CD117 Expression in Fibroblasts-Like Stromal Cells Indicates Unfavorable Clinical Outcomes in Ovarian Carcinoma Patients

The stem cell factor (SCF) receptor CD117 (c-kit), is widely used for identification of hematopoietic stem cells and cancer stem cells. Moreover, CD117 expression in carcinoma cells indicates a poor prognosis in a variety of cancers. However the potential expression in tumor microenvironment and the biological and clinical impact are currently not reported. The expression of CD117 was immunohistochemically evaluated in a serial of 242 epithelial ovarian cancer (EOC) cases. Thirty-eight out of 242 cases were CD117 positive in fibroblast-like stromal cells and 22 cases were positive in EOC cells. Four cases were both positive in fibroblast-like stromal cells and EOC cells for CD117. CD117 expression in fibroblast-like stromal cells in ovarian carcinoma was closely linked to advanced FIGO stage, poor differentiation grade and histological subtype (p<0.05), and it was significantly associated with poor overall survival (OS) and progression free survival (PFS) (Kaplan-Meier analysis; p<0.05, log-rank test). CD117 expression in ovarian carcinoma cells was not associated with these clinicopathological variables. The CD117 positive fibroblast-like stromal cells were all positive for mesenchymal stem/stromal cell (MSC) marker CD73 but negative for fibroblast markers fibroblast activation protein (FAP) and α smooth muscle actin (α-SMA), indicating that the CD117+/CD73+ fibroblast-like stromal cells are a subtype of mesenchymal stem cells in tumor stroma, although further characterization of these cells are needed. It is concluded herewith that the presence of CD117+/CD73+ fibroblast-like stromal cells in ovarian carcinoma is an unfavorable clinical outcome indication.     

Uncoupling of the ERα regulated morphological phenotype from the cancer stem cell phenotype in human breast cancer cell lines

The CD24^low/−CD44⁺EpCAM⁺ phenotype is associated with breast cancer initiating cells. To investigate if these putative breast cancer stem cell markers are regulated by estrogen receptor alpha (ERα) we have determined the expression levels of EpCAM, CD44 and CD24 in several well characterized breast cancer cell lines. The expression levels of the three adhesion proteins were quantitatively different in the cell lines but the composite CD24^low/−CD44⁺EpCAM⁺ breast cancer stem cell phenotype was shown to exist as a small fraction, between 0.1% and 1.2%, in all breast cancer cell lines tested. Experimental silencing of ERα resulted in a reduced epithelial appearance and partial reduction of CD24 mRNA, while levels of CD44 and EpCAM were unaltered. Moreover, knockdown of ERα led to a change in the morphology of the cells similar to the epithelial to mesenchymal transition phenotype and was associated with decreased E-cadherin expression. Our findings offer new insights into the regulation of the breast cancer stem cell phenotype by ERα and suggest that treatments targeting the breast cancer stem cell adhesion molecules and the ERα pathway may be complementary.     

Human umbilical cord Wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro

Umbilical cord mesenchymal stem cells (MSCs) have been shown to inhibit breast cancer cell growth but it is not known whether this effect is specific to only breast cancer cells. We compared the effects of human Wharton's jelly stem cell (hWJSC) extracts [conditioned medium (hWJSC‐CM) and cell lysate (hWJSC‐CL)] on breast adenocarcinoma (MDA‐MB‐231), ovarian carcinoma (TOV‐112D), and osteosarcoma (MG‐63) cells. The cells were treated with either hWJSC‐CM (50%) or hWJSC‐CL (15 µg/ml) for 48–72 h and changes in cell morphology, proliferation, cycle, gene expression, migration, and cell death studied. All three cancer cell lines showed cell shrinkage, blebbing, and vacuolations with hWJSC‐CL and hWJSC‐CM compared to controls. MTT and BrdU assays showed inhibition of cell growth by 2–6% and 30–60%, while Transwell migration assay showed inhibition by 20–26% and 31–46% for hWJSC‐CM and hWJSC‐CL, respectively, for all three cancer cell lines. Cell cycle assays showed increases in sub‐G1 and G2/M phases for all three cancer cell lines suggestive of apoptosis and metaphase arrest. AnnexinV‐FITC and TUNEL positive cells seen in TOV‐112D and MDA‐MB‐231 suggested that inhibition was via apoptosis while the presence of anti‐BECLIN1 and anti‐LC3B antibodies seen with MG‐63 indicated autophagy. Upregulation of pro‐apoptotic BAX and downregulation of anti‐apoptotic BCL2 and SURVIVIN genes were observed in all three cancer cell lines and additionally the autophagy genes (ATG5, ATG7, and BECLIN1) were upregulated in MG‐63 cells. hWJSCs possess tumor inhibitory properties that are not specific to breast cancer cells alone and these effects are mediated via agents in its extracts. J. Cell. Biochem. 113: 2027–2039, 2012. © 2012 Wiley Periodicals, Inc.     

