Hidetada Kawana

Role of p27Kip1 and Cyclin-Dependent Kinase 2 in the Proliferation of Non-Small Cell Lung Cancer

The cell cycle is governed by a family of cyclin-dependent kinases (Cdks). Cdk2 forms a functional complex with cyclin E and plays a pivotal role in the regulation of G1/S transition. Cdk2 activity is negatively regulated by interactions with inhibitors. p27Kip1, one of the most potent inhibitors of Cdk2, was recently identified as a powerful negative prognostic marker in non-small cell lung cancer as well as in colorectal and breast cancer. In the present study, the expression of p27 and Ki-67 antigen in nonneoplastic and cancerous lung tissues was determined by immunohistochemistry. After establishing that the antibody-measured p27 labeling index was a good reflection of the level of p27 expression measured by Western blotting, we show that p27 labeling index is decreased in cancerous lung tissues, compared with nonneoplastic lung tissues, and exhibits a significant inverse relation to the proliferation marker Ki-67 antigen, detected with monoclonal antibody MIB-1. Consistent with these data, all cancerous lung tissues showed enhanced degradation activity of p27 compared with nonneoplastic lung tissues and, in addition, increased levels of the phosphorylated form of Cdk2, as determined with Western blot analysis. The H1 histone kinase activity associated with Cdk2 was also increased in non-small cell lung cancers. Statistical analysis showed that proliferative activity as measured by MIB-1 labeling index was highly correlated with Cdk2 activity (r = 0.767, P < 0.0015). These results suggest that p27 and Cdk2 may play an important role in the proliferation of non-small cell cancer.

CD44 Suppresses TLR-Mediated Inflammation

The cell adhesion molecule CD44, which is the major hyaluronan receptor, has been implicated in the binding, endocytosis, and metabolism of hyaluronan. Previous studies have revealed that CD44 plays crucial roles in a variety of inflammatory diseases. In recent years, TLRs, which are ancient microbial pattern recognition receptors, have been shown to initiate an innate immune response and have been linked to a variety of inflammatory diseases. The present study shows that CD44 negatively regulates in vivo inflammation mediated by TLRs via NF-κB activation, which leads to proinflammatory cytokine production. Furthermore, our results show that CD44 directly associates with TLR2 when stimulated by the TLR2 ligand zymosan and that the cytoplasmic domain of CD44 is crucial for its regulatory effect on TLR signaling. This study indicates that CD44 plays a protective role in TLR-mediated inflammation and is the first to demonstrate a direct association between CD44 and a TLR.

The activity of RhoA is correlated with lymph node metastasis in human colorectal cancer.

Rho family GTPases play a pivotal role in the regulation of numerous cellular functions associated with malignant transformation and metastasis. To evaluate the role of these GTPases in colorectal cancer, the protein expression levels and activities of these proteins in matched sets of tumor and non-tumor tissues of surgical specimens were analyzed. The relationship between the protein levels and activities in tumor tissues to the clinicopathological features was also assessed. The expression levels of RhoA, active RhoA, Rac1, and active Rac1 in tumor tissues were higher than in normal tissues. The amounts of active RhoA protein in primary tumors correlated with lymph nodes metastasis. No relationship was noted between the protein expression levels and other clinicopathological findings. These findings suggest that the Rho family small GTPases are related to malignant transformation and progression of colorectal cancer and the activation of RhoA is associated with the lymph node metastasis.

Human mena associates with Rac1 small GTPase in glioblastoma cell lines

Mammarian enabled (Mena), a member of the Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) family of proteins, has been implicated in cell motility through regulation of the actin cytoskeleton assembly, including lamellipodial protrusion. Rac1, a member of the Rho family GTPases, also plays a pivotal role in the formation of lamellipodia. Here we report that human Mena (hMena) colocalizes with Rac1 in lamellipodia, and using an unmixing assisted acceptor depletion fluorescence resonance energy transfer (u-adFRET) analysis that hMena associates with Rac1 in vivo in the glioblastoma cell line U251MG. Depletion of hMena by siRNA causes cells to be highly spread with the formation of lamellipodia. This cellular phenotype is canceled by introduction of a dominant negative form of Rac1. A Rac activity assay and FRET analysis showed that hMena knock-down cells increased the activation of Rac1 at the lamellipodia. These results suggest that hMena possesses properties which help to regulate the formation of lamellipodia through the modulation of the activity of Rac1.

