C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and prostate cancer stem cells. In breast cancer, CXCR4 signalling via stromal cell-derived factor-1 (SDF-1) has been reported to be a mediator of metastasis, and is linked to poor prognosis. However its role in normal and malignant breast stem cell function has not been investigated. Anoikis-resistant (AR) cells were collected from mammosphere culture from 2 immortalised (MCF10A, 226L) and 3 malignant (MCF7, T47D, SKBR3) breast cell lines. For all cell lines, AR cells had a significantly higher mammosphere forming efficiency (MFE) than unsorted cells. The AR cells of the normal cell lines also demonstrated increased formation of 3D structures using the Matrigel assay. In vivo, MCF7 and T47D AR cells demonstrated increased capacity to form tumours compared with unsorted cells. This suggests that AR cells are enriched for normal and malignant breast stem cells. We performed an Agilent custom gene microarray and demonstrated up-regulation of CXCR4 mRNA expression in AR cells. CXCR4 protein expression was also higher in AR cells, shown by flow cytometry. The effects of AMD3100 (CXCR4 antagonist) and SDF-1 (CXCR4 ligand) on stem cell activity were investigated in the mammosphere assay. In the normal cell lines, SDF-1 significantly reduced MFE and this decrease was rescued by AMD3100. Incubation with AMD3100 increased MFE in the estrogen receptor positive breast cancer cell lines (MCF7 and T47D) and patient-derived metastatic tumour samples. AMD3100 reduced the self-renewal of T47D cells, as assessed by second generation mammospheres. MCF7 cells were retro-virally transfected to over-express CXCR4 or sorted for CXCR4 cell surface expression. Mammosphere formation was significantly increased in CXCR4+ and CXCR4 over-expressing cells compared with CXCR4- and parental cells. There was a greater reduction in self-renewal following AMD3100 treatment in the CXCR4 over-expressing cells compared with parental cells. AMD3100 has been shown to have an agonistic effect on the novel chemokine receptor CXCR7, a scavenging receptor for SDF-1. All cell lines demonstrated cell surface expression of CXCR7, measured by flow cytometry and mRNA expression. Potential interactions between CXCR4, CXCR7 and SDF-1 must be considered in future investigation of the role of CXCR4 signalling. Our data establish that CXCR4 signalling has contrasting effects on normal and malignant breast stem cell activity. CXCR4 influences self-renewal of malignant stem cells which may account for its role in tumorigenesis. CXCR4 signalling may be important in tumour formation at the sites of metastases as well as in cell migration. Its role in stem cell migration merits further investigation. In conclusion, CXCR4-targeted therapy, alongside current standards of care, may improve breast cancer outcomes.

No description available.

Selective killing of tumor cells requires the identification of drug targets critical to pathways that drive or support cancer progression. Protein kinases are an important class of intracellular enzymes involved in the regulation of biochemical pathways, deregulation of these kinases has been strongly implicated in cancer progression. To identify possible oncogenic kinases to which tumor cells might be selectively addicted, we screened the ON108 series of ATP-mimetic small molecule inhibitors in various triple negative breast cancer (TNBC) and normal cell lines. This approach led us to the identification of a novel kinase inhibitor, ON108600. We first examined the in vitro and in vivo effects of ON108600. ON108600 was found to be a potent inhibitor of Casein Kinase 2 (CK2) and the Dual-Specificity-Tyrosine (Y)-Phosphorylation-Regulated-Kinase (DYRK) family of serine-threonine kinases, both of which have been implicated in cancer progression. ON108600 showed broad-spectrum anti-proliferative and cytotoxic activity in multiple TNBC cell lines whilst having little or no effect on normal cells. Treatment of cancer cells with ON108600 resulted in inhibition of downstream signaling mediated by substrates of CK2. Further, ON108600 selectively arrested cancer cells in the mitotic phase of the cell cycle and activated the caspase-signaling cascade. We next performed x-ray crystallographic studies of ON108600-CK2 to determine the structural basis of ON108600-CK2 interaction. The co-crystal structure of ON108600-CK2 revealed that ON108600 binds in the active site pocket of CK2α wherein it mimics the binding of ATP and GTP in the CK2 active site. Notably, ON108600 mimics not only the shape and electrostatics of ATP/GTP, but also their hydration patterns in the CK2 active site pocket. Structural studies further revealed that ON108600 induces a conformational change