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Canonical Wnt signaling has emerged as a critical regulatory pathway for stem cells. The association between ectopic activation of Wnt signaling and many different types of human cancer suggests that Wnt ligands can initiate tumor formation through altered regulation of stem cell populations. Here we have shown that mice deficient for the Wnt co-receptor Lrp5 are resistant to Wnt1-induced mammary tumors,which have been shown to be derived from the mammary stem/progenitor cell population. These mice exhibit a profound delay in tumorigenesis that is associated with reduced Wnt1-induced accumulation of mammary progenitor cells. In addition to the tumor resistance phenotype,loss of Lrp5 delays normal mammary development. The ductal trees of 5-week-old Lrp5-/- females have fewer terminal end buds,which are structures critical for juvenile ductal extension presumed to be rich in stem/progenitor cells. Consequently,the mature ductal tree is hypomorphic and does not completely fill the fat pad. Furthermore,Lrp5-/- ductal cells from mature females exhibit little to no stem cell activity in limiting dilution transplants. Finally,we have shown that Lrp5-/- embryos exhibit substantially impaired canonical Wnt signaling in the primitive stem cell compartment of the mammary placodes. These findings suggest that Lrp5-mediated canonical signaling is required for mammary ductal stem cell activity and for tumor development in response to oncogenic Wnt effectors. View Publication

Cancer-targeting alkylphosphocholine (APC) analogs are being clinically developed for diagnostic imaging,intraoperative visualization,and therapeutic applications. These APC analogs derived from chemically-synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB),APC permeability across intact BBB remains unknown. Three of our APC analogs,CLR1404 (PET radiotracer),CLR1501 (green fluorescence),and CLR1502 (near infrared fluorescence),were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEERtextgreater1500 Ω-cm(2)) and functional expression of drug efflux transporters. Our radioiodinated and fluorescent APC analogs demonstrated fairly low permeability across the iPSC-BMEC (35±5.7 (CLR1404),54±3.2 (CLR1501),and 26±4.9 (CLR1502) x10(-5) cm/min) compared with BBB-impermeable sucrose (13±2.5) and BBB-permeable diazepam (170±29). Only our fluorescent APC analogs (CLR1501,CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model and this efflux can be specifically blocked with pharmacological inhibition. None of our tested APC analogs appeared to undergo substantial P-gp transport. Limited permeability of our APC analogs across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover,addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP,and may affect fluorescence-guided applications. Overall,the characterization of APC analog permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents. View Publication

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge,no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach,we established the genetic relationship between the cell lines and the primary patient cells,and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly,we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity,the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover,these cell lines will provide an invaluable tool to better understand AL,from the combined perspectives of amyloidogenic protein structure and amyloid formation,genetics,and cell biology. View Publication

Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function,as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally,we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines,establish techniques that can facilitate the characterization of regulatory pathways of CSCs,and identify potential stem cell markers and therapeutic targets. View Publication

BACKGROUND: Over-expression of aldehyde dehydrogenase and other stem cell markers is characteristic of cells with tumorigenic potential in NOD/SCID mice. Most of these studies have focused on metastatic cells in bone marrow and on solid tumors. There are no studies on correlation of marker expression with ALDH1 expression in cells from human peripheral blood apheresis (HPC-A) samples. METHODS: HPC-A samples from 44 patients were incubated with Aldefluor with or without the presence of aldehyde dehydrogenase inhibitor DEAB. Cells with high aldehyde dehydrogenase expression (ALDH1(bright)) were analyzed for stem/progenitor markers CD34,CD90,CD117,and CD133. Electronic volume measured by Coulter principal in a Quanta flow analyzer was correlated with ALDH1 and marker expression. RESULTS: In ALDH1(bright)/SSC(low) cells,0.13% of the cells had CD34(+) expression and three distinct populations were seen. Expression of CD90 was dim and the frequency of ALDH1(bright)/SSC(low)/CD90(dim) cells amongst the nonlineage depleted samples was 0.04%. CD117(dim-bright) expression was seen in 0.17% of the samples. Three distinct populations of cells with CD133 expression were seen in ALDH1(bright)/SSC(low) nonlineage depleted cells with a frequency of 0.28%. The ALDH1(bright)/CD90(dim) cells had the smallest mean electronic volume of 264.9 microm(3) when compared with cells with CD34(bright) expression (270.2 microm(3)) and ALDH1(dim)/CD90(dim) cells (223 microm(3)). CONCLUSIONS: ALDH1(bright)/SSC(low) cells show heterogeneity in expression of the four stem cell markers studied. The CD90 cells in both the ALDH1(bright) and ALDH1(dim) populations had the smallest mean electronic volume when compared with similar cells with CD117 expression. View Publication

