Tumors In Immunocompromised Mice Research Articles

Aldehyde Dehydrogenase in Combination with CD133 Defines Angiogenic Ovarian Cancer Stem Cells That Portend Poor Patient Survival

Markers that reliably identify cancer stem cells (CSC) in ovarian cancer could assist prognosis and improve strategies for therapy. CD133 is a reported marker of ovarian CSC. Aldehyde dehydrogenase (ALDH) activity is a reported CSC marker in several solid tumors, but it has not been studied in ovarian CSC. Here we report that dual positivity of CD133 and ALDH defines a compelling marker set in ovarian CSC. All human ovarian tumors and cell lines displayed ALDH activity. ALDH(+) cells isolated from ovarian cancer cell lines were chemoresistant and preferentially grew tumors, compared with ALDH(-) cells, validating ALDH as a marker of ovarian CSC in cell lines. Notably, as few as 1,000 ALDH(+) cells isolated directly from CD133(-) human ovarian tumors were sufficient to generate tumors in immunocompromised mice, whereas 50,000 ALDH(-) cells were unable to initiate tumors. Using ALDH in combination with CD133 to analyze ovarian cancer cell lines, we observed even greater growth in the ALDH(+)CD133(+) cells compared with ALDH(+)CD133(-) cells, suggesting a further enrichment of ovarian CSC in ALDH(+)CD133(+) cells. Strikingly, as few as 11 ALDH(+)CD133(+) cells isolated directly from human tumors were sufficient to initiate tumors in mice. Like other CSC, ovarian CSC exhibited increased angiogenic capacity compared with bulk tumor cells. Finally, the presence of ALDH(+)CD133(+) cells in debulked primary tumor specimens correlated with reduced disease-free and overall survival in ovarian cancer patients. Taken together, our findings define ALDH and CD133 as a functionally significant set of markers to identify ovarian CSCs.

A new approach to delay the development of resistance to docetaxel in the treatment of castration-refractory prostate cancer.

e15177 Background: Resistance to docetaxel is a common problem in the treatment of castration-refractory prostate cancer (CPRC). Pigment epithelium-derived factor (PEDF) is antiangiogenic. We have shown that: (a) PEDF is down-regulated in prostate cancer; and (b) over-expression of PEDF in CRPC cells decreased tumor growth in vivo. Since both increase in interleukin-8 (IL8) production and, plasma membrane Vacuolar H+-ATPases(pmV-ATPases) have been involved in the resistance of cancer cells to chemotherapeutic drugs, we hypothesize that PEDF enhances docetaxel cytotoxicity by modulating IL8 and pmV-ATPases in prostate cancer cells. Methods: CRPC cells (PC3/DU145) were treated with 0-100 nM PEDF alone or in combination with 0-100 nM docetaxel. IL8 was measured in cell conditioned media by ELISA. mRNA levels of the a isoforms that target V-ATPase to the cell surface were investigated by qPCR. pmV-ATPase activity was measured on isolated plasma membranes. To evaluate PEDF anti-tumor properties, we used human CRPC LNCaP-derivative CL1 and LNCaP cell lines that stably express the fluorescent DsRed Express protein in combination with PEDF. Results: We found that PEDF inhibits the proliferation of all CPRC cells tested in vitro associated with a decrease in IL8 production in PC3 and DU145 cells. The effect of PEDF on growth was prominent in PC3 and DU145 cells under PEDF/docetaxel combined treatment. The mRNA levels of the a isoforms of pmV-ATPase were decreased when PEDF is expressed. PEDF expression in vivo decreases by 70% the development of subcutaneous CL1 tumors in immuno-compromised mice. We injected CL1 cells (pre-mixed with 50 nM human recombinant PEDF) into the left lobe of mice dorsal prostate. We found that all control animals die within 61 days after surgery. Importantly, 50% of PEDF-pretreated animals were still alive. Conclusions: These data indicate that inhibition of V-ATPases and over-expression of PEDF are promising new targets to enhance the cytotoxicity of docetaxel drug. Understanding the consequences of V-ATPase inhibition or PEDF over-expression in human prostate cancer cells will contribute significantly to our understanding of prostate cancer progression and treatment.

