Katarzyna Grymula

The role of stromal-derived factor-1--CXCR7 axis in development and cancer.

Cancer metastasis is a major clinical problem that contributes to unsuccessful therapy. Augmenting evidence indicates that metastasizing cancer cells employ several mechanisms that are involved in developmental trafficking of normal stem cells. Stromal-derived factor-1 (SDF-1) is an important alpha-chemokine that binds to the G-protein-coupled seven-transmembrane span CXCR4. The SDF-1-CXCR4 axis regulates trafficking of normal and malignant cells. SDF-1 is an important chemoattractant for a variety of cells including hematopoietic stem/progenitor cells. For many years, it was believed that CXCR4 was the only receptor for SDF-1. However, several reports recently provided evidence that SDF-1 also binds to another seven-transmembrane span receptor called CXCR7, sharing this receptor with another chemokine family member called Interferon-inducible T-cell chemoattractant (I-TAC). Thus, with CXCR7 identified as a new receptor for SDF-1, the role of the SDF-1-CXCR4 axis in regulating several biological processes becomes more complex. Based on the available literature, this review addresses the biological significance of SDF-1s interaction with CXCR7, which may act as a kind of decoy or signaling receptor depending on cell type. Augmenting evidence suggests that CXCR7 is involved in several aspects of tumorogenesis and could become an important target for new anti-metastatic and anti-cancer drugs.

Overlapping and distinct role of CXCR7-SDF-1/ITAC and CXCR4-SDF-1 axes in regulating metastatic behavior of human rhabdomyosarcomas.

We have demonstrated that the α‐chemokine stromal‐derived factor (SDF)‐1‐CXCR4 axis plays an important role in rhabdomyosarcoma (RMS) metastasis. With the recent description of CXCR7, a new receptor for SDF‐1 that also binds the interferon‐inducible T‐cell α chemoattractant (ITAC) chemokine, we became interested in the role of the CXCR7‐SDF‐1/ITAC axis in RMS progression. To address this issue, we evaluated 6 highly metastatic alveolar (A)RMS and 3 less metastatic embryonal (E)RMS cell lines and found that all these cell lines express CXCR7. Although CXCR4 was expressed at a much higher level by highly metastatic ARMS lines, CXCR7 was present at a high level on ERMS lines. We also noticed that CXCR7 expression on RMS cells was downregulated in hypoxic conditions. More importantly, the CXCR7 receptor on RMS cell lines was functional after stimulation with ITAC and SDF‐1 as evidenced by mitogen‐activated protein kinase (MAPK)p42/44 and AKT phosphorylation as well as CXCR7 internalization, chemotaxis, cell motility and adhesion assays. Similarly to CXCR4, signaling from activated CXCR7 was not associated with increased RMS proliferation or cell survival. Moreover, CXCR7+ RMS cells responded to SDF‐1 and I‐TAC in the presence of CXCR4 antagonists (T140, AMD3100). Furthermore, while intravenous injection of RMS cells with overexpressed CXCR7 resulted in increased seeding efficiency of tumor cells to bone marrow, CXCR7 downregulation showed the opposite effect. In conclusion, the CXCR7‐SDF‐1/ITAC axis is involved in the progression of RMS; targeting of the CXCR4‐SDF‐1 axis alone without simultaneous blockage of CXCR7 will be an inefficient strategy for inhibiting SDF‐1‐mediated prometastatic responses of RMS cells.

