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Purpose: Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities.Experimental Design: Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers.Results: Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing–induced apoptosis. K-Ras–mutated, c-Met–amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing–induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways.Conclusion: Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. Clin Cancer Res; 18(2); 380–91. ©2011 AACR.

PDF file - 3.6K, Cancer cells with K-Ras activation mutation (HCT116/Hkh-2 isogenic pair) are more susceptible to Ran silencing-induced apoptosis.

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PDF file - 1.6MB, Percentage of sub-G1 phase apoptotic MCF10a and MDA MB231 cells grown in serum-containing medium after Ran silencing. (A-B) Cells were infected with shRan1 and shRan2. Medium was changed to serum-free medium (DMEM) 24hr post-infection. Cells were harvested at 72hr and 96hr post-infection for (A) cell cycle and (B) Western blot analyses. (A) Significantly more sub-G1 phase cells were observed in MDA MB231 breast cancer cells than in immortalized breast epithelial cells 72 and 96 hr post-infection of the shRNAs. (B) More cleaved PARP, caspase 9 and caspase 3 were observed in MDA MB231 than in MCF10A cells upon Ran silencing, suggesting a higher apoptotic response in MDA MB231 than in MCF10a cells. Levels of Ran knockdown were similar in both cell lines. Actin was used as a loading control. (C-D) Cells were infected with shRan1 and shRan2, and harvested 72hr post-infection for (C) cell cycle and (D) Western blot analyses. (C) More apoptotic cells were observed in MDA MB231 than MCF10a cells upon Ran-silencing 72hr post-infection of shRNAs, when these two cell lines were grown in their normal medium. (D) Higher levels of cleaved PARP, caspase 9 and caspase 3 were observed at 72hr post-infection of shRNAs. Results are plotted as histogram showing the mean � SD from three independent experiments. Key; * represents p < 0.05 and ** represents p

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PDF file - 13.3MB, High level of Ran is correlated with shorter survival time of lung cancer patients.(A) Representative images for IHC staining in lung cancer specimens. Lung cancer specimens stained with different patterns for Ran. Upper panel, 40x magnification; Lower panel, 200x magnification of the same cases. (B) Kaplan-Meier plot of the survival of lung cancer patients using combined nuclear and cytoplasmic expression of Ran; High level, high level expression of both nuclear and cytoplasmc Ran; Intermediate level, high level expression of either nuclear or cytoplasmic Ran; Low level, low level expression of both nuclear and cytoplasmic Ran. Cumulative proportion of lung cancer patients surviving with time is plotted for high, intermediate and low Ran groups. (C) Kaplan-Meier plot of survival of lung cancer patients from seven microarray datasets available in GEO database. Cumulative proportion of lung cancer patients surviving with time is plotted for high (top 25% of patients expressing a high level of Ran) and low (bottom 25% of patients expressing a low level of Ran) Ran groups.

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PDF file - 3.5, MTT assay showing that cancer cells with K-Ras activation mutation (HCT116/Hkh-2 and DLD1/DKO3 isogenic pairs) and c-Met (HCC827 parental/GR5 pair) are more susceptible to Ran silencing-induced apoptosis. (A) In HCT116/Hkh-2 colon cancer isogenic pair, HCT116 cells, which contain K-Ras activation mutation, are more susceptible to apoptosis induction by the 5 shRNAs targeting Ran, compared to the K-Ras wild-type counterpart, Hkh-2. (B) In DLD1/DKO3 colon cancer isogenic pair, DLD1 cells, which contain K-Ras activation mutation, are more susceptible to apoptosis induction by the 2 shRNAs targeting Ran, compared to the K-Ras wild-type counterpart, DKO3. (C) In HCC827 parental/GR5 lung cancer pair, the GR5 cells, which contain c-Met amplification, are more susceptible to apoptosis induction by the 2 shRNAs targeting Ran, compared to the wild-type HCC827 cells. Results are plotted as histogram showing the mean � SD from three independent experiments. Key; *, ** and *** represent p < 0.05