RB1 Status in Triple Negative Breast Cancer Cells Dictates Response to Radiation Treatment and Selective Therapeutic Drugs

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1^met, fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA⁺/CD24^−/low/CD44⁺ cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.     

Endothelial induced EMT in breast epithelial cells with stem cell properties

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.     

Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.     

Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) Regulates Hematopoiesis and Bone Formation In Vivo

Background

Tissue inhibitor of metalloproteinases-3 (TIMP-3) inhibits matrix metalloproteinases and membrane-bound sheddases. TIMP-3 is associated with the extracellular matrix and is expressed in highly remodeling tissues. TIMP-3 function in the hematopoietic system is unknown.

Methodology/Principal Findings

We now report that TIMP-3 is highly expressed in the endosteal region of the bone marrow (BM), particularly by osteoblasts, endothelial and multipotent mesenchymal stromal cells which are all important cellular components of hematopoietic stem cell (HSC) niches, whereas its expression is very low in mature leukocytes and hematopoietic stem and progenitor cells. A possible role of TIMP-3 as an important niche component was further suggested by its down-regulation during granulocyte colony-stimulating factor-induced mobilization. To further investigate TIMP-3 function, mouse HSC were retrovirally transduced with human TIMP-3 and transplanted into lethally irradiated recipients. TIMP-3 overexpression resulted in decreased frequency of B and T lymphocytes and increased frequency of myeloid cells in blood and BM, increased Lineage-negative Sca-1⁺KIT⁺ cell proliferation in vivo and in vitro and increased colony-forming cell trafficking to blood and spleen. Finally, over-expression of human TIMP-3 caused a late onset fatal osteosclerosis.

Conclusions/Significance

Our results suggest that TIMP-3 regulates HSC proliferation, differentiation and trafficking in vivo, as well as bone and bone turn-over, and that TIMP-3 is expressed by stromal cells forming HSC niches within the BM. Thus, TIMP-3 may be an important HSC niche component regulating both hematopoiesis and bone remodeling.     

Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo.

Several murine marrow stromal cells were established from murine bone marrow cultures. Stromal cell lines transfected with a tumor-inducing polyoma virus middle T antigen (MTAg) were inoculated into nude mice subcutaneously. KUSA-MTAg cells, one of these cell lines, led to the rapid local development of bone marrow consisting of trilineage hematopoietic cells and bone; other cell lines produced spindle cell sarcoma or hemangiosarcoma. These results suggested that a single stromal cell line, KUSA-MTAg cells, may induce hematopoietic stem cells or early progenitors of three lineages of hematopoietic cells in vivo. Interestingly, untransfected KUSA cells expressed three new mesenchymal phenotypes, osteocytes, adipocytes, and myotubes, after treatment with 5-azacytidine.     

Autocrine and paracrine growth regulation of human breast cancer

Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vitro. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells, facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation, hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation.     

Enhanced immunoreactivity of TIMP-2 in the stromal compartment of tumor as a marker of favorable prognosis in ovarian cancer patients

Degradation of the extracellular matrix and basement membrane is a critical step in tumor progression. Matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP 2) act in a coordinated manner to form an integrated system involved in ovarian cancer (OC) progression. In this study, the authors describe the expression of TIMP-2 detected by immunohistochemistry in 6 OC cell lines and in 43 malignant epithelial ovarian tumors (in tumor and stromal compartments) in sections originating from primary laparotomies. No significant correlations between overall and progression-free survival and TIMP-2 expression in tumor compartment were observed. The analysis demonstrated a significant association between enhanced stromal expression of TIMP-2 and better clinical response to cisplatin- and paclitaxel-based chemotherapy. Increased expression of TIMP-2 in the stromal compartment and simultaneous overexpression in both stromal and tumor compartments strongly correlated with increased survival. No significant correlations were found in vitro between resistance to cisplatin, paclitaxel, or topotecan and the expression of TIMP-2 in the OC cell lines, suggesting stromal influences on tumor chemoresistance in the physiological environment. This study supports the concept of TIMP-2 expression in the stromal compartment of OC as a promising marker of prognosis and response to cisplatin- and paclitaxel-based chemotherapy in OC patients.     

The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells

ABSTRACT: MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1's functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.     