Abstract 2445: CD24+ liver tumor-initiating cells drives self-renewal and tumor initiation through Stat3-mediated Nanog regulation

Abstract Tumor-initiating cells (TICs) are a subpopulation of chemoresistant tumor cells that have been shown clinically to cause tumor recurrence upon chemotherapy. Identification of T-ICs and their related pathways are therefore priorities for the development of novel therapeutic paradigms. We established chemoresistant hepatocellular carcinoma (HCC) xenograft tumors in immunocompromised mice in which an enriched T-IC population was capable of tumor initiation and self-renewal. Using this model, we found CD24 to be up-regulated in residual chemoresistant tumor tissue when compared with bulk tumor upon cisplatin treatment. The determine whether CD24 is a candidate TIC marker for HCC, cell sorting approach was employed to separate CD24-/CD24+ populations derived from HCC cell lines and clinical samples. CD24+ HCC cells were found to be critical for the maintenance of tumor growth, self-renewal, differentiation, chemoresistance and metastasis of tumors. Using quantitative PCR, CD24 was over-expressed in HCCs when compared with their non-tumor counterparts, and CD24 expression was significantly correlated with poor patients’ survival. Using a lentiviral-based knockdown approach, CD24 was found to be a functional liver T-IC marker that drives tumor initiation and self-renewal. Notably, whether by cell sorting or gene-knockdown approach, it was shown by quantitative PCR that the CD24-enriched population consistently overexpressed Nanog. Upon transfection of Nanog cDNA into CD24-knockdown cells, self-renewal and tumor formation were functionally recovered, suggesting Nanog as the downstream effecter of CD24. By Ingenuity Pathway analysis, ‘acute phase response signaling’ was found to be the most significantly altered upon CD24 knockdown in which phosphorylation of Stat3 at the Tyrosine705 residue is critical. Using Stat3 inhibitor (S3I-201), we found that Nanog promoter and its protein were down-regulated in dose-dependent manner in CD24+ HCC cells. CD24 was found to be a functional liver T-IC marker that drives T-IC genesis through Stat3-mediated Nanog regulation. In conclusion, we identified CD24+ HCC cells within bulky tumor, and they functioned to initiate tumor growth and self-renewal through Stat3-mediated Nanog up-regulation. The identification of novel CD24 signaling pathways provides an attractive therapeutic strategy against this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2445. doi:10.1158/1538-7445.AM2011-2445

Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research.

Cancer stem cells have been hypothesized to drive the growth and metastasis of tumors. Because they need to be targeted for cancer treatment, they have been isolated from many solid cancers. However, cancer stem cells from primary human gastric cancer tissues have not been isolated as yet. For the isolation, we used two cell surface markers: the epithelial cell adhesion molecule (EpCAM) and CD44. When analyzed by flow cytometry, the EpCAM(+)/CD44(+) population accounts for 4.5% of tumor cells. EpCAM(+)/CD44(+) gastric cancer cells formed tumors in immunocompromised mice; however, EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) and EpCAM(-)/CD44(+) cells failed to do so. Xenografts of EpCAM(+)/CD44(+) gastric cancer cells maintained a differentiated phenotype and reproduced the morphological and phenotypical heterogeneity of the original gastric tumor tissues. The tumorigenic subpopulation was serially passaged for several generations without significant phenotypic alterations. Moreover, EpCAM(+)/CD44(+), but not EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) or EpCAM(-)/CD44(+) cells grew exponentially in vitro as cancer spheres in serum-free medium, maintaining the tumorigenicity. Interestingly, a single cancer stem cell generated a cancer sphere that contained various differentiated cells, supporting multi-potency and self-renewal of a cancer stem cell. EpCAM(+)/CD44(+) cells had greater resistance to anti-cancer drugs than other subpopulation cells. The above in vivo and in vitro results suggest that cancer stem cells, which are enriched in the EpCAM(+)/CD44(+) subpopulation of gastric cancer cells, provide an ideal model system for cancer stem cell research.