PDF file - 1.8K, Percentage of sub-G1 phase apoptotic cells and apoptotic pathway induced by Ran silencing in different cell lines. (A) Cells were harvested 72 hours post-infection, fixed and stained with Propidium iodide. Significantly fewer Sub-G1 phase apoptotic cells were observed in the immortalized human embryonic kidney 293 (HEK 293) cell line compared to cancer cell lines from breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (HCT116 and DLD-1) upon Ran silencing (p < 0.01). (B) Western blots of apoptosis mediators in immortalized HEK293, breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (DLD-1 and HCT116) cancer cell lines at 72 hours post-infection with Scr or Ran shRNAs.

PDF file - 1.9MB, Ran silencing induced apoptosis is concentration dependent. Effect of Ran silencing on apoptotic induction was tested using five different shRNAs (shRan1-5) targeting different regions of Ran compared to scramble shRNA (shScr) control in MDA MB231 breast cancer and A549 lung cancer cells. (A) mRNA and (B) protein levels of Ran, and (C) Flow cytometric analysis for DNA content, in MDA MB231 cells infected with different shRNAs targeting Ran. (D) Protein level and (E) Flow analysis for DNA content in A549 cells infected with different shRNAs targeting Ran. Note that the decrease in Ran mRNA level was associated with the decrease in Ran protein levels (r = 0.950, p < 0.01) in MDA MB231 cells infected with shRan1-5. In addition, the percentage of sub-G1 phase apoptotic cells induced by Ran silencing was significantly inversely associated with the levels of Ran in protein levels in MDA MB231 breast cancer cells (r = -0.880, p

PDF file - 4.8, Effect of chemical inhibitors on the activity of signaling pathways in MDA MB231 and A549 cells expressing various levels of Ran. Western blots of phospho- and total Akt, ERK and S6K proteins in MDA MB231 and A549 cells expressing various levels of Ran grown in different growth conditions. Representative blots from two independent experiments are shown.

PDF file - 725K, Ran silencing-induced apoptosis depends on the growth stimulation in MDA MB231 and A549 cells using MTT assay.Percentage of cell survival in (A) MDA MB231 and (B) A549 cells expressing various levels of Ran grown in different conditions as determined by MTT assay. Results are plotted as histograms showing the mean � SD from three independent experiments. Key; *, ** and *** represent p < 0.05

The Fanconi anemia (FA) pathway safeguards genomic stability through cell cycle regulation and DNA damage repair. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established, but their function in mitosis is incompletely understood. Here, we performed a kinome‐wide synthetic lethality screen in FANCA−/− fibroblasts, which revealed multiple mitotic kinases as necessary for survival of FANCA‐deficient cells. Among these kinases, we identified the depletion of the centrosome kinase SIK2 as synthetic lethal upon loss of FANCA. We found that FANCA colocalizes with SIK2 at multiple mitotic structures and regulates the activity of SIK2 at centrosomes. Furthermore, we found that loss of FANCA exacerbates cell cycle defects induced by pharmacological inhibition of SIK2, including impaired G2‐M transition, delayed mitotic progression, and cytokinesis failure. In addition, we showed that inhibition of SIK2 abrogates nocodazole‐induced prometaphase arrest, suggesting a novel role for SIK2 in the spindle assembly checkpoint. Together, these findings demonstrate that FANCA‐deficient cells are dependent upon SIK2 for survival, supporting a preclinical rationale for targeting of SIK2 in FA‐disrupted cancers.