Fragmented hyaluronan is an autocrine chemokinetic motility factor supported by the HAS2-HYAL2/CD44 system on the plasma membrane.

Hyaluronan (HA) is synthesized by HA synthase (HAS) 1, HAS2 and HAS3, and degraded by hyaluronidase (HYAL) 1 and HYAL2 in a CD44-dependent manner. HA and HYALs are intricately involved in tumor growth and metastasis. Random cell movement is generally described as chemokinesis, and represents an important step at the beginning of tumor cell liberation from the primary site. To investigate the roles of HAS2 and HYAL2/CD44 in cell motility, we examined HeLa-S3 cells showing spontaneous chemokinesis. HeLa-S3 cells expressed HAS2 and HAS3. siRNA-mediated knockdown of HAS2 decreased spontaneous chemokinesis of HeLa-S3 cells. Although HeLa-S3 cells secreted 50 ng/ml of high molecular weight (HMW)-HA (peak: 990 kDa) into the culture supernatant after 6 h of culture, exogenously added HMW-HA did not enhance spontaneous chemokinesis of the cells. These observations suggested that HeLa-S3 cells may have a self-degrading system for HA to regulate their spontaneous chemokinesis. To examine this possibility, we investigated the effects of siRNA-mediated knockdown of HYAL2 or CD44 on the spontaneous chemokinesis of HeLa-S3 cells. Knockdown of either molecule decreased the spontaneous chemokinesis of the cells. Low molecular weight (LMW)-HA (23 kDa) reversed the HYAL2 siRNA-mediated reduction in spontaneous chemokinesis of HeLa-S3 cells to the level in control cells stimulated with the same HA. These findings indicate that the HAS2-HYAL2/CD44 system may support spontaneous chemokinesis of human cancer cells through self-degradation of HMW-HA to produce LMW-HA by an autocrine mechanism. Consequently, our study may further expand our understanding of HA functions in cancer.

Expression of Cell Cycle Regulating Proteins in an Unusual Transformation of Mantle Cell Lymphoma

We describe here two patients with mantle cell lymphoma (MCL) who after a few years, developed to the diffuse large cell lymphoma (DLCL) (anaplastic centrocytic lymphoma) growing in a diffuse sheets without the classical MCL component. In both the initial and second biopsy specimens, in each case, tumor cells were positive for cyclin D1, sIgM, sIgD, and CD5, but were negative for CD10 and CD23. In a study of immunoglobulin heavy chain (IgH) gene rearrangement, using the polymerase chain reaction (PCR) method, the products obtained from each paired biopsy tissue sample were the same size, and in one case had an identical sequence to the non-mutated VH gene. Immunohistochemistry was used to examine the expression of p53, p27Kip1 and cyclin E. Interestingly, there was clear overexpression of p53 protein in case 1 but not in case 2, compared with other typical MCL cases. The expression of p27Kip1 in the second biopsies of each case was decreased compared with those in the initial biopsies. In case 2, however, p27Kip1 was clearly expressed in the first and second biopsies, in contrast to other typical MCL cases. Thus these 2 cases demonstrate not only that the variant form of MCL may arise de novo, but also that MCL may transform to DLCL at the time of relapse. Although the mechanism of tumor progression/transformation is still poorly understood, the overexpression of p53 or p27Kip1 may be linked to a cellular mechanism involved in the development of the variant form of MCL.

Visualization of the activity of Rac1 small GTPase in a cell

Rho family G proteins including Rac regulate a variety of cellular functions, such as morphology, motility, and gene expression. Here we developed a fluorescence resonance energy transfer-based analysis in which we could monitor the activity of Rac1. To detect fluorescence resonance energy transfer, yellow fluorescent protein fused Rac1 and cyan fluorescent protein fused Cdc42-Rac1-interaction-binding domain of Pak1 protein were used as intermolecular probes of FRET. The fluorophores were separated with linear unmixing method. The fluorescence resonance energy transfer efficiency was measured by acceptor photobleaching assisted assay. With these methods, the Rac1 activity was visualized in a cell. The present findings indicate that this approach is sensitive enough to achieve results similar to those from ratiometric fluorescence resonance energy transfer analysis.