Shank-interacting protein–like 1 promotes tumorigenesis via PTEN inhibition in human tumor cells

Inactivation of phosphatase and tensin homolog (PTEN) is a critical step during tumorigenesis, and PTEN inactivation by genetic and epigenetic means has been well studied. There is also evidence suggesting that PTEN negative regulators (PTEN-NRs) have a role in PTEN inactivation during tumorigenesis, but their identity has remained elusive. Here we have identified shank-interacting protein-like 1 (SIPL1) as a PTEN-NR in human tumor cell lines and human primary cervical cancer cells. Ectopic SIPL1 expression protected human U87 glioma cells from PTEN-mediated growth inhibition and promoted the formation of HeLa cell-derived xenograft tumors in immunocompromised mice. Conversely, siRNA-mediated knockdown of SIPL1 expression inhibited the growth of both HeLa cells and DU145 human prostate carcinoma cells in vitro and in vivo in a xenograft tumor model. These inhibitions were reversed by concomitant knockdown of PTEN, demonstrating that SIPL1 affects tumorigenesis via inhibition of PTEN function. Mechanistically, SIPL1 was found to interact with PTEN through its ubiquitin-like domain (UBL), inhibiting the phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase activity of PTEN. Furthermore, SIPL1 expression correlated with loss of PTEN function in PTEN-positive human primary cervical cancer tissue. Taken together, these observations indicate that SIPL1 is a PTEN-NR and that it facilitates tumorigenesis, at least in part, through its PTEN inhibitory function.

Derivation and Characterization of an Extra-Axial Chordoma Cell Line (EACH-1) from a Scapular Tumor

Extra-axial chordomas are rare low-grade malignant tumors thought to arise from notochordal remnants in the extra-axial skeleton. Few studies have been done on this neoplasm because of its rarity. In addition, there is a lack of a good in vitro model on which to perform more characterization. We describe a twenty-eight-year-old man with a mass in the right scapula. Cytomorphology and immunohistochemistry, including brachyury staining, were used to formulate the final diagnosis. A fragment of the tumor was placed in culture, and cells obtained were injected subcutaneously in an immunocompromised mouse. From the tumor developed in mice, a cell line has been derived and characterized by fluorescence-activated cell-sorting analysis, karyotyping, clonogenicity, and cell and tumor growth curves. Cytomorphology on the tumor showed nests of round cells with vacuoles and also physaliferous-like cells with uniform nuclei. Immunochemistry revealed a tumor positive for vimentin, moderately positive for S-100 and cytokeratin AE1/AE3, weakly positive for epithelial membrane antigen, and negative for p63 and cytokeratin (CK)-7. Further analysis revealed the tumor was diffusely and strongly positive for brachyury. The cell line derived from the tumor showed rapid doubling-time, a strong expression of mesenchymal cell surface markers, a karyotype of diploid or hypotetraploid clones with numerous chromosomal aberrations, and the ability to form colonies without attachment and to form tumors in immunocompromised mice. The diagnosis of the extra-axial chordoma is difficult but can be resolved by the detection of a strong brachyury expression. In addition, the derivation of a human extra-axial chordoma cell line could be a useful tool for the basic research of this rare neoplasm.

Permanently Blocked Stem Cells Derived From Breast Cancer Cell Lines

Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. However, an unanticipated result was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immunocompromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs.

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

Tumor initiating cells in malignant gliomas: biology and implications for therapy

A rare subpopulation of cells within malignant gliomas, which shares canonical properties with neural stem cells (NSCs), may be integral to glial tumor development and perpetuation. These cells, also known as tumor initiating cells (TICs), have the ability to self-renew, develop into any cell in the overall tumor population (multipotency), and proliferate. A defining property of TICs is their ability to initiate new tumors in immunocompromised mice with high efficiency. Mounting evidence suggests that TICs originate from the transformation of NSCs and their progenitors. New findings show that TICs may be more resistant to chemotherapy and radiation than the bulk of tumor cells, thereby permitting recurrent tumor formation and accounting for the failure of conventional therapies. The development of new therapeutic strategies selectively targeting TICs while sparing NSCs may provide for more effective treatment of malignant gliomas.

