PKCθ Regulates T-Cell Leukemia-Initiating Activity via Reactive Oxygen Species

Reactive oxygen species (ROS), a by-product of cellular metabolism, damage intracellular macromolecules and, in excess, can promote normal hematopoietic stem cell differentiation and exhaustion1–3. However, mechanisms that regulate ROS levels in leukemia-initiating cells (LICs) and the biological role of ROS in these cells remain largely unknown. We show here the ROSlow subset of CD44+ cells in T-cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T-cell progenitors, to be highly enriched in the most aggressive LICs, and that ROS are maintained at low levels by downregulation of protein kinase C theta (PKCθ). Strikingly, primary mouse T-ALLs lacking PKCθ show improved LIC activity whereas enforced PKCθ expression in both mouse and human primary T-ALLs compromised LIC activity. We also demonstrate that PKCθ is positively regulated by RUNX1, and that NOTCH1, which is frequently activated by mutation in T-ALL4–6 and required for LIC activity in both mouse and human models7,8, downregulates PKCθ and ROS via a novel pathway involving induction of RUNX3 and subsequent repression of RUNX1. These results reveal key functional roles for PKCθ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKCθ expression/activity or ROS accumulation

Genomic imbalance of HMMR/RHAMM regulates the sensitivity and response of malignant peripheral nerve sheath tumour cells to aurora kinase inhibition

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatment options as they often resist chemotherapies and have high rates of disease recurrence. Aurora kinase A (AURKA) is an emerging target in cancer and an aurora kinase inhibitor (AKI), termed MLN8237, shows promise against MPNST cell lines in vitro and in vivo. Here, we test MLN8237 against two primary human MPNST grown in vivo as xenotransplants and find that treatment results in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulates in stabilized disease. Targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumour susceptibilities, so we turned to three MPNST cell-lines to further study and modulate the cellular responses to AKI. We find that the sensitivity of cell-lines with amplification of AURKA depends upon the activity of the kinase, which correlates with the expression of the regulatory gene products TPX2 and HMMR/RHAMM. Silencing of HMMR/RHAMM, but not TPX2, augments AURKA activity and sensitizes MPNST cells to AKI. Furthermore, we find that AURKA activity is critical to the propagation and self-renewal of sphere-enriched MPNST cancer stem-like cells. AKI treatment significantly reduces the formation of spheroids, attenuates the self-renewal of spheroid forming cells, and promotes their differentiation. Moreover, silencing of HMMR/RHAMM is sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of HMMR/RHAMM

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer

High-level IGF1R expression is required for leukemia-initiating cell activity in T-ALL and is supported by Notch signaling

Notch-driven expression of IGF1R promotes the growth, viability, and transplantability of T-ALL cells

The regulation of mitotic spindle orientation by BRCA1 controls the proliferation, polarization and growth arrest of human mammary epithelia

Carriers of mutations in the Breast Cancer 1 (BRCA1) gene have an increased risk to develop breast cancer, which tend to be early-onset, lack expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, and resemble basal epithelia by gene expression. The phenotypic resemblance of these tumours to normal stem/progenitor cells suggests that the loss of BRCA1 function may dysregulate stem cell maintenance and differentiation. In model organisms, a mechanism that promotes the generation of daughter cells different from a mother stem cell is the asymmetric segregation of non-genetic factors through mitotic spindle orientation. BRCA1 regulates mitotic spindle assembly through the post-transcriptional degradation of the low-penetrance breast cancer susceptibility gene product RHAMM and the abundance of RHAMM influences mitotic spindle orientation by regulating its movement along the cell cortex. This led to the hypothesis that BRCA1 is necessary for the correct orientation of the mitotic spindle in mammary epithelial cells, which controls their proliferation, polarization and growth arrest. To address this hypothesis, I studied non-malignant human mammary cell-lines and primary human progenitor cell-enriched populations. BRCA1 was silenced by shRNA introduced through lentiviral transduction. Silencing of BRCA1 in cell-lines increased both mitotic and post-mitotic abnormalities, including the loss of spindle orientation with subsequent lagging chromosomes and micronucleus formation in 2D cultures. The consequence of these defects included a significant decrease in colony-forming capacity. I then enquired whether BRCA1 is necessary for MCF10A cells to proliferate, form polarized acini, and growth arrest in 3D cultures. Control cells underwent planar division to form polarized, growth arrested acini, while BRCA1 silenced structures were larger, less polarized and more proliferative. Loss of correct spindle orientation was also observed. These results indicate that BRCA1 plays a role in maintaining the integrity of human mammary cell division. Loss of BRCA1 induces mitotic and post-mitotic consequences that impair cellular proliferative capacity and abolish ability to undergo directional division, polarization and arrest growth. These findings thus raise the possibility that breast cancer treatments aimed at counteracting the BRCA1-mediated loss of polarity may complement drugs that combat the diminished DNA repair characteristic of BRCA1-associated tumours.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.Medicine, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofReviewedFacult

Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma

International audienceRemnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients

Morphology and cellular composition of structures generated in 7-d Matrigel cultures of single fetal and adult mammary cells.

<p>(A) Gross appearance of representative structures from each of the three types of input cells tested. The scale shown indicates 30 µm. (B) Representative photomicrographs of sectioned structures stained with antibodies against the markers shown. Scale, 50 µm. (C) Flow cytometric analysis of dissociated PI⁻ cells obtained from pooled harvests of cultures initiated with the same types of cells.</p

Comparison of CFC and MRU outputs in 7-d Matrigel cultures initiated with single fetal or adult mammary cells.

<p>(A) Correlation between the total number of cells retrieved from individually assessed 7-d cultures with the corresponding number of CFCs detected. (B) Distribution of clonal CFC outputs in 7-d cultures initiated with different types of input cells. The dotted line indicates the median values of positive clones: 1,000 for clones derived from basal adult cells (<i>n</i> = 44), 140 for clones from CD61⁺ adult luminal cells (<i>n</i> = 35), and 13,000 for clones from fetal cells (<i>n</i> = 55). (C) Comparison of MRU outputs determined by LDA in 7-d Matrigel cultures initiated with single adult and fetal mammary cells. The value for fetal cells is significantly higher than either of the adult values (<i>p</i><0.01, Chi-square <i>p</i> value, pair-wise comparison of stem cell frequencies, ELDA, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001630#pbio.1001630.s009" target="_blank">Table S7</a>).</p