Eun Y. Lee

mTORC1 and mTORC2 regulate EMT, motility and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways

Activation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with growth and progression of colorectal cancer (CRC). We have previously shown that the mTOR kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis of CRC. However, the contribution of mTOR and its interaction partners toward regulating CRC progression and metastasis remains poorly understood. We found that increased expression of mTOR, Raptor, and Rictor mRNA was noted with advanced stages of CRC, suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor, and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastases compared with normal colon. Inhibition of mTOR signaling, using rapamycin or stable inhibition of mTORC1 (Raptor) and mTORC2 (Rictor), attenuated migration and invasion of CRCs. Furthermore, knockdown of mTORC1 and mTORC2 induced a mesenchymal-epithelial transition (MET) and enhanced chemosensitivity of CRCs to oxaliplatin. We observed increased cell-cell contact and decreased actin cytoskeletal remodeling concomitant with decreased activation of the small GTPases, RhoA and Rac1, upon inhibition of both mTORC1 and mTORC2. Finally, establishment of CRC metastasis in vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating epithelial-mesenchymal transition (EMT), motility, and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC patients.

Targeted inhibition of mammalian target of rapamycin signaling inhibits tumorigenesis of colorectal cancer

Purpose: The mammalian target of rapamycin (mTOR) kinase acts downstream of phosphoinositide 3-kinase/Akt to regulate cellular growth, metabolism, and cytoskeleton. Because ∼60% of sporadic colorectal cancers (CRC) exhibit high levels of activated Akt, we determined whether downstream mTOR signaling pathway components are overexpressed and activated in CRCs. Experimental Design: HCT116, KM20, Caco-2, and SW480 human CRC cells were used to determine the effects of pharmacologic (using rapamycin) or genetic (using RNAi) blockade of mTOR signaling on cell proliferation, apoptosis, cell cycle progression, and subcutaneous growth in vivo. Results: We show that the mTOR complex proteins mTOR, Raptor, and Rictor are overexpressed in CRC. Treatment with rapamycin significantly decreased proliferation of certain CRC cell lines (rapamycin sensitive), whereas other cell lines were resistant to its effects (rapamycin resistant). Transient siRNA-mediated knockdown of the mTORC2 protein, Rictor, significantly decreased proliferation of both rapamycin-sensitive and rapamycin-resistant CRC cells. Stable shRNA-mediated knockdown of both mTORC1 and mTORC2 decreased proliferation, increased apoptosis, and attenuated cell cycle progression in rapamycin-sensitive CRCs. Moreover, stable knockdown of both mTORC1 and mTORC2 decreased proliferation and attenuated cell cycle progression, whereas only mTORC2 knockdown increased apoptosis in rapamycin-resistant CRCs. Finally, knockdown of both mTORC1 and mTORC2 inhibited growth of rapamycin-sensitive and rapamycin-resistant CRCs in vivo when implanted as tumor xenografts. Conclusions: Targeted inhibition of the mTORC2 protein, Rictor, leads to growth inhibition and induces apoptosis in both rapamycin-sensitive and rapamycin-resistant CRCs, suggesting that selective targeting of mTORC2 may represent a novel therapeutic strategy for treatment of CRC.(Clin Cancer Res 2009;15(23):7207–16)

S-adenosylmethionine deficiency and TNF-α in lipopolysaccharide-induced hepatic injury

S-adenosylmethionine (Adomet) is a substrate for de novo synthesis of choline. Adomet deficiency occurs in certain types of liver injury, and the injury is attenuated by exogenous Adomet. Tumor necrosis factor-α (TNF-α) is also a mediator of these models of hepatotoxicity. We investigated the role of Adomet in lipopolysaccharide (LPS)-induced liver injury in rats made deficient in both Adomet and choline. Rats were maintained on either a methionine-restricted and choline-deficient (MCD) diet or a diet containing sufficient amounts of all nutrients [methionine and choline sufficient (MCS)] and then administered either LPS or saline. MCS-LPS rats had normal liver histology and no change in serum transaminases compared with the MCS-saline control group. MCD-saline rats had hepatosteatosis but no necrosis, and a five- to sevenfold increase in transaminases vs. the MCS-saline group. MCD-LPS rats additionally had hepatonecrosis and a 30- to 50-fold increase in transaminases. Exogenous Adomet administration to MCD-LPS rats corrected the hepatic deficiency of Adomet but not of choline, prevented necrosis but not steatosis, and attenuated transaminases. Serum TNF-α was sixfold higher in MCD rats even without LPS challenge and 300-fold higher with LPS challenge. Exogenous Adomet attenuated increased serum TNF-α in MCD-LPS rats.