Increasingly transformed MCF-10A cells have a progressively tumor-like phenotype in three-dimensional basement membrane culture

BackgroundMCF-10A cells are near diploid and normal human mammary epithelial cells. In three-dimensional reconstituted basement membrane culture, they undergo a well-defined program of proliferation, differentiation, and growth arrest, forming acinar structures that recapitulate many aspects of mammary architecture in vivo. The pre-malignant MCF-10AT cells and malignant MCF-10CA1a lines were sequentially derived from the MCF-10A parental cell line first by expression of a constitutively active T24 H-Ras generating the MCF-10AT cell line. This was followed by repeated selection for increasingly aggressive tumor formation from cells recovered from xenograft tumors in immuno-compromised mice, generating the MCF-10CA1a cell line. When inoculated subcutaneously into the flanks of immuno-compromised mice, MCF-10AT cells occasionally form tumors, whereas MCF-10CA1a cells invariably form tumors with a shorter latency than MCF-10AT derived tumors.ResultsMCF-10AT cells grown in three-dimensional basement membrane culture form complex multi-acinar structures that produce a basement membrane but undergo delayed cell cycle arrest and have incomplete luminal development. MCF-10CA1a cells grown in three-dimensional basement membrane culture form large, hyper-proliferative masses, that retain few characteristics of MCF10A acini and more closely resemble tumors.ConclusionHere we report on the growth and differentiation properties of these three matched cell lines in three-dimensional basement membrane culture. Features of tissue morphogenesis were assessed, including proliferation, basement membrane formation, polarization of alpha-6 beta-4 integrin to the basement membrane, formation of cell:cell junctions, and apoptosis for luminal clearance. The matched series of normal MCF-10A, pre-malignant MCF-10AT, and malignant MCF-10CA1a cells offers a unique opportunity to study the mechanisms of malignant progression both in a three-dimensional microenvironment and in the same cell background.

Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage

Mutation of the RB-1 and p53 tumor suppressors is associated with the development of human osteosarcoma. With the goal of generating a mouse model of this disease, we used conditional and transgenic mouse strains to inactivate Rb and/or p53 specifically in osteoblast precursors. The resulting Rb;p53 double mutant (DKO) animals are viable but develop early onset osteosarcomas with complete penetrance. These tumors display many of the characteristics of human osteosarcomas, including being highly metastatic. We established cell lines from the DKO osteosarcomas to further investigate their properties. These immortalized cell lines are highly proliferative and they retain their tumorigenic potential, as judged by their ability to form metastatic tumors in immunocompromised mice. Moreover, they can be induced to differentiate and, depending on the inductive signal, will adopt either the osteogenic or adipogenic fate. Consistent with this multipotency, a significant portion of these tumor cells express Sca-1, a marker that is typically associated with stem cells/uncommitted progenitors. By assaying sorted cells in transplant assays, we demonstrate that the tumorigenicity of the osteosarcoma cell lines correlates with the presence of the Sca-1 marker. Finally, we show that loss of Rb and p53 in Sca-1-positive mesenchymal stem/progenitor cells is sufficient to yield transformed cells that can initiate osteosarcoma formation in vivo.

Abstract 4998: Molecular profiling reveals heterogeneity of active self-renewal pathways in bladder cancer stem cells

Abstract The objective of the current study is to investigate the functional involvement of self-renewal related genes in patient bladder cancer stem cells. First, by fractionating primary patient bladder transitional cell carcinomas (TCCs) into subpopulations with flow cytometry, a unique tumor-initiating subpopulation expressing CD44 was identified in 5 independent TCCs, based on their enriched potential (10-200 folds) to induce xenograft tumors in immunocompromised mice. To extend this finding to a larger sample set, we analyzed a tissue array containing more than 300 bladder TCCs by immunohistochemistry. Approximately 40.4% of TCCs contain CD44+ cells; these cells usually express cytokeratin 5 (CK5) (P<0.0001) but not cytokeratin 20 (P=0.8160). In addition, cellular morphologies of CD44+ tumor cells are often small and round shape. These data suggest that CD44+ tumor cells retain cytokeratin markers and cellular morphologies resembling that of urothelial basal cells. Further, molecular profiling of oncogenic proteins implicated in the self-renewal of stem cells (i.e. Bmi1, Stat3, ß-catenin, Gli1, Oct4 and Nanog) revealed heterogeneity in bladder TCCs. Different subsets of CD44+ bladder TCCs express either one or more of different self-renewal proteins in their active forms (i.e. ~10% with nuclear Bmi1, 30% with nuclear Stat3, 5% with nuclear ß-catenin, 85% with active Gli1 and 0% with active Oct4 nor Nanog). In particular, in situ hybridization demonstrated that Gli1 mRNA was present in over 85% of bladder TCC specimens analyzed. Therefore, we examined the functional significance of the sonic hedgehog pathway in bladder tumor development. In patient bladder xenografts that originally contained active Gli1 in the cancer stem cells, preliminary experiment demonstrated that inhibition of this pathway with cyclopamine resulted in partial reduction of tumor volume (4 out of 6 xenografts). Previous reports demonstrated that genetic mutations in the pathway components (i.e. PTCH or Gli1) leading to constitutive Gli1 activation is rare in bladder TCCs. Preliminary microRNA (miRs) profiling of CD44+ bladder cancer stem cells using a real-time PCR based approach revealed a panel of differentially expressed miRs. Interestingly, in several highly elevated miRs in CD44+ bladder cancer stem cells, the negative regulators of the Gli1 pathways (i.e. PTCH and SUFU) are among the top predicted targets for these miRs. Further experiments are ongoing to validate the possible involvement of miRs in regulating the Gli1 pathways in bladder cancer stem cells. In conclusion, we demonstrated the existence of a functionally unique, CD44+ tumorigenic subpopulation in a subset of bladder TCCs. Diverse “self-renewal” proteins, including Gli1, possibly contribute to the tumorigenic properties of cancer stem cells in different subset of patient bladder TCCs.

