Mai Mochizuki

Synergistic cytotoxicity of afatinib and cetuximab against EGFR T790M involves Rab11-dependent EGFR recycling.

EGFR is an important therapeutic target for non-small cell lung cancers (NSCLCs). Tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, are effective in cases with EGFR-activating mutations. However, most such cases become resistant through a secondary EGFR mutation, T790M. While the second-generation TKI afatinib has a higher affinity for double-mutant EGFRs, better efficacy is needed. Combining afatinib with the anti-EGFR monoclonal antibody cetuximab improves clinical outcomes, but the mechanism is unclear. Here we examined this effect using erythroleukemic K562 cells. The activating EGFR mutation L858R is sensitive to first-generation TKIs, and adding T790M confers resistance to these drugs. This double-mutant EGFR was moderately sensitive to afatinib, but responded weakly to cetuximab. Combined afatinib and cetuximab synergistically increased their cytotoxicity for K562 cells expressing the double-mutant EGFR. Apoptosis in these cells followed induction of the pro-apoptotic protein BIM. Unexpectedly, afatinib caused redistribution of EGFR to the cell surface through Rab11a-dependent recycling. Cetuximab reduced cell-surface EGFR, and total EGFR decreased synergistically when cetuximab was combined with afatinib. Our results suggest that the synergistic effect exerted by afatinib and cetuximab on NSCLCs is associated with BIM induction and alterations in EGFR status.

Suppressive expression of CD274 increases tumorigenesis and cancer stem cell phenotypes in cholangiocarcinoma

Cholangiocarcinoma is an aggressive malignant tumor originating from intrahepatic or extrahepatic bile ducts. Its malignant phenotypes may be assumed by cancer stem cells (CSC). Here, we demonstrate that CD274 (PD‐L1), known as an immunomodulatory ligand, has suppressive effects on CSC‐related phenotypes of cholangiocarcinoma. Using two human cholangiocarcinoma cell lines, RBE and HuCCT1, we attempted to isolate the CD274low and CD274high cells from each cell line, and xenografted them into immunodeficient NOD⁄scid⁄γcnull (NOG) mice. We found that the CD274low cells isolated from both RBE and HuCCT1 are highly tumorigenic in NOG mice compared with CD274high cells. Furthermore, the CD274low cells possess several CSC‐related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle. Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity. These findings are compatible with our observation that clinical cholangiocarcinoma specimens are classified into low and high groups for CD274 expression, and the CD274 low group shows poorer prognosis when compared with the CD274 high group. These results strongly suggest that CD274 has a novel function in the negative regulation of CSC‐related phenotypes in human cholangiocarcinoma, which is distinct from its immunomodulatory actions.

CD271 regulates the proliferation and motility of hypopharyngeal cancer cells.

CD271 (p75 neurotrophin receptor) plays both positive and negative roles in cancer development, depending on the cell type. We previously reported that CD271 is a marker for tumor initiation and is correlated with a poor prognosis in human hypopharyngeal cancer (HPC). To clarify the role of CD271 in HPC, we established HPC cell lines and knocked down the CD271 expression using siRNA. We found that CD271-knockdown completely suppressed the cells’ tumor-forming capability both in vivo and in vitro. CD271-knockdown also induced cell-cycle arrest in G0 and suppressed ERK phosphorylation. While treatment with an ERK inhibitor only partially inhibited cell growth, CDKN1C, which is required for maintenance of quiescence, was strongly upregulated in CD271-depleted HPC cells, and the double knockdown of CD271 and CDKN1C partially rescued the cells from G0 arrest. In addition, either CD271 depletion or the inhibition of CD271-RhoA signaling by TAT-Pep5 diminished the in vitro migration capability of the HPC cells. Collectively, CD271 initiates tumor formation by increasing the cell proliferation capacity through CDKN1C suppression and ERK-signaling activation, and by accelerating the migration signaling pathway in HPC.

Long non-coding RNA HOTAIR promotes cell migration by upregulating insulin growth factor–binding protein 2 in renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers. About one-third of RCC patients already have distal metastasis at the time of diagnosis. There is growing evidence that Hox antisense intergenic RNA (HOTAIR) plays essential roles in metastasis in several types of cancers. However, the precise mechanism by which HOTAIR enhances malignancy remains unclear, especially in RCC. Here, we demonstrated that HOTAIR enhances RCC-cell migration by regulating the insulin growth factor-binding protein 2 (IGFBP2) expression. HOTAIR expression in tumors was significantly correlated with nuclear grade, lymph-node metastasis, and lung metastasis. High HOTAIR expression was associated with a poor prognosis in both our dataset and The Cancer Genome Atlas dataset. Migratory capacity was enhanced in RCC cell lines in a HOTAIR-dependent manner. HOTAIR overexpression accelerated tumorigenicity and lung metastasis in immunodeficient mice. Microarray analysis revealed that IGFBP2 expression was upregulated in HOTAIR-overexpressing cells compared with control cells. The enhanced migration activity of HOTAIR-overexpressing cells was attenuated by IGFBP2 knockdown. IGFBP2 and HOTAIR were co-expressed in clinical RCC samples. Our findings suggest that the HOTAIR-IGFBP2 axis plays critical roles in RCC metastasis and may serve as a novel therapeutic target for advanced RCC.

Xenotransplantation elicits salient tumorigenicity of adult T-cell leukemia-derived cells via aberrant AKT activation.

