Agata Poniewierska-Baran

Hematopoietic Stem/Progenitor Cells Express Several Functional Sex Hormone Receptors—Novel Evidence for a Potential Developmental Link Between Hematopoiesis and Primordial Germ Cells

Evidence has accumulated that hematopoietic stem progenitor cells (HSPCs) share several markers with the germline, a connection supported by reports that prolactin, androgens, and estrogens stimulate hematopoiesis. To address this issue more directly, we tested the expression of receptors for pituitary-derived hormones, such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on purified murine bone marrow (BM) cells enriched for HSPCs and tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. We also tested whether administration of pituitary- and gonad-derived sex hormones (SexHs) increases incorporation of bromodeoxyuridine (BrdU) into HSPCs and expansion of hematopoietic clonogenic progenitors in mice and promotes recovery of blood counts in sublethally irradiated animals. We report for the first time that HSPCs express functional FSH and LH receptors and that both proliferate in vivo and in vitro in response to stimulation by pituitary SexHs. Furthermore, based on our observations that at least some of CD45(-) very small embryonic-like stem cells (VSELs) may become specified into CD45(+) HSPCs, we also evaluated the expression of pituitary and gonadal SexHs receptors on these cells and tested whether these quiescent cells may expand in vivo in response to SexHs administration. We found that VSELs express SexHs receptors and respond in vivo to SexHs stimulation, as evidenced by BrdU accumulation. Since at least some VSELs share several markers characteristic of migrating primordial germ cells and can be specified into HSPCs, this observation sheds new light on the BM stem cell hierarchy.

Very small embryonic-like stem cells as a novel developmental concept and the hierarchy of the stem cell compartment

Our current understanding of stem cells suffers from a lack of precision, as the stem cell compartment is a broad continuum between early stages of development and adult postnatal tissues, and it is not fully understood how this transition occurs. The definition of stem cell pluripotency is adapted from embryology and excludes the possibility that some early-development stem cells with pluri- and/or multipotential differentiation potential may reside in postnatal tissues in a dormant state in which they are protected from uncontrolled proliferation and thus do not form teratomas or have the ability to complement blastocyst development. We will discuss the concept that a population of very small embryonic-like stem cells (VSELs) could be a link between early-development stages and adult stem cell compartments and reside in a quiescent state in adult tissues. The epigenetic mechanism identified that changes expression of certain genes involved in insulin/insulin-like growth factor signaling (IIS) in VSELs, on the one hand, keeps these cells quiescent in adult tissues and, on the other hand, provides a novel view of the stem cell compartment, IIS, tissue/organ rejuvenation, aging, and cancerogenesis.

Expression of the erythropoietin receptor by germline-derived cells - further support for a potential developmental link between the germline and hematopoiesis

BackgroundExpressing several markers of migrating primordial germ cells (PGCs), the rare population of quiescent, bone marrow (BM)-residing very small embryonic-like stem cells (VSELs) can be specified like PGCs into hematopoietic stem/progenitor cells (HSPCs). These two properties of VSELs support the possibility of a developmental origin of HSPCs from migrating PGCs.MethodsTo address a potential link between VSELs and germ line cells we analyzed by RT-PCR and FACS expression of erythropoietin receptor (EpoR) on murine bone marrow- and human umbilical cord blood-derived VSELs, murine and human teratocarcinoma cell lines and human ovarian cancer cells. A proper gating strategy and immunostaining excluded from FACS analysis potential contamination by erythroblasts. Furthermore, the transwell chemotaxis assays as well as adhesion and signaling studies were performed to demonstrate functionality of erythropoietin - EpoR axes on these cells.ResultsWe report here that murine and human VSELs as well as murine and human teratocarcinoma cell lines and ovarian cancer cell lines share a functional EpoR.ConclusionsOur data provide more evidence of a potential developmental link between germline cells, VSELs, and HSCs and sheds more light on the developmental hierarchy of the stem cell compartment in adult tissues.

