelial cells, and MCF10A, MCF7, SUM149, SUM159, SUM1315 and MDA.MB.231 cells) were analyzed using flow cytometry for CD44, CD24, and epithelial-specific antigen (ESA) expression. Limiting dilution orthotopic injections were used to evaluate tumor initiation, while serial colony-forming unit, reconstitution and tumorsphere assays were performed to assess self-renewal and differentiation. Pulse-chase bromodeoxyuridine (5-bromo-2-deoxyuridine [BrdU]) labeling was used to examine cell cycle and label-retention of cancer stem cells. Cells were treated with paclitaxol and 5-fluorouracil to test selective resistance to chemotherapy, and gene expression profile after chemotherapy were examined.

Results

The percentage of CD44⁺/CD24^- cells within cell lines does not correlate with tumorigenicity, but as few as 100 cells can form tumors when sorted for CD44⁺/CD24^-/low/ESA⁺. Furthermore, CD44⁺/CD24^-/ESA⁺ cells can self-renew, reconstitute the parental cell line, retain BrdU label, and preferentially survive chemotherapy.

Conclusion

These data validate the use of cancer cell lines as models for the development and testing of novel therapeutics aimed at eradicating cancer stem cells.     

Prolyl isomerase Pin1 is highly expressed in Her2-positive breast cancer and regulates erbB2 protein stability

Background

Development of therapies for patients with BRCA1 mutations has been hampered by lack of readily available in vitro and in vivo models. We recently showed that transplantation of transgenic mammary tumors as cell suspensions into naïve recipients generates reproducible tumors with remarkable stability of gene expression profile. We examined the expression profiles of original and serially transplanted mammary tumors from Brca1 deficient mice, and tumor derived cell lines to validate their use for preclinical testing and studies of tumor biology.

Methods

Original tumors, serially transplanted and multiple cell lines derived from Brca1 mammary tumors were characterized by morphology, gene and protein expression, and cell surface markers.

Results

Gene expression among Brca1 tumors showed more heterogeneity than among previously characterized tumors from MMTV-PyMT and -Wnt1 models. Gene expression data segregated Brca1 tumors into 3 distinct types: basal, mixed luminal, and tumors with epithelial-to-mesenchymal transition (EMT). Serial transplantation of individual tumors and multiple cell lines derived from the original tumors recapitulated the molecular characteristics of each tumor of origin. One tumor had distinct features of EMT and gave rise to cell lines that contained a distinct CD44⁺/CD24^-/low population that may correlate with human breast cancer stem cells.

Conclusion

Although individual tumors expanded by transplantation maintain the genomic profile of the original tumors, the heterogeneity among Brca1 tumors limits the extent of their use for preclinical testing. However, cell lines offer a robust material for understanding tumor biology and response to therapies driven by BRCA1 deficiency.     

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.     

CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells

Introduction

Breast cancer is thought to arise in mammary epithelial stem cells. There is, therefore, a large amount of interest in identifying these cells. The breast is a complex tissue consisting of two epithelial layers (an outer myoepithelial/basal layer and an inner luminal epithelial layer) as well as a large non-epithelial component (fibroblasts, endothelial cells, lymphocytes, adipocytes, neurons and myocytes). The definitive identification of a mammary epithelial stem cell population is critically dependent on its purity. To date, this has been hampered by the lack of suitable markers to separate out the two epithelial layers, and to remove contaminating non-epithelial cells.

Methods

Mouse mammary glands were dissociated and stained with CD24. Cells were sorted into separate populations based on CD24 expression and assessed for luminal epithelial and myoepithelial/basal markers by direct fluorescent microscopy and real time PCR. The stem/progenitor potential of these cell populations was assessed in vivo by cleared mammary fat pad transplantation.