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL‐2Rγcnull (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T‐cell leukemia‐derived cell lines, ST1‐N6 and TL‐Om1‐N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL‐Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1‐N6 and TL‐Om1‐N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK‐2206 attenuated the progression of tumors induced by ST1‐N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T‐cell leukemia‐derived cells.

Periostin is a negative prognostic factor and promotes cancer cell proliferation in non-small cell lung cancer

Periostin is a matricellular protein that is secreted by fibroblasts and interacts with various cell-surface integrin molecules. Although periostin is known to support tumor development in human malignancies, little is known about its effect on lung-cancer progression. We here demonstrate that periostin is a negative prognostic factor that increases tumor proliferation through ERK signaling in non-small cell lung carcinoma. We classified 189 clinical specimens from patients with non-small cell lung-cancer according to high or low periostin expression, and found a better prognosis for patients with low rather than high periostin, even in cases of advanced-stage cancer. In a syngenic implantation model, murine Ex3LL lung-cancer cells formed smaller tumor nodules in periostin−/− mice than in periostin+/+ mice, both at the primary site and at metastatic lung sites. An in vitro proliferation assay showed that stimulation with recombinant periostin increased Ex3LL-cell proliferation. We also found that periostin promotes ERK phosphorylation, but not Akt or FAK activation. These findings suggest that periostin represents a potential target in lung-cancer tumor progression.

Periostin attenuates tumor growth by inducing apoptosis in colitis-related colorectal cancer

Inflammatory bowel diseases, which are multifactorial autoimmune colitis diseases, are occurring with increasing prevalence. One of the most serious complications of these diseases is colorectal cancer. Here we investigated the role of periostin (Postn), a matricellular protein that interacts with various integrin molecules on the cell surface, in colitis-induced colorectal cancer. Immunohistochemistry of mouse and human colorectal cancer samples revealed that Postn was expressed in the stroma and was upregulated in close proximity to the cancer cells. The colonic tumorigenesis in an inflammation-related colon carcinogenesis mouse model was increased in Postn knock-out (Postn−/−) mice compared to Postn+/+ mice. Although no difference was found in the degree of colitis between Postn+/+ and Postn−/− mice, Postn inhibited tumor growth and induced the apoptosis of mouse rectal cancer cells in vitro. Furthermore, fewer apoptotic colorectal cancer cells were observed in Postn−/− than in Postn+/+ mice. These data suggested that Postn has an anti-tumor effect on colitis-induced colorectal cancer.

Long non-coding RNA HOTAIR up‑regulates chemokine (C‑C motif) ligand 2 and promotes proliferation of macrophages and myeloid‑derived suppressor cells in hepatocellular carcinoma cell lines

Accumulating evidence demonstrated that Hox antisense intergenic RNA (HOTAIR) serves essential roles in the development and metastasis of several types of cancer. In hepatocellular carcinoma (HCC), high expression of HOTAIR is associated with poor prognosis, and HOTAIR regulates cell migration and proliferation. However, the downstream molecular targets of HOTAIR depend on the cancer cell types, and little is known about the precise molecular mechanisms of HOTAIR involved in cancer development. The present study investigated the role of HOTAIR in HCC cell lines. Notably, the overexpression of HOTAIR in HCC cell lines did not affect cell migration and proliferation capability. In the microarray analysis, C-C motif chemokine ligand (CCL)2 was identified to be differentially expressed in HOTAIR-overexpressing cells, and it was confirmed that HOTAIR promotes the secretion of CCL2. Furthermore, it was revealed that the proportion of macrophages and myeloid-derived suppressor cells (MDSCs) were increased when peripheral blood mononuclear cells were co-cultured with HOTAIR-overexpressing cells. Collectively, these data suggest that HOTAIR regulates CCL2 expression, which may be involved in the recruitment of macrophages and MDSCs to the tumor microenvironment.

Abstract 2331: CD271 defines a cancer-initiating cell population in hypopharyngeal cancer

Cancer stem cells (CSCs) or Cancer initiating cells (CICs) contribute to the malignant phenotypes of a variety of cancers, but markers to identify human hypopharyngeal cancer (HPC) stem cells remain poorly understood. We found that the CD271+ but not CD271- cell population sorted from xenotransplanted HPCs possesses tumor-initiating capability in immunodeficient mice. Tumors generated from the CD271+ cells contained both CD271+ and CD271- cells, indicating that the population could undergo differentiation. Immunohistological analyses of the tumors revealed that the CD271+ cells localized to a perivascular niche near CD34+ vasculature, to invasive fronts, and to the basal layer. In accordance with these characteristics, a stemness marker, Nanog, and matrix metaloproteinases (MMPs), which are implicated in cancer invasion, were significantly up-regulated in the CD271+ compared to the CD271- cell population. Furthermore, using primary HPC specimens, we demonstrated that high CD271 expression was correlated with a poor prognosis for patients. Furthermore, RNA interference of CD271 in CD271+ cells decreased efficiency of tumor initiation and migration phenotypes, suggesting that CD271 is indeed functional. Taken together, our findings indicate that CD271 is a novel marker for cancer initiating cells and for HPC prognosis. Citation Format: Nobuyuki Tanaka, Takayuki Imai, Mai Mochizuki, Keiichi Tamai, Kazunori Yamaguchi, Kennichi Satoh, Kazuto Matsuura, Ikuro Sato, Shigeru Saijo, Kazuo Sugamura. CD271 defines a cancer-initiating cell population in hypopharyngeal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2331. doi:10.1158/1538-7445.AM2015-2331