Collision of advanced gastric adenocarcinoma and gastrointestinal stromal tumour: a case report.

We present a case of an 83-year-old female patient with a collision tumour of an advanced Borrmann type 4 gastric cancer and a large gastric gastrointestinal stromal tumour (GIST). According to the deformity of the gastric wall caused by the GIST, type 4 cancer was difficult to identify by oesophagogastroduodenoscopy (OGD). The patient died of progressive gastric cancer related disease. While the mechanism of histogenesis of the simultaneous adenocarcinoma and GIST remains to be determined, the present case suggests that gastric adenocarcinoma has a more adverse effect on prognosis than does GIST. Additionally, this case suggests that thorough inspection of GIST patients is required at the OGD and at the pathology facility, in order to avoid overlooking the underlying cancer.

Abstract C74: Activated RhoA, Rac1 and ERK1/2 participate in chemokinesis triggered by fragmented 23 kDa hyaluronan

Tumors generally possess high levels of hyaluronan (HA), but have a reduced size compared with normal tissue. Experimentally increased HA induces tumor growth and metastasis in xenograft models (review by Toole, BP, 2004). Our study sought to elucidate the possible molecular mechanism of HA‐mediated cell motility in a human epithelial cell line, HeLaS3, using a Boyden chamber system. We showed that among the different molecular masses of HA (3, 23, 230 and 940 kDa) (Seikagaku Kogyo Co.) 23 kDa HA was the most potent in chemokinesis, while 230 and 940 kDa HAs had no detectable effect on chemokinesis. With a drop of 23‐kDa HA, sparsely adhered HeLaS3 cells spread, exhibiting multiple surface extensions in various directions, and altered lamellipodia. HeLaS3 cells exhibited fine cortical F‐actin at the lamellipodia and formed many small focal complexes instead of large focal adhesions at the base of membrane protrusions at 1–20 minutes after 23 kDa HA stimulation. These morphological alterations are reminiscent of Rac1 activated fibroblasts (Rottner, 1999. To properly address the modulation of Rho GTPases in HA‐induced chemokinesis, we evaluated cells carrying dominant negative mutants of either RhoA cDNA (dnRhoA) or Rac1 cDNA (dnRac1). The transduction of either dnRhoA or dnRac1 into HeLaS3 cells caused a dramatic decrease in chemokinesis with or without HA stimulation, compared with mock‐transduced control. To biochemically confirm Rho GTPase activation, we measured GTP‐RhoA and GTP‐Rac1 levels after stimulation with 23 kDa HA using ELISA based Rho GTPases activation assay kits (Cytoskeleton, Inc.). GTP‐Rho A increased after 23kD HA ligation, had a small peak at 1 minute, decreased, and then returned to baseline by 60 min. After 23kD HA stimulation, Rac1 activation was observed in dual peaks of 1.8–2 fold increases at 3 and 15 minutes; thereafter, it became near to baseline by 60 minutes. It has been shown that HA can modulate cell migration involving hyaladherin‐mediated signaling through ERK1/2 (Vigetti, 2008, Tolg, 2006). Initially, we examined the effect of the ERK inhibitor PD98059 in our system. A treatment of 6.6 µg/ ml PD98059 suppressed 23 kDa HA‐induced chemokinesis compared with the control group. Western blotting of the cell lysates showed sustained ERK1/2 phosphorylation over 120 minutes. This study indicates that sustained ERK1/2 phosphorylation is required for 23 kDa HA‐induced chemokinesis. Here we show that fragmented 23 kDa HA elicits enhanced chemokinesis and changes the shape of epithelial cells by weak Rho A activation prior to the recurrent Rac1 activation and the sustained phosphorylation of ERK1/2. Our unpublished study also indicates that hyaluronidase‐2‐mediated catabolism could play a significant role in HA oligosaccharide generation with the association of CD44 in epithelial cells. This indicates the possibility that fragmented HA acts as a mediator in the autocrine/paracrine mechanism. Our study showed that signals initiated by fragmented HA‐hyaladherin interactions induce chemokinesis of epithelial cells, expanding our understanding of the functions of HA in cancer cell biology. Citation Information: Cancer Res 2009;69(23 Suppl):C74.