FGFR2 Promotes Breast Tumorigenicity through Maintenance of Breast Tumor-Initiating Cells

<div><p>Emerging evidence suggests that some cancers contain a population of stem-like TICs (tumor-initiating cells) and eliminating TICs may offer a new strategy to develop successful anti-cancer therapies. As molecular mechanisms underlying the maintenance of the TIC pool are poorly understood, the development of TIC-specific therapeutics remains a major challenge. We first identified and characterized TICs and non-TICs isolated from a mouse breast cancer model. TICs displayed increased tumorigenic potential, self-renewal, heterogeneous differentiation, and bipotency. Gene expression analysis and immunostaining of TICs and non-TICs revealed that FGFR2 was preferentially expressed in TICs. Loss of FGFR2 impaired self-renewal of TICs, thus resulting in marked decreases in the TIC population and tumorigenic potential. Restoration of FGFR2 rescued the defects in TIC pool maintenance, bipotency, and breast tumor growth driven by FGFR2 knockdown. In addition, pharmacological inhibition of FGFR2 kinase activity led to a decrease in the TIC population which resulted in suppression of breast tumor growth. Moreover, human breast TICs isolated from patient tumor samples were found enriched in a FGFR2+ population that was sufficient to initiate tumor growth. Our data suggest that FGFR2 is essential in sustaining the breast TIC pool through promotion of self-renewal and maintenance of bipotent TICs, and raise the possibility of FGFR2 inhibition as a strategy for anti-cancer therapy by eradicating breast TICs.</p> </div

CD29^highCD24⁺ Cells Have Self-Renewal Capacity and Contain Bipotent Precursor-like Cells.

<p>(A) Mammosphere assay for the four FACS-sorted subpopulations from MMTV-PyMT breast tumors. The number of spheres (>50 µm in diameter) formed from 2000 or 4000 seeded cells was measured 3 weeks after seeding. Statistical comparison with CD29^highCD24⁺ subpopulation (*<i>P</i><0.02; **<i>P</i><0.01). Data (n = 3) represent mean ± SEM. Scale bars in the sphere images represent 50 µm. (B) Differentiation potential of breast tumor subpopulations assessed by immunofluorescence. The breast tumor cells from the four FACS-sorted subpopulations were cultured under the differentiation condition. The sorted cells from various populations were plated at the same cell density on collagen-coated plates. The differentiated cells were stained for the luminal epithelial marker (K18, green), the myoepithelial markers (K14 and SMA, red), and DAPI (nuclei, blue). A significant portion of CD29^highCD24⁺ cells contains K18⁺K14⁺ (bipotent precursor-like), whereas the majority of cells from the other subpopulations contain lineage-restricted cells. Magnifications of the boxed regions are shown below the figure. Scale bars represent 32 µm. (C) Myoepithelial lineage-specific differentiation assessed by immunofluorescence. The differentiated tumor cells from FACS-sorted subpopulations were stained for myoepithelial markers (SMA, red), and DAPI (nuclei, blue). Scale bars represent 120 µm. (D) Quantification of the immunofluorescence images (B and C) for the frequency of K18⁺K14⁻ (luminal), K18⁻K14⁺ (myoepithelial), K18⁺K14⁺ (bipotent precursor-like), and SMA⁺ (myoepithelial) cells. Image quantification was achieved using the multi-wavelength cell scoring analysis module on the ImageXpress Ultra confocal microscope. An average of three values from three images (different positions) per group is presented. Statistical comparison with CD29^highCD24⁺ subpopulation (*<i>P≤</i>0.05; **<i>P≤</i>0.01; ***<i>P</i><0.001). Data (n = 3) represent mean ± SEM.</p

Gene Expression Profiles of Breast TICs and Non-TICs Revealed that FGFR2 Is Preferentially Expressed in Breast TICs.

<p>(A) Heat map of microarray analysis depicting the expression levels of the mRNAs of 7 genes that are significantly upregulated in the TIC population compared with the three other non-TIC populations. RNA was prepared from the four FACS-sorted populations of primary MMTV-PyMT breast tumors. Red and green indicate high and low mRNA expression levels, respectively. The expression values were normalized by Z score. (B) The expression levels of the mRNAs of 7 genes in TICs and non-TICs, as determined by quantitative real-time PCR. cDNA isolated from non-TICs was used to normalize data for each primer of gene and generate RQ (relative quantity). (C) The expression levels FGFR2 protein in the subpopulations by flow cytometry. This figure represents a typical result of three independent experiments. Dashed line shows isotype labeling.</p

Loss of FGFR2 Impaired Self-Renewal of Breast TICs, Resulting in a Decreased TIC Pool.

