Hiroko Endo

Retaining cell–cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer

Primary culture of the cancer cells from patients’ tumors can provide crucial information of individual tumors, yet the technology has not been optimized until now. We developed an innovative culture method for primary colorectal cancer cells, based on the principle that cell–cell contact of cancer cells was maintained throughout the process. When tumor tissue was dissociated into cell clusters, in which cell–cell contact was retained, they rapidly formed spheroids that we termed cancer tissue-originated spheroids (CTOSs). CTOSs of colorectal cancer consisted of highly purified and viable cancer cells, and they were prepared with high efficiency. In immunodeficient mice, CTOSs formed xenograft tumors that retained the features of the parental tumors. Moreover, CTOSs were able to be cultured and expanded in vitro using a 3D culture system and stem cell culture medium. This method allowed evaluation of chemosensitivity and signal pathway activation in cancer cells from individual patients. Easy preparation and culture of pure primary cancer cells provides an innovative platform for studying cancer biology and developing personalized medicine.

A congenital mutation of the novel gene LRRC8 causes agammaglobulinemia in humans.

A girl with congenital agammaglobulinemia and minor facial anomalies lacked B cells in peripheral blood: karyotypic analysis of white blood cells showed balanced translocation, t(9;20)(q33.2;q12). In the current study, we isolated a novel gene, leucine-rich repeat-containing 8 (LRRC8), at the translocation site on chromosome 9. It has four transmembrane helices with one isolated and eight sequentially located leucine-rich repeats (LRRs) and constitutes a new protein family. It is expressed on T cells as well as on B-lineage cells. Translocation truncates the LRRC8 gene, resulting in deletion of the eighth, ninth, and half of the seventh LRR domains located close to the C-terminal. The truncated form of the LRRC8 gene is transcribed with sequences from the noncoding region adjacent to the truncated seventh LRR. Protein products derived from the truncated gene are coexpressed on white blood cells with the intact LRRC8 protein from the untranslocated allele. Transplantation experiments with murine bone marrow cells that were forced to express the truncated LRRC8 show that expression of the truncated protein inhibited B cell development. These results indicate that LRRC8 is responsible for the B cell deficiency in this patient and is required for B cell development.

Cellular Hypoxia of Pancreatic β-Cells Due to High Levels of Oxygen Consumption for Insulin Secretion in Vitro

Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic β-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic β-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated β-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.

Downregulation of c-MYC Protein Levels Contributes to Cancer Cell Survival under Dual Deficiency of Oxygen and Glucose

The c-MYC protein participates in energy-consuming processes such as proliferation and ribosome biosynthesis, and its expression is often dysregulated in human cancers. Cancer cells distant from blood vessels in solid tumors are in short supply of oxygen and nutrition yet can adapt to the microenvironment and survive under metabolic stress. The role and regulation of c-MYC protein in the tumor microenvironment of limited energy sources are poorly understood. Here, we show that c-MYC protein levels in cancer cells are strikingly reduced in the area distant from the blood vessels in vivo and also under oxygen- and glucose-deprived conditions in vitro. The rapid reduction of c-MYC protein levels requires low levels of both oxygen and glucose, and under these conditions, downregulation is mainly achieved by enhanced degradation. Suppression of c-MYC protein levels by small hairpin RNA decreases the necrotic cell death induced by oxygen and glucose deprivation. Thus, the environmental milieu regulates c-MYC protein levels, and downregulation of c-MYC might be a strategy for cancer cells to survive under conditions of limited energy sources.

Spheroid Culture of Primary Lung Cancer Cells with Neuregulin 1/HER3 Pathway Activation