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.

Human rhabdomyosarcoma cells express functional pituitary and gonadal sex hormone receptors: therapeutic implications

Evidence has accumulated that sex hormones play an important role in several types of cancer. Because they are also involved in skeletal muscle development and regeneration, we were therefore interested in their potential involvement in the pathogenesis of human rhabdomyosarcoma (RMS), a skeletal muscle tumor. In the present study, we employed eight RMS cell lines (three fusion positive and five fusion negative RMS cell lines) and mRNA samples obtained from RMS patients. The expression of sex hormone receptors was evaluated by RT-PCR and their functionality by chemotaxis, adhesion and direct cell proliferation assays. We report here for the first time that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors are expressed in established human RMS cell lines as well as in primary tumor samples isolated from RMS patients. We also report that human RMS cell lines responded both to pituitary and gonadal sex hormone stimulation by enhanced proliferation, chemotaxis, cell adhesion and phosphorylation of MAPKp42/44 and AKT. In summary, our results indicate that sex hormones are involved in the pathogenesis and progression of RMS, and therefore, their therapeutic application should be avoided in patients that have been diagnosed with RMS.

Human rhabdomyosarcoma cells express functional erythropoietin receptor: Potential therapeutic implications.

The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression.

Evidence that vitronectin is a potent migration-enhancing factor for cancer cells chaperoned by fibrinogen: a novel view of the metastasis of cancer cells to low-fibrinogen lymphatics and body cavities

Diluted (1%) plasma induces migration of malignant cell lines much more strongly than potent pro-metastatic factors. To characterize the factor(s) present in diluted plasma responsible for this phenomenon we performed i) heat inactivation, ii) dialysis, iii) proteinase K treatment, and iv) molecular size filtration studies. We found that this remarkable pro-migratory activity of diluted normal plasma is associated with a ~50–100-kD protein that interacts with GαI protein-coupled receptors and activates p42/44 MAPK and AKT signaling in target cells. Since this pro-migratory activity of 1% plasma decreases at higher plasma concentrations (> 20%), but is retained in serum, we hypothesized that fibrinogen may be involved as a chaperone of the protein(s). To identify the pro-migratory protein(s) present in diluted plasma and fibrinogen-depleted serum, we performed gel filtration and hydrophobic interaction chromatography followed by mass spectrometry analysis. We identified several putative protein candidates that were further tested in in vitro experiments. We found that this pro-migratory factor chaperoned by fibrinogen is vitronectin, which activates uPAR, and that this effect can be inhibited by fibrinogen. These results provide a novel mechanism for the metastasis of cancer cells to lymphatics and body cavities, in which the concentration of fibrinogen is low, and thus suggests that free vitronectin stimulates migration of tumor cells.

Molecular mechanisms regulating metastasis of cancer cells with special emphasis on rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a malignant tumor of soft tissue derived from embryonic mesenchymal and/or neuroectodermal tissues. It is most often associated with other genetic syndromes such as Li-Fraumeni or Bechwith-Wiedeman. RMS cells show morphological similarities to striated muscle and the presence of specific markers of muscle tissue. At the histological level, it is divided into two subtypes (alveolar RMS - ARMS and embryonal RMS - ERMS), which differ in their genetic background, and prognosis. In recent years there has been significant progress in understanding the mechanisms that regulate RMS cell growth and metastasis. Recently, a number of several chemokines, cytokines or growth factors and their receptors were identified involved in RMS pathogenesis as well as animal models of this tumor have been developed. This knowledge is of great importance in the development of potential therapeutic strategies not only in RMS, but also other types of cancer. This paper will discuss the theories of the origin of this rare tumor and the molecular mechanisms involved in its growth and metastasis. The processes and mechanisms described herein, such as chemotaxis, adhesion, proliferation, intracellular signal transduction, seem to universal for number of cancer types.