IQGAP1 Stimulates Proliferation and Enhances Tumorigenesis of Human Breast Epithelial Cells

The scaffold protein IQGAP1 integrates signaling pathways and participates in diverse cellular activities. IQGAP1 is overexpressed in a number of human solid neoplasms, but its functional role in tumorigenesis has not been previously evaluated. Here we report that IQGAP1 contributes to neoplastic transformation of human breast epithelial cells. The amount of IQGAP1 in breast carcinoma is greater than that in normal tissue, with highly metastatic breast epithelial cells expressing the highest levels. Overexpression of IQGAP1 enhances proliferation of MCF-7 breast epithelial cells. Reduction of endogenous IQGAP1 by RNA interference impairs both serum-dependent and anchorage-independent growth of MCF-7 cells. Consistent with these in vitro observations, immortalized MCF-7 cells overexpressing IQGAP1 form invasive tumors in immunocompromised mice, whereas tumors derived from MCF-7 cells with stable knockdown of IQGAP1 are smaller and less invasive. In vitro analysis with selected IQGAP1 mutant constructs and a chemical inhibitor suggests that actin, Cdc42/Rac1, and the mitogen-activated protein kinase pathway contribute to the mechanism by which IQGAP1 increases cell invasion. Collectively, our data reveal that IQGAP1 enhances mammary tumorigenesis, suggesting that it may be a target for therapeutic intervention.

Mutation of Prkar1a Causes Osteoblast Neoplasia Driven by Dysregulation of Protein Kinase A

Carney complex (CNC) is an autosomal dominant neoplasia syndrome caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A (PKA). This genetic defect induces skin pigmentation, endocrine tumors, myxomas, and schwannomas. Some patients with the complex also develop myxoid bone tumors termed osteochondromyxomas. To study the link between the PRKAR1A mutations and tumor formation, we generated a mouse model of this condition. Prkar1a(+/-) mice develop bone tumors with high frequency, although these lesions have not yet been characterized, either from human patients or from mice. Bone tumors from Prkar1a(+/-) mice were heterogeneous, including elements of myxomatous, cartilaginous, and bony differentiation that effaced the normal bone architecture. Immunohistochemical analysis identified an osteoblastic origin for the abnormal cells associated with islands of bone. To better understand these cells at the biochemical level, we isolated primary cultures of tumoral bone and compared them with cultures of bone from wild-type animals. The tumor cells exhibited the expected decrease in Prkar1a protein and exhibited increased PKA activity. At the phenotypic level, we observed that tumor cells behaved as incompletely differentiated osteoblasts and were able to form tumors in immunocompromised mice. Examination of gene expression revealed down-regulation of markers of bone differentiation and increased expression of locally acting growth factors, including members of the Wnt signaling pathway. Tumor cells exhibited enhanced growth in response to PKA-stimulating agents, suggesting that tumorigenesis in osteoblast precursor cells is driven by effects directly mediated by the dysregulation of PKA.