Introduction: Primary culture of cancer cells is expected to be useful for investigating the biology of cancer and predicting chemosensitivity for individual patients, yet has been hampered by technical difficulties. We recently developed the cancer tissue–originated spheroid (CTOS) method for the primary culture of colorectal cancer cells. In the present study, we applied this system to the primary culture of non–small-cell lung cancer. Methods: We used 125 surgical specimens and 18 pleural effusions for CTOS preparation. Partially digested tumor fragments were cultured in a medium for embryonic stem cells. CTOSs were subjected to sensitivity assay and signal transduction assay for the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib. We also investigated the effects of growth factors in culturing lung cancer CTOS. Results: The success rate of CTOS preparation from surgical specimens was 80.0%. The CTOS method was also suitable for culturing tumor spheroids from pleural effusions. CTOSs from lung cancer consisted mostly of pure cancer cells. CTOSs and CTOS-derived xenografts retained the characteristics of the original tumors. In vitro assay results showed that EGFR mutation status and expression levels corresponded with erlotinib sensitivity, confirming previous clinical findings. Furthermore, we found that neuregulin 1, a ligand of HER3, potently induced CTOS growth. Conclusions: The CTOS method enables us to obtain primary lung tumor cells of high viability and purity. CTOS could be a new platform for studying lung cancer biology.

Inactivation of Rho GTPases by p190 RhoGAP reduces human pancreatic cancer cell invasion and metastasis

A number of small GTPases are involved in cancer cell proliferation, migration and invasion, acting as molecular switches that cycle between GTP‐ and GDP‐bound states. GTPase‐activating proteins (GAPs) have been established as a major class of negative regulators of Rho GTPase signaling. To investigate the biological function of p190 RhoGAP toward RhoA in cancer cell invasion and metastasis, we generated a chimera made of the RhoGAP domain of p190 and the C‐terminus of RhoA (p190‐RhoA chimera), and transfected it into human pancreatic cancer cells, AsPC‐1. Epidermal growth factor (EGF)‐induced activation of RhoA, as well as RhoB and RhoC, to a lesser extent, was significantly inhibited in p190‐RhoA chimera‐transfected AsPC‐1 cells compared with that of control cells (mock‐infected), when assessed by pull‐down assay for GTP‐bound RhoA, RhoB, and RhoC, respectively. EGF‐induced invasion of p190‐RhoA chimera transfectants was significantly inhibited compared with that of mock‐infected cells in a modified Boyden chamber assay. Furthermore, the mice injected intrasplenically with AsPC‐1 cells that overexpressed the p190‐RhoA chimera had a marked reduction in the number and size of metastatic nodules in the liver. These data suggest that the inhibitory action of p190 RhoGAP toward RhoA offers a novel approach to the treatment of invasion and metastasis of cancer cells. (Cancer Sci 2006; 97: 848–853)

Activation of Insulin-like Growth Factor Signaling Induces Apoptotic Cell Death Under Prolonged Hypoxia by Enhancing Endoplasmic Reticulum Stress Response

Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Mammalian target of rapamycin (mTOR), one of the downstream molecules of the insulin-like growth factor (IGF) pathway, is a key regulator of translation, integrating multiple environmental and nutritional cues. The activity of mTOR is known to be suppressed under hypoxic conditions in cancer cells, whereas the contribution of this suppression to cell survival has not yet been clarified. We show that stimulating IGF signaling provoked caspase-dependent apoptosis under low oxygen tension in two cancer cell lines, COLO 320 and AsPC-1. In concurrence with increased levels of BAD phosphorylation, cell death was not accompanied by cytochrome c release from mitochondria. The cells were rescued from apoptosis when phosphatidylinositol 3-kinase (PI3K) or mTOR activity was inhibited, suggesting that these signals are critical in the observed cell death. IGFs and insulin enhanced the endoplasmic reticulum (ER) stress response as monitored by induction of the CCAAT/enhancer binding protein homologous protein (CHOP) proteins and the X box protein-1 splicing under hypoxic conditions, and this response was suppressed by inhibiting PI3K and mTOR activity. IGF-induced cell death under hypoxic conditions was prevented by treatment with cycloheximide, suggesting that de novo protein synthesis is required. Indeed, suppression of CHOP protein levels with small hairpin RNA reduced cell death. Taken together, the data suggest that stimulating IGF signaling under hypoxic conditions provokes apoptosis by enhancing the ER stress response.

Cross talk between apoptosis and invasion signaling in cancer cells through caspase-3 activation.