Uterine sarcomas express KIT protein but lack mutation(s) in exon 11 or 17 of c-KIT.

OBJECTIVE Several tumors express the protein product of the protooncogene c-KIT. Some of these respond to imatinib mesylate, a tyrosine kinase inhibitor. The tumors that respond frequently have mutation(s) in exon 11 of c-KIT that encodes for the regulatory juxtamembrane helix. Some tumors that express KIT protein have mutation(s) in exon 17 of c-KIT; however, these do not respond to imatinib mesylate. This investigation was performed to determine the expression of KIT protein and mutational status of exons 11 and 17 of c-KIT in uterine sarcomas. METHODS Twenty-five uterine sarcomas treated from 1990 to 2002 were evaluated. These included 14 malignant mullerian mixed tumors (MMMT), 7 leiomyosarcomas (LMS), 2 endometrial stromal sarcomas (ESS), and 2 high-grade heterologous sarcomas (HGHS). Formalin-fixed, paraffin-embedded tissue sections were immunostained with anti-KIT antibody (Santa Cruz Biotechnology, Santa Cruz, CA) with a semiquantitative assessment. Normal myometrium when present in the section was used as an internal negative control. Areas of tumor were microdissected followed by DNA extraction, polymerase chain reaction (PCR) amplification of exons 11 and 17, single-strand conformational polymorphism (SSCP), and DNA sequencing to detect the presence of mutation(s). RESULTS All 25 tumors expressed KIT protein at varying levels as assessed by immunohistochemistry. The staining was diffuse and of moderate to strong intensity in 22 tumors. In three tumors (one of each type except MMMT) the staining intensity was weak. In MMMT the epithelial and sarcomatous foci stained similarly. No mutation(s) in exons 11 or 17 of c-KIT were identified in 24/25 tumors. One LMS had deletion of both exons 11 and 17. CONCLUSIONS Although uterine sarcomas express KIT protein, they lack KIT-activating mutation(s) in exon 11 or 17 of c-KIT. Therefore, these tumors are unlikely to respond to imatinib mesylate.

Inhibition of Fatty Acid Synthase Attenuates CD44-Associated Signaling and Reduces Metastasis in Colorectal Cancer

Fatty acid synthase (FASN) and ATP-citrate lyase, key enzymes of de novo lipogenesis, are significantly upregulated and activated in many cancers and portend poor prognosis. Even though the role of lipogenesis in providing proliferative and survival advantages to cancer cells has been described, the impact of aberrant activation of lipogenic enzymes on cancer progression remains unknown. In this study, we found that elevated expression of FASN is associated with advanced stages of colorectal cancer (CRC) and liver metastasis, suggesting that it may play a role in progression of CRC to metastatic disease. Targeted inhibition of lipogenic enzymes abolished expression of CD44, a transmembrane protein associated with metastases in several cancers including CRC. In addition, inhibition of lipogenic enzymes and reduced expression of CD44 attenuated the activation of MET, Akt, FAK, and paxillin, which are known to regulate adhesion, migration, and invasion. These changes were consistent with an observed decrease in migration and adhesion of CRC cells in functional assays and with reorganization of actin cytoskeleton upon FASN inhibition. Despite the modest effect of FASN inhibition on tumor growth in xenografts, attenuation of lipogenesis completely abolished establishment of hepatic metastasis and formation of secondary metastasis. Together, our findings suggest that targeting de novo lipogenesis may be a potential treatment strategy for advanced CRC.

Regulation of the Potential Marker for Intestinal Cells, Bmi1, by β-Catenin and the Zinc Finger Protein KLF4 IMPLICATIONS FOR COLON CANCER

Background: Bmi1 is a potential marker for the intestinal stem cells. Results: Wnt regulates Bmi1 indirectly, while KLF4 directly inhibits Bmi1, as well as Bmi1-mediated histone ubiquitination in colon cancer cells. Conclusion: Bmi1 is required for colon cancer cell proliferation, and it is up-regulated in colon cancer tissues. Significance: Study of the mechanisms of Bmi1 regulation suggests potential targets for cancer therapeutics. B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear β-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/β-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.