The Effect of Radiation on Tumorigenesis and Clonogenic Survival of Breast Cancer Stem Cells

Solid tumors consist of a heterogeneous mixture of cells that display large variations in their capacity to drive tumor growth. Breast cancer stem cells (referred here as breast cancer-initiating cells, or BCICs) are a subpopulation of cells identified within primary tumors that have an enhanced ability to form tumors in immunocompromised mice. These tumorigenic cells have been prospectively identified and isolated based on the CD44+/CD24−/lo/ESA+/lo surface marker profile. An implication of the presence of a highly tumorigenic subpopulation is that total elimination of this cell fraction is imperative for tumor control. Radiotherapy (RT) is an integral component of breast cancer treatment; therefore, understanding the response of BCICs to radiation is of particular importance. This current study sought to further characterize the effect of radiation on BCICs. To measure BCIC survival following radiation, SUM149, SUM159 and SUM1315 breast cancer cell lines were irradiated as adherent cultures and seeded on ultra-low adherence plates for propagation of surviving BCICs as tumorspheres. Likewise, the tumorigenic capacity of these cells was tested directly by assaying xenograft tumor formation in NOD/SCID mice. In addition, the percent of BCICs was analyzed 24 hours following radiation by flow cytometry. Finally, BCICs were isolated from the SUM159 cell line using fluorescently-labeled antibodies against the BCICs surface markers and sorted into BCICs and non-BCIC subpopulations. The radiosensitivity of these two populations was compared by assaying clonogenic survival. Irradiation of all three cell lines resulted in a significant decrease in tumorsphere formation (mean tumorsphere formation {95% CI}; SUM149: 0 Gy = 84.5 {72.3–97.4}, 9 Gy = 23.6 {11.1–36.3}, 5 × 3 Gy = 13.4 {9.7–21.0}; SUM159: 0 Gy = 517.8 {460.3–575.4}, 9 Gy = 181.8 {124.3–239.4}, 5 × 3 Gy = 198.8 {165.4–232.3}; SUM1315: 0 Gy = 86.0 {64.6–107.4}, 9 Gy 1.8 {0–23.3}), 5 × 3 Gy = no tumorspheres {0–21.4} and p < 0.001 for unirradiated vs. irradiated samples in all three cell lines. Xenograft tumor formation with 1000 cells per injection was also found to be decreased by pre-treatment of these cell lines with radiation (0 Gy = 33.3% tumor formation, n = 6 vs. 9 Gy = no tumor formation, n = 6, 80 days post-injection). There was no significant difference in radiosensitivity of BCICs vs non-BCICs in the clonogenic survival assay. However, cells with the CD44+/CD24−/lo/ESA+/lo cell surface phenotype, which have been used to identify a subpopulation enriched for BCICs, were found to be enriched 24 hours following irradiation as measured by flow cytometric analysis (mean %CD44+/CD24−/lo/ESA+/lo {95% CI}; SUM149: 0 Gy = 0.50% {0.30–0.70%}, 5 × 3 Gy = 1.45% {1.23–1.64%}, p = 001; SUM159: 0 Gy = 0.92% {0.66–1.19%}, 5 × 3 Gy = 2.57% {2.30–2.83%}, p < 0.001; SUM1315: 0 Gy = 0.83% {0.42–1.24%}), 5 × 3 Gy = 2.23% {1.82–2.64%}, p < 0.001. The tumorigenic capacity of breast cancer-initiating cells can be effectively eradicated by radiation. This effect was seen with both single fraction and fractionated delivery of the radiation. Finally, although BCICs may show an initial increase in their proportion of the cell fraction following radiation, this did not translate into an increase in clonogenic survival as adherent colonies, tumorspheres or xenograft tumors.