in the β4-β5 loop of the catalytic subunit, which is known to interact with the β-regulatory subunit of CK2 and is critical for substrate recognition and activation. Lastly, we examined the efficacy of ON108600 in Triple Negative Breast Cancer (TNBC) and its ability to target and eliminate chemo-resistant Tumor-Initiating Stem Cells (TI-SCs) in TNBC. Clonogenic survival and sphere forming ability of purified CD44high CD24-/low TI-SCs from MDAMB-231 and Hs578t cells was potently inhibited by ON108600 treatment. We also observed that paclitaxel-resistant MDAMB-231 cells had increased levels of the CD44high CD24-/low stem cell- like population that correlated with increased expression of kinases CK2α2, DYRK1A and DYRK1B and these cells were sensitive to ON108600 treatment. Significantly, ON108600 showed robust antitumor efficacy as a single agent in a highly aggressive orthotopic TNBC xenograft model showing ~60% tumor growth inhibition. Immunohistochemical analysis of ON108600 treated tumors showed that a significant percentage of cells were apoptotic, indicating that activation of caspase mediated apoptosis contributes to the mechanism of action of ON108600 in vivo. Taken together, our results demonstrate that ON108600 is a novel and potent inhibitor of the CK2α1, CK2α2, DYRK1A and DYRK1B kinases. ON108600 binds in the active site pocket of CK2α and mimics ATP-GTP binding. ON108600 inhibits CK2-mediated signaling; arrests cancer cells in mitosis and induces apoptotic cell death via activation of caspases. Importantly, ON108600 is able to effectively kill the CD44high CD24-/low breast-cancer stem cell like population from TNBC cells. Finally, taxol-resistant MDAMB-231 TNBC cells express high levels of CD44, CK2α2, DYRK1a and DYRK1b and are sensitive to ON108600 treatment. Our study represents the first attempt to associate protein kinase CK2, DYRK1A and DYRK1B with TNBC and TI-SCs in TNBC and identifies a novel kinase inhibitor, ON108600 which effectively kills TI-SCs and taxol-resistant cells in TNBC. / Molecular Biology and Genetics

In the United States, metastatic breast cancer kills approximately 40,000 women and 400 men annually, and approximately 200,000 new cases of breast cancer are diagnosed each year. Worldwide, breast cancer is the leading cause of cancer deaths among women. Despite advances in the detection and treatment of metastatic breast cancer, mortality rates from this disease remain high because the fact is that once metastatic, it is virtually incurable. It is widely accepted that a major reason breast cancer continues to exhibit recurrence after remission is that current therapies are insufficient for targeting and eliminating therapy-resistant cancer cells. Emerging research has demonstrated that these therapy-resistant cells possess stem cell-like properties and are therefore commonly referred to as breast cancer stem cells (BCSCs). A major hallmark of BCSCs is the cell surface expression of CD44 and lack of expression of CD24, the so-called CD24-/CD44+ phenotype. Research indicates that this dangerous and rare subpopulation of BCSCs may be responsible for cancer onset, recurrence, and ultimately metastasis that leads to death. Two different model systems were utilized in this research. The first was the MCF7 cell line, a luminal A tumor subtype representative of a mildly invasive breast ductal carcinoma with an ER+/PR+/-/HER2- immunoprofile. The second was the MCF10A breast cancer progression model, which consists of three cell lines: MCF10A, MCF10AT1, and MCF10CA1a. In this system, spontaneously immortalized, non-malignant MCF10A cells were transfected with constitutively active H-Ras to form pre-malignant MCF10AT1 cells, which were then subcutaneously injected into mice and allowed to metastasize in order to form the oncogenic MCF10ACA1a cell line. This thesis presents evidence of a CD24low/-/CD44+ BCSC subpopulation within the MCF10A breast cancer progression model system. Findings indicate that RUNX1 and RUNX2 expression levels are involved in maintaining the BCSC phenotype. Across two different model systems, qRT-PCR analysis revealed that decreased levels of RUNX1 expression and increased levels of RUNX2 expression are essential for the maintenance of the BCSC subpopulation. It was also shown that low expression levels of RUNX1 and high expression levels of RUNX2 are present in CD24low/-/CD44+ BCSCs as compared to CD24+/CD44+ non-BCSCs. Furthermore, shRNA knockdown of RUNX1 was shown to enhance tumorigenicity, while shRNA knockdown of RUNX2 repressed tumorigenicity in BCSCs, as measured by the tumorsphere-formation assay. This research lays the groundwork for future investigations into the roles of RUNX1 and RUNX2 in regulating stemness in breast cancer.