Results

Three populations of CD24 expressing cells were identified: CD24^Negative, CD24^Low and CD24^High. Staining of these cells with cytokeratin markers revealed that these populations correspond to non-epithelial, myoepithelial/basal and luminal epithelial cells, respectively. Cell identities were confirmed by quantitative PCR. Cleared mammary fat pad transplantation of these cell populations revealed that extensive mammary fat pad repopulation capacity segregates with the CD24^Low cells, whilst CD24^High cells have limited repopulation capacity.

Conclusion

Differential staining of mammary epithelial cells for CD24 can be used to simultaneously isolate pure populations of non-epithelial, myoepithelial/basal and luminal epithelial cells. Furthermore, mammary fat pad repopulation capacity is enriched in the CD24^Low population. As separation is achieved using a single marker, it will be possible to incorporate additional markers to further subdivide these populations. This will considerably facilitate the further analysis of mammary epithelial subpopulations, whilst ensuring high purity, which is key for understanding mammary epithelial stem cells in normal tissue biology and carcinogenesis.     

CD44+/CD24− breast cancer cells isolated from MCF-7 cultures exhibit enhanced angiogenic properties

Background

Recent studies suggest that the relationship between cancer stem cells (CSCs) and the vascular niche may be bidirectional; the niche can support the growth and renewal of CSCs, and CSCs may contribute to the maintenance of the niche. There is little knowledge concerning the role of breast cancer stem cells in promoting tumor angiogenesis.

Aim

For human breast cancers, CSCs have been shown to be associated with a CD44⁺/CD24 ^? phenotype. We investigated the potential activities of CD44⁺/CD24 ^? breast cancer stem cells in promoting tumor angiogenesis.

Methods

The expression of pro-angiogenic genes was determined by quantitative real-time RT-PCR. Endothelial cell migration assays were employed to evaluate effects of conditioned media from CD44⁺/CD24 ^? on human umbilical vein endothelial cells. A chorioallantoic membrane (CAM) assay was used to study the potential of CD44⁺/CD24 ^? cells to promote angiogenesis.

Results

In our study, CD44⁺/CD24 ^? cells expressed elevated levels of pro-angiogenic factors compared with CD44⁺/CD24⁺ cells. CD44⁺/CD24 ^? cell-conditioned media significantly increased endothelial cell migration. Breast cancer cell lines enriched with CD44⁺/CD24 ^? cells were more pro-angiogenic in the CAM assay than those lacking a CD44⁺/CD24 ^? subpopulation. CD44⁺/CD24 ^? cells sorted from MCF-7 cell lines were more pro-angiogenic in a CAM assay than CD44⁺/CD24⁺ cells. Furthermore, the VEGF concentration was significantly higher in CD44⁺/CD24 ^? cell-conditioned media than in CD44⁺/CD24⁺ cell-conditioned media. The pro-angiogenic effect of CD44⁺/CD24 ^? cells on endothelial cells was abolished by bevacizumab.

Conclusion

Our findings demonstrate that CD44⁺/CD24 ^? breast cancer stem cells have substantial pro-angiogenic potential and activity. This provides new insights to explore in the development of targeted therapies.     

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.     

Characterization of the conversion between CD133+ and CD133- cells in colon cancer SW620 cell line

The state of cancer stem cells (CSC) under reversible fluctuations, which has been revealed in breast cancer cells most recently, suggests that subpopulations with distinct phenotypes and functions within cancer cells can undergo inter-conversion. To investigate the possibility in colon cancer cells, we employed CD133 as the CSC marker, and characterized CD133 expression pattern and the biological features of the CD133⁺ and CD133^- subsets. Flow cytometry revealed that CD133 was bimodally expressed in SW620 cells among eight colon cancer cell lines. The CD133⁺ clonal SW620 cells displayed a differential gene expression profile, higher cellular reactive oxygen species (ROS), enhanced tumorigenesis and resistance to 5-fluorouracil. The conversion in term of the CD133 phenotype of the sorted cells was observed in vitro and in vivo. The fraction of the CD133⁺ cells decreased from 99% to 80% in the sorted CD133⁺ population while rising from 5 to 10% in the sorted CD133^- population during the first 20-day cultivation and then stayed almost unchanged. A fraction (about 20%) of the CD133⁺ clonal cells lost their CD133 marker while about 10% of the CD133^- clonal cells acquired the CD133 marker. 5-Azacytidine enhanced the fraction of the CD133⁺ cells in both of the CD133⁺ and CD133^- clonal cells. Our data demonstrate that CD133 expression is dynamic and reversible, and reveal the inter-conversion between the CD133⁺ and the CD133^- SW620 cells, suggesting that the CD133 phenotype of SW620 cell population is retained by the conversion between the two cell subsets.     

CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell lines HCT-116

Background and purpose

CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133^+/− subpopulations of one cell line of interest.

Materials and methods

Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133⁺ and CD133⁻ HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice.

Results and conclusions

CRC cell lines could be classified into three groups: (i) CD133⁻, (ii) CD133⁺ and (iii) those with two distinct CD133⁺ and CD133⁻ subpopulations. Isolated CD133^+/− HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133⁻ HCT-116 showed a striking enrichment in the CD133⁺ fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell lines. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.     

CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell line HCT-116

Background and purpose

CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133^+/− subpopulations of one cell line of interest.

Materials and methods

Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133⁺ and CD133⁻ HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice.

Results and conclusions

CRC cell lines could be classified into three groups: (i) CD133⁻, (ii) CD133⁺ and (iii) those with two distinct CD133⁺ and CD133⁻ subpopulations. Isolated CD133^+/− HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133⁻ HCT-116 showed a striking enrichment in the CD133⁺ fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell line. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.     

ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas

A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDH^highCD44^high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDH^lowCD44^low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.     

Cancer stem cell markers are enriched in normal tissue adjacent to triple negative breast cancer and inversely correlated with DNA repair deficiency

Introduction

We hypothesized that cells present in normal tissue that bear cancer stem cell markers may represent a cancer cell of origin or a microenvironment primed for tumor development, and that their presence may correlate with the clinically defined subtypes of breast cancer that show increased tumorigenicity and stem cell features.

Methods

Normal tissues sampled at least 5 cm from primary tumors (normal adjacent tissue) were obtained from 61 chemotherapy-naive patients with breast cancer treated with mastectomy. Samples were stained simultaneously with immunofluorescence for CD44/CD49f/CD133/2 stem cell markers. We assessed the association between CD44⁺CD49f⁺CD133/2⁺ staining in normal adjacent tissue and breast cancer receptor subtype (defined by the expression of the estrogen (ER), progesterone (PR), or human epidermal growth factor-2 (Her2) receptors). We also examined the correlation between CD44⁺CD49f⁺CD133/2⁺ immunofluorescence and each of two previously published gene signatures, one derived from stem-cell enriched tissue and one from BRCA mutated tissue expected to have defective DNA repair.

Results

Patients with triple negative breast cancer (ER^–/PR^–/HER2^–) expressed CD44⁺CD49f⁺CD133/2⁺ in 9 of 9 normal adjacent tissue samples compared with 7 of 52 ER⁺ and/or Her2⁺ tumors (P < 0.001). Further, expression of CD44⁺CD49f⁺CD133/2⁺ by normal adjacent tissue correlated positively with a stem cell-derived tumorigenic signature (P <0.001) and inversely with a defective DNA-repair signature (P <0.001).

Conclusion

Normal cells bearing cancer stem cell markers are associated with the triple negative receptor subtype of breast cancer. This study suggests stem cell staining and gene expression signatures from normal breast tissues represent novel tissue-based risk biomarkers for triple negative breast cancer. Validation of these results in additional studies of normal tissue from cancer-free women could lay the foundation for future targeted triple negative breast cancer prevention strategies.     

The prognostic value of androgen receptors in breast cancer subtypes

Introduction

We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype.