Interleukin-1 directly regulates hormone-dependent human breast cancer cell proliferation in vitro

Direct in vitro effects of IL-1 on hormone-dependent (MCF-7 and ZR-75-B) and independent (HS-578-T and MDA-231) human breast cancer cell proliferation were investigated in short-term and long-term cell cultures. For short-term (48 h) studies [3H]thymidine uptake was used as an index of proliferation, while for long-term (12 day) cultures actual cell numbers were determined. Initial studies, conducted with MCF-7 cells, demonstrated that both forms of recombinant human IL-1 (alpha and beta) at 10(-11) M inhibited [3H]thymidine uptake by MCF-7 by 70%, and by day 7 of the long-term study alpha and beta IL-1 at 10(-11) M inhibited MCF-7 cell growth by 80%. IL-1, while inhibiting the growth of another hormone-dependent breast cancer cell line; ZR-75-B, had no effect on the hormone-independent cell lines MDA-231 and HS-578-T. The differing proliferative responses of the hormone-dependent and independent cells to IL-1 may, in part, be due to the expression of IL-1 receptors on these cells, in that MCF-7 cells express IL-1 receptors [dissociation constant (Kd) = 2.0 x 10(-10) M; receptor density = 2,500 sites per cell and mol wt = 80,000] while the hormone-independent MDA-231 cells do not.     

Effects of SDF-1-CXCR4 signaling on microRNA expression and tumorigenesis in estrogen receptor-alpha (ER-α)-positive breast cancer cells

The majority of breast cancer cases ultimately become unresponsive to endocrine therapies, and this progression of breast cancer from hormone-responsive to hormone-independent represents an area in need of further research. Additionally, hormone-independent carcinomas are characterized as being more aggressive and metastatic, key features of more advanced disease. Having previously shown the ability of the stromal-cell derived factor-1 (SDF-1)–CXCR4 signaling axis to promote primary tumorigenesis and hormone independence by overexpressing CXCR4 in MCF-7 cells, in this study we further examined the role of SDF-1/CXCR4 in the endogenously CXCR4-positive, estrogen receptor α (ER-α)-positive breast carcinoma cell line, MDA–MB-361. In addition to regulating estrogen-induced and hormone-independent tumor growth, CXCR4 signaling stimulated the epithelial-to-mesenchymal transition, evidenced by decreased CDH1 expression following SDF-1 treatment. Furthermore, inhibition of CXCR4 with the small molecule inhibitor AMD3100 induced CDH1 gene expression and inhibited CDH2 gene expression in MDA–MB-361 cells. Further, exogenous SDF-1 treatment induced ER-α-phosphorylation in both MDA–MB-361 and MCF-7–CXCR4 cells, demonstrating ligand-independent activation of ER-α through CXCR4 crosstalk. qPCR microRNA array analyses of the MDA–MB-361 and MCF-7–CXCR4 cell lines revealed changes in microRNA expression profiles induced by SDF-1, consistent with a more advanced disease phenotype and further supporting our hypothesis that the SDF-1/CXCR4 signaling axis drives ER-α-positive breast cancer cells to a hormone independent and more aggressive phenotype. In this first demonstration of SDF-1–CXCR4-induced microRNAs in breast cancer, we suggest that this signaling axis may promote tumorigenesis via microRNA regulation. These findings represent future potential therapeutic targets for the treatment of hormone-independent and endocrine-resistant breast cancer.     

Breast cancer stem cell-derived extracellular vesicles transfer ARRDC1-AS1 to promote breast carcinogenesis via a miR-4731-5p/AKT1 axis-dependent mechanism

ObjectivesDeregulation of long non-coding RNAs (lncRNAs) has been frequently reported in breast cancer (BC). This goes to show the importance of understanding its significant contribution towards breast carcinogenesis. In the present study, we clarified a carcinogenic mechanism based on the ARRDC1-AS1 delivered by breast cancer stem cells-derived extracellular vesicles (BCSCs-EVs) in BC.MethodsThe isolated and well characterized BCSCs-EVs were co-cultured with BC cells. The expression of ARRDC1-AS1, miR-4731-5p, and AKT1 was determined in BC cell lines. BC cells were assayed for their viability, invasion, migration and apoptosis in vitro by CCK-8, Transwell and flow cytometry, as well as tumor growth in vivo after loss- and gain-of function assays. Dual-luciferase reporter gene, RIP and RNA pull-down assays were performed to determine the interactions among ARRDC1-AS1, miR-4731-5p, and AKT1.ResultsElevation of ARRDC1-AS1 and AKT1 as well as miR-4731-5p downregulation were observed in BC cells. ARRDC1-AS1 was enriched in BCSCs-EVs. Furthermore, EVs containing ARRDC1-AS1 enhanced the BC cell viability, invasion and migration and glutamate concentration. Mechanistically, ARRDC1-AS1 elevated the expression of AKT1 by competitively binding to miR-4731-5p. ARRDC1-AS1-containing EVs were also found to enhance tumor growth in vivo.ConclusionCollectively, BCSCs-EVs-mediated delivery of ARRDC1-AS1 may promote the malignant phenotypes of BC cells via the miR-4731-5p/AKT1 axis.