<p>(A) Flow cytometry analysis of CD24 and CD29 expression for TIC and non-TIC subpopulation frequencies in shRNA-transduced primary MMTV-PyMT breast tumor cells. (B) Quantification of TIC and non-TIC subpopulation frequencies in shRNA transduced breast tumor cells determined by FACS analysis (A). Data (n = 2) represent mean ± s.d. Statistical comparison with shNT (*<i>P</i><0.027; **<i>P</i><0.015). (C) Reduced TIC sphere formation (mean ± SEM., n = 2) of breast tumor cells upon FGFR2 knockdown. Ten thousand FACS-sorted TICs were transduced with lentiviral shRNAs and then subjected to a mammosphere (>50 µm in diameter) forming assay. (D) Differentiation potential of shRNA-transduced breast tumor cells as assessed by immunofluorescence. The shRNA-transduced tumor cells were cultured with a selective drug for 7 days after infection and then were plated them at the same cell density under the differentiation condition for 6 days. The cells were stained for the luminal epithelial marker (K18, green), myoepithelial markers (K14 and SMA, red), and DAPI (nuclei, blue). The scale bars for K18/K14 and SMA represent 40 µm and 140 µm, respectively. (E) Quantification of the immunofluorescence images (D) for the frequency of K18⁺K14⁻ (luminal), K18⁻K14⁺ (myoepithelial), K18⁺K14⁺ (bipotent precursor-like), and SMA⁺ (myoepithelial) cells in shRNA transduced tumor cells. Image quantification was achieved using the multi-wavelength cell scoring analysis module on the ImageXpress Ultra confocal microscope. Statistical comparison with mock infection (*<i>P</i><0.05; **<i>P≤</i>0.01; ***<i>P</i><0.0003). Data (n = 3) represent mean ± SEM.</p

Antitumor Activity of FGFR Inhibitor as a Result of a Decrease in the Breast TIC Subpopulation In Vivo.

<p>(A) Antitumor activity of FGFR inhibitor, TKI258. Daily oral administration of TKI258 at 50 mg/kg was initiated in tumor-bearing NOD/SCID mice (n = 5 per group) when the MMTV-PyMT breast tumors reached ∼150 mm³ in volume. <i>P</i> value at day 32 is indicated. (B) Reduced phosphorylation of FGFR2 and Erk1/2 upon treatment of breast tumors with TKI258. Breast tumors were collected at predose, 4 and 24 hours after administration of a single oral dose of TKI258. Tumors were homogenized, immunoprecipitated for FGFR2 and immunoblotted with anti-phosphotyrosine to evaluate phosphorylation of FGFR2. The membranes were reprobed to evaluate total level of FGFR2 protein. (C) Flow cytometry analysis of CD24 and CD29 expression for TIC and non-TIC subpopulation frequencies in TKI258- or control-treated breast tumors. (D) Quantification of TIC and non-TIC subpopulations in TKI258- or control-treated breast tumors determined by flow cytometry analysis (C). Data (n = 3) represent mean ± SEM. Statistical comparison with control treatment (*<i>P</i><0.04; **<i>P</i><0.02; ***<i>P</i><0.002).</p

Small Molecule Mediated Proliferation of Primary Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells <i>ex vivo</i> could provide a renewable source of cells for transplantation. Here, we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration

Enrichment of Breast Tumor-Initiating Cells in the CD29^hiCD24⁺ Population.

<p>(A) Isolation of five populations from MMTV-PyMT breast tumors based on the expression of cell-surface markers CD29 and CD24 by FACS. (B) Limiting dilution analyses of tumors to calculate the frequency of TICs in primary breast tumor subpopulations isolated from the primary tumors during secondary tumor formation. The number of tumors was measured 12–16 weeks after injection of 50–5000 tumor cells of five subpopulations into the breast of NOD/SCID mice. The significance of difference in TIC frequencies for each subpopulations against the CD29^highCD24⁺ population is indicated by <i>P</i> values (<i>P difference</i><0.05). The single hit hypothesis was validated by likelihood ratio tests and accepted for all dilution series (<i>P fit</i>>0.05). (C) Comparison of the growth of secondary breast tumors which arose from purified primary tumor cells of different subpopulations. The number of cells injected is indicated next to the population. (D) Flow cytometry analysis of CD24 and CD29 expression after serial transplantation demonstrates that secondary and tertiary tumors initiated from the primary and secondary CD29^hiCD24⁺ population, respectively are as heterogeneous as the primary tumors. (E) Limiting dilution analyses of tumors to calculate the frequency of TICs in secondary subpopulations isolated from the secondary tumors during tertiary tumor formation. The CD29^highCD24⁺ population isolated from the secondary tumors displayed significantly (<i>P = </i>3.6×10⁻¹²) higher frequency of TICs compared to non-CD29^highCD24⁺ populations. (F) Comparison of the growth of tertiary breast tumors in NOD/SCID mice which were generated from purified tumor cells of the secondary CD29^highCD24⁺ or non-CD29^highCD24⁺ populations.</p

Suppression of Breast Tumor Growth and Inhibition of Oncogenic Signaling by Loss of FGFR2.