Adult stem cells are a small population present within several tissues of an individual, possessing two unique properties: one, the ability to differentiate to give rise to all the cell types of the tissue, and second, the ability to self-renew and make more of their own kind. Owing to these two properties, stem cells underlie the process of organogenesis during development and tissue homeostasis in adult life. In the past decade a small sub-population of cells having phenotypic and functional properties similar to normal stem cells have been identified within several tumors. Only this sub-population of cancer cells seems to have the ability to both initiate and maintain tumors. These cells have been termed as ‘cancer stem cells’ (CSCs) owing to their striking similarities with the normal stem cells of the tissue. It is therefore of fundamental importance to understand normal stem cell biology in order to understand tumorigenesis. The rarity of normal stem cells within adult tissues, the absence of specific cell surface markers to identify and isolate them, and the absence of suitable culture conditions to maintain them has marred our understanding of stem cell behaviour. Recently, growth of mammary cells in serum free suspension cultures resulted in the generation of floating spheroids termed “mammospheres” that were shown to be enriched in stem/progenitor cell population. We established the mammosphere system in our laboratory using mastectomy samples obtained from the Kidwai Memorial Institute of Oncology. In order to understand the composition of the spheres, the stem cell characteristics within them, and the long term self renewal potential of human mammary epithelial stem cells, a detailed phenotypic and functional characterization of the mammospheres was carried out. Phenotypic Characterization: Confocal microscopy of propidium iodide stained mammospheres demonstrated that these spheres are cellular and not hollow structures. Immunostaining revealed that primary mammospheres expressed the epithelial markers like E Cadherin, ESA, CK14, CK18 and CK 19, but failed to express nestin or CD34, indicating their epithelial origin, devoid of contamination from haematopoeitic or neural stem cells. The sizes of mammospheres ranged from 40 to 110 μm, while that of the cells within them ranged from 9-15 μm. Although the sizes of the largest and smallest spheres through subsequent passages remained consistent, the proportion of small spheres increased in later passages. These results indicate the difference in the sphere initiating cells. While a large sphere might be generated by a stem cell, a smaller sphere might be originating from a progenitor. Thus, heterogeneity exists within mammospheres, with respect to size and composition. Unique cell surface markers coupled with flow cytometry serves as useful tools to isolate stem cells. However, no specific marker profile has been reported for normal human breast stem cells. In several tissues, like blood, brain etc, markers of normal stem cells have been successfully used to isolate cancer stem cells within that tissue. Since breast cancer stem cells have already been identified as CD24low/-44high cells, we explored if the same marker profile would hold true to identify normal breast stem cells as well. Two-colour based flow cytometry revealed that only the CD24low/-44high subpopulation of mammospheres could re-generate mammospheres, as well as give rise to all the other cellular fractions. These data demonstrated that normal and cancerous breast stem cells share identical marker profile. Functional Characterization: In addition to cell surface markers, a Hoechst dye based strategy used to isolate stem cells, exploits their unique property to efflux certain lipophilic drugs and small molecules due to the overexpression of ABC family of cell surface transporters. Cells effluxing Hoechst appear as a low fluorescing ‘Side population’ (SP) in a bivariate FACS plot. We detected a small, but distinct SP in human breast cells, which had a CD24low44low profile, and failed to initiate new mammospheres. Thus, the SP cells in mammospheres failed to correspond to the stem cell subpopulation. The hallmark feature of a stem cell is its long term self renewal ability, given that it is