Arsenic and chromium in drinking water promote tumorigenesis in a mouse colitis-associated colorectal cancer model and the potential mechanism is ROS-mediated Wnt/β-catenin signaling pathway

Exposure to carcinogenic metals, such as trivalent arsenic [As(III)] and hexavalent chromium [Cr(VI)], through drinking water is a major global public health problem and is associated with various cancers. However, the mechanism of their carcinogenicity remains unclear. In this study, we used azoxymethane/dextran sodium sulfate (AOM/DSS)-induced mouse colitis-associated colorectal cancer model to investigate their tumorigenesis. Our results demonstrate that exposure to As(III) or Cr(VI), alone or in combination, together with AOM/DSS pretreatment has a promotion effect, increasing the colorectal tumor incidence, multiplicity, size, and grade, as well as cell inflammatory response. Two-dimensional differential gel electrophoresis coupled with mass spectrometry revealed that As(III) or Cr(VI) treatment alone significantly changed the density of proteins. The expression of β-catenin and phospho-GSK was increased by treatment of carcinogenic metals alone. Concomitantly, the expression of NADPH oxidase1 (NOX1) and the level of 8-OHdG were also increased by treatment of carcinogenic metals alone. Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, were decreased. Similarly, in an in vitro system, exposure of CRL-1807 to carcinogenic metals increased reactive oxygen species (ROS) generation, the expression of β-catenin, phospho-GSK, and NOX1. Inhibition of ROS generation by addition of SOD or catalase inhibited β-catenin expression and activity. Our study provides a new animal model to study the carcinogenicity of As(III) and Cr(VI) and suggests that As(III) and Cr(VI) promote colorectal cancer tumorigenesis, at least partly, through ROS-mediated Wnt/β-catenin signaling pathway.

VEGFR-2 expression in carcinoid cancer cells and its role in tumor growth and metastasis

Carcinoid tumors are slow growing and highly vascular neuroendocrine neoplasms that are increasing in incidence. Previously, we showed that carcinoid tumors express vascular endothelial growth factor receptor 2 (VEGFR‐2) in the epithelial compartment of carcinoid tumor sections; yet, its role is not completely understood. The purpose of our study was to: (i) assess the expression of VEGFR‐2 in the novel human carcinoid cell line BON, (ii) to determine the role of PI3K/Akt signaling on VEGFR‐2 expression and (iii) to assess the effect of VEGFR‐2 on BON cell invasion, migration and proliferation. We found that, although VEGFR‐2 is expressed in BON cells, reduction in VEGFR‐2 expression actually enhanced proliferation, invasion, and migration of the BON cell line. Also, expression of VEGFR‐2 was inversely related to PI3K signaling. Carcinoid liver metastases in mice demonstrated decreased VEGFR‐2 expression. Furthermore, the expression of a truncated, soluble form of VEGFR‐2 (sVEGFR‐2), a protein demonstrated to inhibit cell growth, was detected in BON cells. The presence of VEGFR‐2 in the epithelial component of carcinoid tumors and in the BON cell line suggests an alternate role for VEGFR‐2, in addition to its well‐defined role in angiogenesis. The expression of sVEGFR‐2 may explain the inverse relationship between VEGFR‐2 expression and PI3K/Akt signaling and the inhibitory effect VEGFR‐2 has on BON cell proliferation, migration and invasion.

Delivery of RNA Nanoparticles into Colorectal Cancer Metastases Following Systemic Administration

The majority of deaths from all cancers, including colorectal cancer (CRC), is a result of tumor metastasis to distant organs. To date, an effective and safe system capable of exclusively targeting metastatic cancers that have spread to distant organs or lymph nodes does not exist. Here, we constructed multifunctional RNA nanoparticles, derived from the three-way junction (3WJ) of bacteriophage phi29 motor pRNA, to target metastatic cancer cells in a clinically relevant mouse model of CRC metastasis. The RNA nanoparticles demonstrated metastatic tumor homing without accumulation in normal organ tissues surrounding metastatic tumors. The RNA nanoparticles simultaneously targeted CRC cancer cells in major sites of metastasis, such as liver, lymph nodes, and lung. Our results demonstrate the therapeutic potential of these RNA nanoparticles as a delivery system for the treatment of CRC metastasis.

Resorption Controls Bone Anabolism Driven by Parathyroid Hormone (PTH) Receptor Signaling in Osteocytes

Background: Contribution of resorption to bone anabolism by PTH receptor signaling in osteocytes is unknown. Results: Pharmacologic/genetic approaches demonstrated that remodeling- or modeling-based bone formation differentially operate in specific surfaces. Conclusion: Resorption is critical for anabolism in periosteal/endocortical bone surfaces, but tempers bone gain in cancellous bone. Significance: Targeting bone compartment-specific actions of PTH receptor signaling could enhance the therapeutic potential of the pathway. The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.