Materials and methods

Normal breast parenchyma?≥?5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44⁺CD49f⁺CD133/2⁺ mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples.

Results

8 of 8 IBC samples expressed isolated CD44⁺CD49f⁺CD133/2⁺ stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p?=?0.001). Similarly, the median number of CD44⁺CD49f⁺CD133/2⁺ cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p?=?0.007). 7 of 8 IBC samples expressed CD68?+?histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p?=?0.001). In the validation cohort, the median number of CD68?+?cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p?=?0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p?=?0.02) and with a 79-gene IBC signature (p?<?0.001).

Conclusions

Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

     

The CD44<Superscript>+</Superscript>/CD24<Superscript>-</Superscript>phenotype is enriched in basal-like breast tumors

Introduction  

Human breast tumors are heterogeneous and consist of phenotypically diverse cells. Breast cancer cells with a CD44⁺/CD24^- phenotype have been suggested to have tumor-initiating properties with stem cell-like and invasive features, although it is unclear whether their presence within a tumor has clinical implications. There is also a large heterogeneity between tumors, illustrated by reproducible stratification into various subtypes based on gene expression profiles or histopathological features. We have explored the prevalence of cells with different CD44/CD24 phenotypes within breast cancer subtypes.     

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.     

CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis

Introduction  

A subpopulation (CD44⁺/CD24^-) of breast cancer cells has been reported to have stem/progenitor cell properties. The aim of this study was to investigate whether this subpopulation of cancer cells has the unique ability to invade, home, and proliferate at sites of metastasis.     

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.     

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.     

Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers

Introduction

To understand which signalling pathways become deregulated in breast cancer, it is necessary to identify functionally significant gene expression patterns in the stem, progenitor, transit amplifying and differentiated cells of the mammary epithelium. We have previously used the markers 33A10, CD24 and Sca-1 to identify mouse mammary epithelial cell subpopulations. We now investigate the relationship between cells expressing these markers and use gene expression microarray analysis to identify genes differentially expressed in the cell populations.

Methods

Freshly isolated primary mouse mammary epithelial cells were separated on the basis of staining with the 33A10 antibody and an α-Sca-1 antibody. The populations identified were profiled using gene expression microarray analysis. Gene expression patterns were confirmed on normal mouse and human mammary epithelial subpopulations and were examined in a panel of breast cancer samples and cell lines.

Results

Analysis of the separated populations demonstrated that Sca-1^- 33A10^High stained cells were estrogen receptor α (Esr1)^- luminal epithelial cells, whereas Sca-1⁺ 33A10^Low/- stained cells were a mix of nonepithelial cells and Esr1⁺ epithelial cells. Analysis of the gene expression data identified the gene Rgs2 (regulator of G-protein signalling 2) as being highly expressed in the Sca-1^- 33A10^Low/- population, which included myoepithelial/basal cells. RGS2 has previously been described as a regulator of angiotensin II receptor signalling. Gene expression analysis by quantitative real-time RT-PCR of cells separated on the basis of CD24 and Sca-1 expression confirmed that Rgs2 was more highly expressed in mouse myoepithelial/basal mammary cells than luminal cells. This expression pattern was conserved in normal human breast cells. Functional analysis demonstrated RGS2 to be a modulator of oxytocin receptor signalling. The potential significance of RGS2 expression in breast cancer was demonstrated by semi-quantitative RT-PCR analysis, data mining and quantitative real-time RT-PCR approaches, which showed that RGS2 was expressed in the majority of solid breast cancers at much higher levels than in normal human mammary cells.

Conclusion

Molecular analysis of prospectively isolated mammary epithelial cells identified RGS2 as a modulator of oxytocin receptor signalling, which is highly expressed in the myoepithelial cells. The RGS2 gene, but not the oxytocin receptor, was also shown to be over-expressed in the majority of breast cancers, identifying the product of this gene, or the pathway(s) it regulates, as potentially significant therapeutic targets.     

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.