A novel potential role of pituitary gonadotropins in the pathogenesis of human colorectal cancer

Background Colorectal cancer (CRC) is a leading cause of death in the western world, and its incidence increases with patient age. It is also known that with age there occur changes in the levels of certain hormones, including an increase in the secretion of pituitary gonadotropins (PtGs) as a result of the loss of gonadal hormone feedback. We recently reported that functional PtG receptors are expressed in human lung cancer cells, rhabdomyosarcoma cells, and malignant hematopoietic stem cells. Findings Here we report for the first time that the receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are expressed in primary tumor samples isolated from CRC patients as well as in the established human CRC cell lines HTC116 and HTB37. Moreover, we also report that PtGs stimulate chemotaxis, adhesion, and proliferation of these cell lines. Conclusions Our results suggest that PtGs play an important and underappreciated role in CRC pathogenesis, and we call for further studies to better define their role in gastrointestinal malignancies and their direct effect on putative CRC cancer stem cells.

Abstract 1698: Novel evidence that blood plasma vitronectin is a major chemoattractant for cancer cells and its pro-migratory activity is suppressed/chaperoned after binding to fibrinogen

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Background. One of the crucial problems with cancer therapy is migration of cancer cells that leave primary tumor and migrate to vital organs where they form metastases. Throughout the years several factors have been identified that direct metastatic process including growth factors, chemokines, bioactive lipids or extracellular nucleotides. To our surprise however, we noticed that highly diluted plasma (1%) possess remarkable chemokinetic activity against several cancer cell lines that highly exceeds those observed for optimal doses of chemokines (e.g. SDF-1) and growth factors (e.g. HGF/SF), that are considered as a main metastatic factors present in plasma. Aim of the study. Based on this observation our aim was to identify the “factor/s” responsible for this remarkable chemokinetic activity of normal diluted 1% plasma. Experimental strategies. We employed several human cancer cell lines and different dilutions of normal human, murine and bovine plasma and serum in Transwell migration assays, adhesion and cell signaling studies. We tested effect of heat inactivation, protease exposure, dialysis and molecular filtration on chemotactic activity of plasma. We also used gel filtration followed by hydrophobic interaction chromatography (HIC) to identified plasma fractions with the most potent chemotactic activity that were further analyze using MassSpec. Results. We found that remarkable chemokinetic activity of highly diluted 1% plasma against malignant cells, rapidly decreased at higher plasma concentration (>5%). Our initial characterization studies revealed that this “activity” is sensitive to proteolytic treatment, is not removed from plasma by dialysis and is temperature sensitive. The similar effect has been observed for diluted 1% serum however chemotactic responsiveness of serum was maintained with its higher concentrations. Based on this we hypothesized possible involvement of inhibitory effect of fibrinogen, which was subsequently confirmed in experiments where fibrinogen was removed from plasma or it was added to serum. Finally, gel filtration followed by HIC and MasSpec analysis allow us to identify several possible candidates that were further tested in in vitro experiments. These studies revealed that vitronectin (VTN) is a main factor responsible for observed chemotactic response and that its effect can be inhibited by fibrinogen. Conclusions. Our data indicate that VTN present in normal plasma is a main migration inducing factor responsible for metastasis of malignant cells and that VTN is more potent chemoattractant than already known pro-metastatic chemokines or growth factors. Pro-migratory effect of VTN is neutralized/chaperoned by fibrinogen what may explain preference in migration of tumor cells into lymphatic vessels and body cavities, where concentration of fibrinogen is low. Citation Format: Gabriela Schneider, Nichola C. Garbett, Ewa Bryndza, Agata Poniewierska-Baran, Michael L. Merchant, Karol Serwin, Zachariah P. Sellers, Barbara Dolegowska, Mariusz Z. Ratajczak. Novel evidence that blood plasma vitronectin is a major chemoattractant for cancer cells and its pro-migratory activity is suppressed/chaperoned after binding to fibrinogen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1698.