CXCL12 Activates a Robust Transcriptional Response in Human Prostate Epithelial Cells

CXCL12 is a CXC-type chemokine that plays important roles in hematopoiesis, development, and organization of the immune system and supports the survival or growth of a variety of normal or malignant cell types. Our laboratory recently showed that CXCL12 is secreted by aging stromal fibroblast cells and is a major paracrine factor that specifically stimulates the proliferation of prostate epithelial cells. The current study shows that this CXCL12-mediated proliferative response may be either ERK-dependent or ERK-independent. Moreover, CXCL12 initiates a previously undefined and complex global transcriptional response in prostate epithelial cells. This CXCL12-mediated transcriptional response directly stimulates the expression of genes encoding proteins that are involved in the promotion of cellular proliferation and progression through the cell cycle, tumor metastasis, and cellular motility, and directly represses the transcription of genes encoding proteins involved in cell-cell adhesion and resistance to apoptosis. Thus, CXCL12 may play a major role in the etiology of benign proliferative disease in the context of an aging tissue microenvironment.

Kinetic Analysis of Epidermal Growth Factor Receptor Somatic Mutant Proteins Shows Increased Sensitivity to the Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Erlotinib

We show that two commonly occurring epidermal growth factor receptor (EGFR) somatic mutations, L858R and an in-frame deletion mutant, Del(746-750), exhibit distinct enzymatic properties relative to wild-type EGFR and are differentially sensitive to erlotinib. Kinetic analysis of the purified intracellular domains of EGFR L858R and EGFR Del(746-750) reveals that both mutants are active but exhibit a higher K(M) for ATP and a lower K(i) for erlotinib relative to wild-type receptor. When expressed in NR6 cells, a cell line that does not express EGFR or other ErbB receptors, both mutations are ligand dependent for receptor activation, can activate downstream EGFR signaling pathways, and promote cell cycle progression. As expected from the kinetic analysis, the EGFR Del(746-752) is more sensitive to erlotinib inhibition than the EGFR L858R mutant. Further characterization shows that these mutations promote ligand-dependent and anchorage-independent growth, and cells harboring these mutant receptors form tumors in immunocompromised mice. Analysis of tumor lysates reveals that the tumorigenicity of the mutant EGFR cell lines may be due to a differential pattern of mutant EGFR autophosphorylation as compared with wild-type receptor. Significant inhibition of tumor growth, in mice harboring wild-type EGFR receptors, is only observed at doses of erlotinib approaching the maximum tolerated dose for the mouse. In contrast, the growth of mutant tumors is inhibited by erlotinib treatment at approximately one third the maximum tolerated dose. These findings suggest that EGFR somatic mutations directly influence both erlotinib sensitivity and cellular transformation.

Nup88 expression in hepatocellular carcinogenesis indicates interaction of hepatitis B virus with the nuclear pore

Levels of atonal homolog 8 (ATOH8) are reduced in 48% of hepatitis B virus-associated hepatocellular carcinoma cells (HCCs). ATOH8 downregulation is associated with loss of tumor differentiation, indicating an effect mediated by cancer stem cells. We investigated the effects of loss of ATOH8 in human hepatocellular carcinoma (HCC) cells and cell lines.HCC and adjacent nontumor tissues were collected, from 2001 through 2012, from 242 patients undergoing hepatectomy at Sun Yat-Sen University Cancer Center in China; 83% of HCCs were associated with hepatitis B virus (HBV) infection. CD133+ cells were isolated from tumor tissues by flow cytometry. Experiments were performed in HBV-positive and HBV-negative HCC cell lines, the immortalized liver cell line LO2, and 8 other HCC cell lines. ATOH8 was expressed from lentiviral vectors in PLC8024 and Huh7 cells; levels were knocked down with small interfering RNAs in QSG7701 cells. Cells carrying empty vectors were used as controls. Gene regulation by ATOH8 was assessed in mobility shift and luciferase reporter assays. Cells were analyzed in proliferation, foci formation, and colony formation assays. The tumorigenic and chemo-resistant potential of cells were investigated by assessing growth of xenograft tumors in immunocompromised mice. Metastatic features of cells were assessed in Matrigel invasion assays and wound healing analyses.Levels of ATOH8 mRNA were reduced by more than 4-fold, compared to nontumor tissues, in 118 of 242 HCC samples (48.8%). Patients with tumor reductions in ATOH8 had significantly shorter times of disease-free survival (mean, 41.4 months) than patients with normal tissue levels (mean, 52.6 months). ATOH8 expression was reduced in HepG2, Huh7, PLC8024 and CRL8064 HCC cells, as well as CD133+ cells isolated from human HCC samples. Transgenic expression of ATOH8 in HCC cell lines significantly reduced proliferation and foci colony formation, as well as their invasive and migratory abilities. Transgenic expression of ATOH8 reduced the ability of HBV-positive PLC8024 cells to form tumors in mice, compared to control cells. Cells with ATOH8 knockdown formed xenograft tumors more rapidly, in more mice, than control cells. ATOH8 repressed transcription of stem-cell associated genes including OCT4, NANOG, and CD133. Knockdown of ATOH8 in CD133-negative QSG7701 cells caused them to express CD133; acquire self-renewal, differentiation, chemo-resistance properties; form more xenograft tumors in mice; and generate induced pluripotent stem cells (based on staining for alkaline phosphatase and their ability to form embryoid bodies and teratomas). Alternatively, expression of ATOH8 in PLC8024 and Huh7 cells significantly reduced the numbers of cells expressing CD133, and increased the chemo-sensitivity of Huh7 cells to 5-fluorouracil (5-FU) and cisplatin, in vitro and in mice.ATOH8 appears to be a tumor suppressor that induces stem-cell features and chemoresistance in HCC cells. Strategies to restore its levels and activities might be developed to treat patients with liver cancer.