<p>(A) Quantitative real-time PCR analysis of expression levels of FGFR2 mRNA in MMTV-PyMT breast tumor cells stably transduced with lentiviral shRNAs targeting FGFR2 (shFGFR2) and non-targeting shRNA (shNT). shFGFR2-2 and shFGFR2-3, targeting different regions of <i>FGFR2</i> gene, were used for knockdown of FGFR2. Mock (no infection) and shNT infection were used as negative controls. (B) Reduced proliferation of breast tumor cells upon FGFR2 knockdown <i>in vitro</i>. Proliferation of tumor cells was determined by cell viability using CellTiter-Glo reagent (Promega). Statistical comparison with shNT (*<i>P</i><0.05). (C) Suppression of anchorage-independent growth (mean ± SEM., n = 2) of breast tumor cells upon FGFR2 loss was assessed by the ability of cells forming colonies (>100 µm in diameter) in soft agar. The number of colonies from 10000 cells seeded is shown. (D) Inhibition of downstream target activation upon FGFR2 knockdown. shFGFR2 efficiently knocked down the expression of FGFR2 protein and strongly inhibited phosphorylation of Erk1/2, as evidenced by immunoblotting with anti-phospho-ERK1/2 (p-ERK1/2). The bands with lower molecular weights are either cleaved forms or alternative spliced variants of endogenous FGFR2 protein (e.g. sliced variants of carboxyl terminal domains). The membranes were reprobed for actin and Erk1/2 as loading controls. (E) Immunoblotting analysis of expression levels of FGFR2 protein in the breast tumor cells transduced with a lentiviral shFGFR2 and/or a retroviral vector encoding FGFR2. Restored FGFR2 expression was shown in the tumor cells transduced with a lentiviral shFGFR2 and a retroviral FGFR2. Anti-FGFR2 immunoblotting was reprobed for actin as a loading control. (F) Effect of FGFR2 loss and restoration on breast tumor growth <i>in vivo</i>. The breast tumor cells transduced with the lentiviral shFGFR2 and/or retroviral FGFR2 vector were injected in the mammary fat pad of the NOD/SCID mice. Mock, shNT, and empty vector were used as negative controls for infection, lentiviral shRNA, and retroviral cDNA, respectively. Data (n = 4) represent mean ± SEM. Statistical comparison with shNT (*<i>P</i><0.037).</p

Human Breast TICs Were Enriched in FGFR2+ Population that Was Sufficient to Initiate Tumor Growth.

<p>(A) The expression levels of FGFR2 mRNA in patient-derived breast tumors. Quantitative real-time PCR was performed using cDNA generated from RNA isolated from 26 primary human breast cancer specimens. cDNA isolated from a normal breast tissue was used to normalize data and generate RQ. (B) Flow cytometry analysis of FGFR2 protein expression in primary human breast tumors. High (BT5 and BT12) or low level (BT8 and BT25) of FGFR2 protein corresponded to high or low level of <i>FGFR2</i> mRNA (A). This figure represents a typical result of three independent experiments. Dashed line shows an unstained control for each tumor sample. (C) Effect of FGFR2 expression on primary human breast tumor growth. FGFR2−overexpressing primary human breast tumor cells (BT5) were FACS sorted based on the expression of FGFR2. The two isolated populations (FGFR+ and FGFR2−) were injected in the mammary fat pad of the NOD/SCID mice (n = 5 per group). <i>P</i> value at day 70 is indicated. Data represent mean ± SEM. (D) Flow cytometry analysis of ALDH activity in FGFR2+ and FGFR2− subpopulations of BT5 tumors. Higher ALDH activity was found in the FGFR2+ population compared to the FGFR2− population. Dashed line shows a specific inhibitor of ALDH (DEAB)-treated labeling.</p

The legacy of ICTY and its impact upon the Statue and the practice of the ICC

Legacy of ICTY and its Impact on Statute and Case Law of ICC Establishing of International Criminal Court is a significant moment in evolution of international criminal law. It is the first permanent institution of international justice with personal jurisdiction and it was founded for one purpose only - to finally end the era of impunity for war criminals all over the world. But ICC didn't appear from nowhere. There was a long road towards its establishment and who knows if it would ever happen without influence of so called ad hoc tribunals, International Criminal Tribunal for the former Yugoslavia and International Criminal Tribunal for Rwanda. It is a sad truth that international criminal law made its biggest evolution as a reflection of the world's most horrible conflicts. The purpose of this thesis is to analyze a legacy of one of these tribunals, International Criminal Tribunal for the former Yugoslavia, and to explore its influence not only on the Statute of International Criminal Court, but also on its case law. International Criminal Court is not operating for long, but we can still notice many references on ICTY case law in its judgments. The reason for this research is my particular relation towards Balkan region as well as my interest in international justice in general. The thesis is composed..