Macrophage Migration Inhibitory Factor Is Secreted by Rhabdomyosarcoma Cells, Modulates Tumor Metastasis by Binding to CXCR4 and CXCR7 Receptors and Inhibits Recruitment of Cancer-Associated Fibroblasts

The overexpression of macrophage migration inhibitory factor (MIF) has been observed in many tumors and is implicated in oncogenic transformation and tumor progression. MIF activates CXCR2 and CD74 receptors and, as recently reported, may also bind to the stromal-derived factor-1 (SDF-1)–binding receptor CXCR4. Here, we report that human rhabdomyosarcoma (RMS) cell lines secrete MIF and that this chemokine (a) induces phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, (b) stimulates RMS cell adhesion, (c) enhances tumor vascularization, but surprisingly (d) decreases recruitment of cancer-associated fibroblasts (CAF). Because RMS cells used in our studies do not express CXCR2 and CD74 receptors, the biological effects of MIF on RMS cells depend on its interaction with CXCR4, and as we report here for the first time, MIF may also engage another SDF-1–binding receptor (CXCR7) as well. Interestingly, downregulation of MIF in RMS cells inoculated into immunodeficient mice led to formation of larger tumors that displayed higher stromal cell support. Based on these observations, we postulate that MIF is an important autocrine/paracrine factor that stimulates both CXCR4 and CXCR7 receptors to enhance the adhesiveness of RMS cells. We also envision that when locally secreted by a growing tumor, MIF prevents responsiveness of RMS to chemoattractants secreted outside the growing tumor (e.g., SDF-1) and thereby prevents release of cells into the circulation. On the other hand, despite its obvious proangiopoietic effects, MIF inhibits in CXCR2/CD74-dependent manner recruitment of CAFs to the growing tumor. Our data indicate that therapeutic inhibition of MIF in RMS may accelerate metastasis and tumor growth. Mol Cancer Res; 8(10); 1328–43. ©2010 AACR.The overexpression of macrophage migration inhibitory factor (MIF) has been observed in many tumors and is implicated in oncogenic transformation and tumor progression. MIF activates CXCR2 and CD74 receptors and, as recently reported, may also bind to the stromal-derived factor-1 (SDF-1)–binding receptor CXCR4. Here, we report that human rhabdomyosarcoma (RMS) cell lines secrete MIF and that this chemokine (a) induces phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, (b) stimulates RMS cell adhesion, (c) enhances tumor vascularization, but surprisingly (d) decreases recruitment of cancer-associated fibroblasts (CAF). Because RMS cells used in our studies do not express CXCR2 and CD74 receptors, the biological effects of MIF on RMS cells depend on its interaction with CXCR4, and as we report here for the first time, MIF may also engage another SDF-1–binding receptor (CXCR7) as well. Interestingly, downregulation of MIF in RMS cells inoculated into immunodeficient mice led to formation of larger tumors that displayed higher stromal cell support. Based on these observations, we postulate that MIF is an important autocrine/paracrine factor that stimulates both CXCR4 and CXCR7 receptors to enhance the adhesiveness of RMS cells. We also envision that when locally secreted by a growing tumor, MIF prevents responsiveness of RMS to chemoattractants secreted outside the growing tumor (e.g., SDF-1) and thereby prevents release of cells into the circulation. On the other hand, despite its obvious proangiopoietic effects, MIF inhibits in CXCR2/CD74dependent manner recruitment of CAFs to the growing tumor. Our data indicate that therapeutic inhibition of MIF in RMS may accelerate metastasis and tumor growth. Mol Cancer Res; 8(10); OF1–16. ©2010 AACR.

Regulation of Expression of Stromal-Derived Factor-1 Receptors: CXCR4 and CXCR7 in Human Rhabdomyosarcomas