Characterization of a novel rearrangement from the ret/PTC family in a case of radiation-associated human thyroid papillary carcinoma

In the tumor tissue of papillary thyroid carcinoma (PTC) developed in an externally irradiated patient, we identified an undescribed yet structural mutation which comprised the juxtapositioning of the 5′ portion of an rfp gene upstream of the fragment encoding the tyrosine kinase domain of RET. At the genomic level, the formation of a chimeric gene occurred as a result of balanced chromosomal translocation of the short arm of chromosome 6 and long arm of chromosome 10, t(6;10)(p21.3;q11.2). The presence of the mutation was confirmed by extensive molecular, cytogenetic, and functional analysis. The product of the fusion gene, termed Δrfp/RET, was able to form homodimers as demonstrated by a yeast two-hybrid system, thus being in compliance with an essential condition of the constitutive activation of ret/PTC tyrosine kinases. In addition, the newly revealed rearrangement was shown to be tumorigenic, as NIH 3T3 cells stably transfected with a mammalian expression vector encoding full-length cDNA of the Δrfp/RET yielded rapidly growing tumors in immunocompromised mice. The finding provides an additional line of evidence of facilitated susceptibility of the ret protooncogene to structural mutations in irradiated human thyroid cells and pathogenic role of the ret rearrangements in human papillary thyroid cancer.

Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression.

Telomerase activation is a common feature of most advanced human cancers and is postulated to restore genomic stability to a level permissive for cell viability and tumor progression. Here, we used genetically defined transformed mouse embryonic fibroblast (MEF) cultures derived from late generation mTerc(-/-) Ink4a/Arf(-/-) mice to explore more directly how telomere-based crisis relates to the evolution of cancer cell genomes and to tumor biology. An exhaustive serial analysis of cytogenetic profiles over extensive passage in culture revealed that the emergence of chromosomal fusions (including dicentrics) coincided with onset of deletions and complex nonreciprocal translocations (NRTs), whereas mTerc-transduced cultures maintained intact chromosomes and stable genomes. Despite a high degree of telomere dysfunction and genomic instability, transformed late passage mTerc(-/-) Ink4a/Arf(-/-) cultures retained the capacity to form subcutaneous tumors in immunocompromised mice. However, even moderate levels of telomere dysfunction completely abrogated the capacity of these cells to form lung metastases after tail-vein injection, whereas mTerc reconstitution alone conferred robust metastatic activity in these cells. Finally, serial subcutaneous tumor formation using late passage transformed mTerc(-/-) Ink4a/Arf(-/-) cultures revealed clear evidence of telomerase-independent alternative lengthening of telomeres (ALT). Significantly, despite a marked increase in telomere reserve, cells derived from the ALT+ subcutaneous tumors were unable to generate lung metastases, indicating in vivo functional differences in these principal mechanisms of telomere maintenance. Together, these results are consistent with the model that although telomere dysfunction provokes chromosomal aberrations that initiate carcinogenesis, telomerase-mediated telomere maintenance enables such initiated cells to efficiently achieve a fully malignant endpoint, including metastasis.