The phosphatidylinositol 3' kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway,which is a critical regulator of cell proliferation and survival,is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens,and hypothesised that KP372-1,an Akt inhibitor,would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells,leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling,further preclinical evaluation of this compound for treatment of thyroid cancer is warranted. View Publication

Multiple myeloma is characterized by the proliferation of clonal plasma cells that have a heterogeneous expression of various cell surface markers,precluding successful use of monoclonal antibodies for therapeutic targeting of the tumor cell. Thymoglobulin (rabbit-derived polyclonal anti-thymocyte globulin),by virtue of its method of preparation,contains antibodies against several B-cell and plasma cell antigens and offers an attractive option for immunotherapy of myeloma. Here,we demonstrate potent anti-myeloma activity of the rabbit anti-thymocyte globulin preparation Thymoglobulin in vitro and in vivo in an animal model of myeloma. Thymoglobulin was able to induce dose- and time-dependent apoptosis of several myeloma cell lines,including those resistant to conventional anti-myeloma agents. Importantly,the anti-myeloma activity was preserved even when myeloma cells were grown with different cytokines demonstrating the ability to overcome microenvironment-mediated resistance. Thymoglobulin induced apoptosis of freshly isolated primary myeloma cells from patients. Using a competitive flow cytometric analysis,we were able to identify the potential antigen targets for Thymoglobulin preparation. Finally,in a plasmacytoma mouse model of myeloma,Thymoglobulin delayed the tumor growth in a dose-dependent manner providing convincing evidence for continued evaluation of this agent in the clinic in patients with myeloma,either alone or in combination with other agents. View Publication

This study was to evaluate the enhancement value of chloroquine (CQ) in cancer cell killing when used in combination with Akt inhibitors. The results showed that the combination of CQ and Akt inhibitors is much more effective than either one alone. Importantly,the CQ-mediated chemosensitization of cell killing effects by Akt inhibitors is cancer specific. In particular,when combined with 10 microM CQ,1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one (an Akt1 and 2 inhibitor; compound 8) killed cancer cells 10-120 times more effectively than normal cells. Thus,CQ is a very effective and cancer-specific chemosensitizer when used in combination with Akt inhibitors. View Publication

EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma,its expression in other tumor types remains controversial. In this study,we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII,but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial-mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically,EGFRvIII mediated its effects through the Wnt/β-catenin pathway,leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together,our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis. View Publication

BACKGROUND Cancer development results from the progressive accumulation of genomic abnormalities that culminate in the neoplastic phenotype. Cytogenetic alterations,mutations and rearrangements may be considered as molecular legacy which trace the clonal history of the disease. Concomitant tumors are reported and they may derive from a common or divergent founder clone. B-cell chronic lymphocytic leukemia (B-CLL) and plasma cell myeloma (PCM) are both mature B-cell neoplasms,and their concomitancy,albeit rare,is documented. CASE PRESENTATION Here,we described a patient with prior B-CLL with secondary development of PCM. Cytogenetic and multi parametric flow cytometry analyses were performed. The B-CLL population presented chromosome 12 trisomy,unlikely the arisen PCM population. CONCLUSION The close follow up of B-CLL patients is important for early intervention in case of development of other malignancy,such as myeloma. Our observation suggests these two diseases may have arisen from different clones. We understand that the investigation of clonal origin may provide important information regarding therapeutic decisions,and should be considered in concomitant neoplasm. View Publication

In CD34(+) acute myeloid leukemia (AML),the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously,we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population,containing the CD34(+)CD38(-)CLL-1(+) cells,does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission,both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future. View Publication

Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress,cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62),but its role in the regulation of autophagy is controversial. Here,we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK,thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly,p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV,and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy. View Publication