Rhabdomyosarcomas (RMS) express CXCR4 and CXCR7 receptors that bind prometastatic α-chemokine stromal-derived factor-1 (SDF-1). In this report, we analyzed the activity of both promoters in a model of less metastatic human embryonal-RMS cell line (RD) and more metastatic alveolar-like RMS (RD cells transduced with paired box gene 3/forkhead homologue; PAX3-FKHR fusion gene). First, CXCR4 is barely detectable in RD and becomes upregulated in RD/PAX3-FKHR cells. In contrast, CXCR7 highly expressed in RD becomes downregulated in RD/PAX3-FKHR cells. Next, promoter deletion and mutation studies revealed that whereas (a) expression of CXCR4 in RD and RD/PAX3-FKHR cells required nuclear respiratory factor-1 (NRF-1) binding site and (b) was additionally upregulated by direct interaction of NRF-1 with PAX3-FKHR, CXCR7 promoter activity required a proximal nuclear factor-κB–binding motif. The requirement of these factors for CXCR4 and CXCR7 promoter activities was additionally supported after blocking NRF-1 and nuclear factor-κB. Furthermore, CXCR4 expression in PAX3-FKHR+ RMS cells seems to be enhanced because of the interaction of PAX3-FKHR and NRF-1 proteins in the proximal part of the promoter that prevents access of the negative regulator of transcription YY1 to its binding site. Finally, although hypoxia enhances CXCR4 and CXCR7 promoter activity and receptor expression in RD cells, it inhibits CXCR7 expression in RD/PAX3-FKHR cells. In conclusion, SDF-1 binding receptors CXCR4 and CXCR7 are differently regulated in RMS cells. The upregulation of CXCR4 and downregulation of CXCR7 expression by PAX3-FKHR or hypoxia may give SDF-1 an advantage to better engage the CXCR4 receptor, thus increasing RMS motility. Mol Cancer Res; 8(1); 1–14

Human Rhabdomyosarcomas Secrete Macrophage Migration Inhibitory Factor That Modulates Metastatic Behavior of Tumor Cells and Inhibits Recruitment of Cancer-Associated Fibroblasts

The overexpression of macrophage migration inhibitory factor (MIF) has been observed in many tumors and is implicated in oncogenic transformation and tumor progression. MIF activates CXCR2 and CD74 receptors and, as recently reported, may also bind to the stromal-derived factor-1 (SDF-1)–binding receptor CXCR4. Here, we report that human rhabdomyosarcoma (RMS) cell lines secrete MIF and that this chemokine (a) induces phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, (b) stimulates RMS cell adhesion, (c) enhances tumor vascularization, but surprisingly (d) decreases recruitment of cancer-associated fibroblasts (CAF). Because RMS cells used in our studies do not express CXCR2 and CD74 receptors, the biological effects of MIF on RMS cells depend on its interaction with CXCR4, and as we report here for the first time, MIF may also engage another SDF-1–binding receptor (CXCR7) as well. Interestingly, downregulation of MIF in RMS cells inoculated into immunodeficient mice led to formation of larger tumors that displayed higher stromal cell support. Based on these observations, we postulate that MIF is an important autocrine/paracrine factor that stimulates both CXCR4 and CXCR7 receptors to enhance the adhesiveness of RMS cells. We also envision that when locally secreted by a growing tumor, MIF prevents responsiveness of RMS to chemoattractants secreted outside the growing tumor (e.g., SDF-1) and thereby prevents release of cells into the circulation. On the other hand, despite its obvious proangiopoietic effects, MIF inhibits in CXCR2/CD74-dependent manner recruitment of CAFs to the growing tumor. Our data indicate that therapeutic inhibition of MIF in RMS may accelerate metastasis and tumor growth. Mol Cancer Res; 8(10); 1328–43. ©2010 AACR.The overexpression of macrophage migration inhibitory factor (MIF) has been observed in many tumors and is implicated in oncogenic transformation and tumor progression. MIF activates CXCR2 and CD74 receptors and, as recently reported, may also bind to the stromal-derived factor-1 (SDF-1)–binding receptor CXCR4. Here, we report that human rhabdomyosarcoma (RMS) cell lines secrete MIF and that this chemokine (a) induces phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, (b) stimulates RMS cell adhesion, (c) enhances tumor vascularization, but surprisingly (d) decreases recruitment of cancer-associated fibroblasts (CAF). Because RMS cells used in our studies do not express CXCR2 and CD74 receptors, the biological effects of MIF on RMS cells depend on its interaction with CXCR4, and as we report here for the first time, MIF may also engage another SDF-1–binding receptor (CXCR7) as well. Interestingly, downregulation of MIF in RMS cells inoculated into immunodeficient mice led to formation of larger tumors that displayed higher stromal cell support. Based on these observations, we postulate that MIF is an important autocrine/paracrine factor that stimulates both CXCR4 and CXCR7 receptors to enhance the adhesiveness of RMS cells. We also envision that when locally secreted by a growing tumor, MIF prevents responsiveness of RMS to chemoattractants secreted outside the growing tumor (e.g., SDF-1) and thereby prevents release of cells into the circulation. On the other hand, despite its obvious proangiopoietic effects, MIF inhibits in CXCR2/CD74dependent manner recruitment of CAFs to the growing tumor. Our data indicate that therapeutic inhibition of MIF in RMS may accelerate metastasis and tumor growth. Mol Cancer Res; 8(10); OF1–16. ©2010 AACR.