In solid tumors, cancer cells are exposed to various microenvironmental stresses such as hypoxia, nutritional depletion, and low pH. Cancer cells adapt to these stresses and circumvent cell death. When the antiapoptotic signals overcome the stress, cancer cells might acquire physiologic functions, such as invasiveness, instead of cell death. Here, we report that tumor cells acquire an invasive capacity from apoptotic signals through caspase activation. We treated rat ascites hepatoma MM1 cells with an apoptosis-inducing drug, etoposide, or hypoxia, and assessed the invasion capacity with an in vitro bioassay. Although MM1 cells hardly showed invasiveness in serum-free medium, under stress conditions, invasive capacity accompanied with morphologic change was induced with caspase-3 activation. Such stress-induced invasion as well as morphologic change was suppressed by blocking caspase-3 activity with caspase inhibitors or by RNA interference of caspase-3. In contrast, lysophosphatidic acid-induced invasiveness was not affected by caspase-3 inhibition. These results suggest that caspase-3 activation contributes to the stress-induced invasive capacity of these cancer cells.

Involvement of Heregulin/HER3 in the Primary Culture of Human Urothelial Cancer

PURPOSEnWe previously established a novel method of human colorectal cancer primary culture. This method, termed the cancer tissue originated spheroid method, involves the preparation of multicellular spheroids of primary cancer cells that are cultured so that cell-cell contact is maintained. We applied this method to human urothelial cancer.nnnMATERIALS AND METHODSnCancer tissue originated spheroids were prepared from xenografts or primary human bladder urothelial cancer tumors following the same protocol used for human colorectal cancer. Cancer tissue originated spheroids were characterized using immunohistochemistry, Western blot and polymerase chain reaction.nnnRESULTSnWe established a xenograft from a primary bladder urothelial cancer, and isolated and cultured cancer tissue originated spheroids from the xenograft tumor. Cancer tissue originated spheroids retained the characteristics of the original tumor and those of the xenograft. Heregulin promoted cancer tissue originated spheroid growth, and inhibitors of PI3K and mTOR inhibited heregulin induced growth, as did lapatinib but not erlotinib. We also prepared cancer tissue originated spheroids from primary bladder urothelial cancer. The success rate of establishing primary cancer tissue originated spheroids from nonmuscle invasive urothelial cancer was 90.7% and that from muscle invasive cancer was 68.2%. The overall success rate was 84.2%. Heregulin promoted the growth of primary cancer tissue originated spheroids from 4 of 7 patients.nnnCONCLUSIONSnWe report a method of establishing primary cultures of human urothelial cancer cells. Growth stimulation by heregulin in cancer tissue originated spheroids from xenografts and primary tumors suggests the possibility of molecular targeting therapy against HER3 signaling for human urothelial cancer. The cancer tissue originated spheroid method might be useful for selecting patients for molecular targeting drugs such as lapatinib.

CKAP4 is a Dickkopf1 receptor and is involved in tumor progression

Dickkopf1 (DKK1) is a secretory protein that antagonizes oncogenic Wnt signaling by binding to the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). DKK1 may also regulate its own signaling to promote cancer cell proliferation, but the mechanism is not understood. Here, we identified cytoskeleton-associated protein 4 (CKAP4) as a DKK1 receptor and evaluated CKAP4-mediated DKK1 signaling in cancer cell proliferation. We determined that DKK1 binds CKAP4 and LRP6 with similar affinity but interacts with these 2 receptors with different cysteine-rich domains. DKK1 induced internalization of CKAP4 in a clathrin-dependent manner, further supporting CKAP4 as a receptor for DKK1. DKK1/CKAP4 signaling activated AKT by forming a complex between the proline-rich domain of CKAP4 and the Src homology 3 domain of PI3K, resulting in proliferation of normal cells and cancer cells. Expression of DKK1 and CKAP4 was frequent in tumor lesions of human pancreatic and lung cancers, and simultaneous expression of both proteins in patient tumors was negatively correlated with prognosis and relapse-free survival. An anti-CKAP4 antibody blocked the binding of DKK1 to CKAP4, suppressed AKT activity in a human cancer cell line, and attenuated xenograft tumor formation in immunodeficient mice. Together, our results suggest that CKAP4 is a potential therapeutic target for cancers that express both DKK1 and CKAP4.