the longest lived cell in the body. Long term culture of mammospheres was carried out by passaging the spheres every week. We failed to observe mammosphere formation beyond four passages though there were live, proliferating and undifferentiated cells in fourth passage spheres. These results suggested that either the mammopsheres didn’t contain stem cells to begin with, or their stemness is restricted to four in vitro passages. In order to assess if mammospheres contained stem cells to begin with, we assayed for telomerase activity, since in the adult tissue, only stem cells retain telomerase activity. Telomerase, an enzyme that maintains the length of telomeres through multiple rounds of cell division, is not active in somatic cells. We detected the expression and activity of this enzyme in primary mammospheres, suggesting that the spheres may contain stem cells withinthem Another unique property of a stem cell is its ‘quiescence’, owing to their infrequent divisions. This property is studied by chasing a label (like BrdU or H3-Thymidine), which is taken up by the cells at an earlier time point and retained within the cell after prolonged periods, like weeks or months. In long term culture of mammospheres, using BrdU as the label, 1-2 distinct cells could be detected within late passage spheres which had retained the label, indicating that stem cells may be present within the fourth passage mammospheres as well. Staining for β-Galactosidase activity revealed that almost 70% cells derived from fourth passage spheres were senescent. We speculated that this senescent environment might be one of the inhibitory reasons for further mammosphere formation. Alteration of mammosphere culture conditions for long term maintenance of stem cells. A high level of atmospheric O2 is known to be one of the reasons for inducing senescence in cells. Culturing cells in conventional tissue culture conditions exposes them to high levels of O2 (21%) as against the physiological levels of 1-3% O2. Therefore, to assess the effects of lowered, or physiologically relevant levels of O2 on mammosphere stem cell biology, the mammospheres were cultured in 3% O2. Under this altered condition, a close to 3-fold increase was observed in the number of mammospheres formed coupled with a significant increase in their survival and proliferation. In order to understand the molecular basis of this observation, a microarray based global gene expression profiling was carried out. We observed a significant upregulation of VEGF, a gene responsive to hypoxia; three growth factor related genes, namely adrenomedullin, cMET and osteopontin. Upregulation of β Catenin, the downstream effector of the Wnt signaling pathway was also observed, indicating a possible mechanism for the increase in self renewal seen in 3% O2. We also observed downregulation of the cell cycle inhibitor, Chk1, which in part might explain the observed increase in proliferation. The increase in the number of proliferating cells might be one of the reasons for an increase in the number of spheres, as observed in 3% O2. Even though a significant decrease in the number of senescent cells was detected at 3% O2, mammosphere formation was not seen beyond four passages. It is therefore possible that there are other physico-chemical parameters, comprising the niche of the mammospheres, coupled to the O2 level, which need to be improvised for long term culture of human mammary epithelial stem cells. To summarize, this work reports for the first time that human mammary epithelial stem cells have an identical marker profile as breast cancer stem cells, which is CD24low/-CD44high. It has also been demonstrated for the first time that in long term mammosphere culture, the number of self renewal divisions of human mammary stem cells is restricted to four in vitro passages, at which most of the cells undergo senescence. Altering one of the parameters of the niche, by culturing mammospheres at physiological O2 level failed to prolong the in vitro lifespan of the spheres, although cell survival, proliferation and sphere formation increased, indicating that the niche requirements of human mammary epithelial stem cells for their long term self renewal needs to be further characterized.

No description available.