Different populations of circulating endothelial cells in patients with age-related macular degeneration: a novel insight into pathogenesis.

PURPOSE Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) may serve as novel markers of endothelial dysfunction. The presence and clinical implications of CECs and the expression of endothelin (ET)-1, one of the most potent vasoconstrictors, have not been evaluated in patients with the neovascular form of age-related macular degeneration (AMD). This study was conducted to determine the different populations of endothelial cells (ECs) in the peripheral blood of AMD patients and to correlate these findings with the expression of ET-1 and the cytokines and growth factors responsible for EC migration and function. METHODS Peripheral blood samples were collected from 29 patients with diagnosed neovascular AMD and from 38 healthy control subjects. CD133(-)CD144(+) CECs and CD34(+)CD133(+)CD144(+) EPCs were counted and analyzed by flow cytometry. The intracellular expression of ET-1 in peripheral blood nuclear cells (PBNCs) was studied by using qRT-PCR, Western blot, and immunocytofluorescence assays, and ET-1, IGF-1, VEGF, SDF-1, and HGF plasma concentrations were measured in enzyme-linked immunosorbent assays. RESULTS Increased CECs and EPCs were found in the AMD patients compared with the counts in healthy individuals. The expression of intracellular ET-1 was significantly elevated in PBNCs from the AMD patients compared with the control subjects. In addition a significantly higher plasma concentration of IGF-1 was observed, but a lower SDF-1 level in the group of AMD patients. CONCLUSIONS These findings suggest that circulating endothelial cells, together with high ET-1 content, may contribute to the development of AMD. Further prospective investigations on the mechanism involved may be relevant to the potential treatment of this disease.

The influence of 3,3′,5-triiodo-l-thyronine on human haematopoiesis

Abstract.  Objectives: Thyroid hormones mediate many physiological and developmental functions in humans. The role of the 3,3′,5‐triiodo‐l‐thyronine (T3) in normal human haematopoiesis at the cellular and molecular levels has not been determined. In this study, it was revealed that the human haematopoietic system might be directly depended on T3 influence. Materials and methods: We detected the TRα1 and TRβ1 gene expression at the mRNA level in human cord blood, peripheral blood and bone marrow CD34+‐enriched progenitor cells, using the RT‐PCR method. Furthermore, we performed Western blotting to prove TRα1 and TRβ1 expression occurs at the protein level in human cord blood, peripheral blood and bone marrow CD34+ cells. In addition, the examined populations of cells were exposed in serum‐free conditions to increasing doses of T3 and were subsequently investigated for clonogenic growth of granulocyte‐macrophage colony‐forming unit and erythrocyte burst‐forming unit in methylcellulose cultures, and for the level of apoptosis, by employing annexin V staining and the terminal deoxynucleotidyltransferase‐mediated dUTP nick‐end labelling method. We investigated expression levels of apoptosis‐related Bax and antiapoptotic Bcl‐2 and Bcl‐xL genes in the examined cells. Results: We found that exposure to higher and lower than normal concentration of thyroid hormone significantly influenced clonogenecity and induced apoptosis in human haematopoietic progenitor cells. Conclusions: This study expands the understanding of the role of thyroid disorders in normal human haematopoiesis and indicates a direct influence of T3 on this process.

Evidence that the population of quiescent bone marrow-residing very small embryonic/epiblast-like stem cells (VSELs) expands in response to neurotoxic treatment

The concept that bone marrow (BM)‐derived cells may participate in neural regeneration remains controversial, and the identity of the specific cell type(s) involved remains unknown. We recently reported that the adult murine BM contains a highly mobile population of Sca‐1+Lin−CD45− cells known as very small embryonic/epiblast‐like stem cells (VSELs) that express several markers of pluripotency such as Oct‐4. In the BM microenvironment, these cells are kept quiescent because of epigenetic modification of certain paternally imprinted genes. However, as reported, these cells can be mobilized in mice in an experimental model of stroke and express several genes involved in neurogenesis while circulating in peripheral blood (PB). In the current work, we employed a model of toxic brain damage, which is induced by administration of kainic acid, to see not only whether VSELs can be mobilized into PB in response to this neurotoxin, but, more importantly, whether they proliferate and expand in BM tissue. We report here for the first time that brain damage leads to activation and expansion of the BM pool of quiescent VSELs, which precedes their subsequent egress into PB. Harnessing these cells in neural tissue regeneration is currently one of the challenges in regenerative medicine.

5‑Azacytidine inhibits human rhabdomyosarcoma cell growth by downregulating insulin‑like growth factor 2 expression and reactivating the H19 gene product miR‑675, which negatively affects insulin‑like growth factors and insulin signaling

Insulin-like growth factor 2 (IGF2) and 1 (IGF1) and insulin (INS) promote proliferation of rhabdomyosarcoma (RMS) cells by interacting with the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor (INSR). Loss of imprinting (LOI) by DNA hypermethylation at the differentially methylated region (DMR) for the IGF2‑H19 locus is commonly observed in RMS cells and results in an increase in the expression of proliferation-promoting IGF2 and downregulation of proliferation-inhibiting non-coding H19 miRNAs. One of these miRNAs, miR‑675, has been reported in murine cells to be a negative regulator of IGF1R expression. To better address the role of IGF2 and 1, as well as INS signaling in the pathogenesis of RMS and the involvement of LOI at the IGF2‑H19 locus, we employed the DNA demethylating agent 5‑azacytidine (AzaC). We observed that AzaC‑mediated demethylation of the DMR at the IGF2‑H19 locus resulted in downregulation of IGF2 and an increase in the expression of H19. This epigenetic change resulted in a decrease in RMS proliferation due to downregulation of IGF2 and, IGF1R expression in an miR‑675‑dependent manner. Interestingly, we observed that miR‑675 not only inhibited the expression of IGF1R in a similar manner in human and murine cells, but we also observed its negative effect on the expression of the INSR. These results confirm the crucial role of LOI at the IGF2‑H19 DMR in the pathogenesis of RMS and are relevant to the development of new treatment strategies.

Caloric restriction increases ratio of estrogen to androgen receptors expression in murine ovaries--potential therapeutic implications.

Both estrogens and androgens are involved in the development and normal functioning of the ovaries. It is also known that ovarian function is regulated by diet. The goal of this study was to estimate the expression of sex hormone receptors in ovaries of mice that were on a 9-month caloric restriction (alternate-day feeding) as compared to normal control animals fed ad libitum. We found that prolonged caloric restriction in mouse ovaries led to increased expression of estrogen receptors (ERs) but did not affect expression of the androgen receptor (AR). This increase in ER:AR ration as result of caloric restriction may lead to higher sensitivity to estrogens and upon return to normal diet may increase ovulation. Thus our observation shed more light on a role of beneficial effect of calorie restriction on female reproduction.