Background Gestational trophoblastic disease (GTD) is a heterogeneous group of diseases developed from trophoblasts. ASPP (Ankyrin-repeat, SH3-domain and proline-rich region containing protein) family proteins, ASPP1 and ASPP2, have been reported to be dysregulated in GTD. They modulate p53 activities and are responsible for multiple cellular processes. Nevertheless, the functional role of the ASPP family inhibitory member, iASPP, is not well characterized in GTD. Methods To study the functional role of iASPP in GTD, trophoblastic tissues from normal placentas, hydatidiform mole (HM) and choriocarcinoma were used for immunohistochemistry, whereas siRNAs were used to manipulate iASPP expression in choriocarcinoma cell lines and study the subsequent molecular changes. Results We demonstrated that iASPP was overexpressed in both HM and choriocarcinoma when compared to normal placenta. Progressive increase in iASPP expression from HM to choriocarcinoma suggests that iASPP may be related to the development of trophoblastic malignancy. High iASPP expression in HM was also significantly associated with a high expression of autophagy-related protein LC3. Interestingly, iASPP silencing retarded the growth of choriocarcinoma through senescence instead of induction of apoptosis. LC3 expression decreased once iASPP was knocked down, suggesting a downregulation on autophagy. This may be due to iASPP downregulation rendered decrease in Atg5 expression and concomitantly hindered autophagy in choriocarcinoma cells. Autophagy inhibition per se had no effect on the growth of choriocarcinoma cells but increased the susceptibility of choriocarcinoma cells to oxidative stress, implying a protective role of iASPP against oxidative stress through autophagy in choriocarcinoma. Conclusions iASPP regulates growth and the cellular responses towards oxidative stress in choriocarcinoma cells. Its overexpression is advantageous to the pathogenesis of GTD. (266 words).

Ovarian clear cell carcinoma (OCCC) is a type of epithelial ovarian cancer that is strongly associated with endometriosis, resistance against conventional chemotherapy and thus poorer prognosis. The expression of inhibitory member of the ASPP family proteins (iASPP) and Polo-like kinase (PLK)1 were significantly higher in OCCC compared to benign cystadenomas and endometriosis. Both protein expressions were found to correlate with chemoresistance in patients with OCCC while high iASPP expression alone was significantly associated with a poor patient survival. The growth of OCCC cell lines, OVTOKO and KK, were inhibited after iASPP silencing. Such effect was related to senescence triggering as evidenced by increased SA-β-Gal staining and p21WAF1/Cip1 expression. Moreover, knockdown of iASPP induced PLK1 downregulation, whereas either genes' silencing sensitized the cells in response to cisplatin treatment. More prominent apoptosis was induced by cisplatin in OCCC cells after the knockdown of either iASPP or PLK1 as evidenced by the formation of more cleaved caspase 3. Heightened chemosensitivity to cisplatin after iASPP knockdown was further demonstrated in in vivo xenograft model. Additionally, both iASPP and PLK1 were shown to regulate autophagic flux as the induction of LC3B-II and LC3 puncta were much less in OCCC cells with either knockdown. Importantly, inhibition of autophagy also enhanced chemosensitivity to cisplatin in OCCC cells. These findings strongly imply that iASPP and PLK1 affect the chemoresistance of OCCC via the regulation of autophagy and apoptosis. Both iASPP and PLK1 can be potential therapeutic targets for treating OCCC in combination with conventional chemotherapy.

Hydatidiform mole (HM), an unusual pregnancy with pure or predominant paternal genetic contribution, is the most common form of gestational trophoblastic disease. Most HM regress after uterine evacuation but some will develop into persistent disease or even frank malignancy. Although p53 is highly expressed in HM, TP53 mutations have rarely been detected in previous studies. Here we screened for specific missense mutations on several TP53 hotspots in 49 HMs using a highly sensitive pyrosequencing approach and revealed the significant existence of such mutations in HM tissues. A particularly high frequency (∼59% of the cases) of p53 inactivating mutation on exon 7 has been detected. Our identification of hitherto unreported TP53 mutations in HM suggests the presence of p53 mutants and reflects the advantages of using pyrosequencing for point mutation detection in clinical samples. Traditional sequencing method may have overlooked such mutations that only occur in a small population of trophoblasts.

Additional file 1. A list of average immunoscores for patients and their individual diagnosis.

It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival.Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator.

Breast cancer is one of the most prevalent malignancies worldwide. It consists of a group of tumor cells that have the ability to grow uncontrollably, overcome replicative senescence (tumor progression) and metastasize within the body. Metastases are processes that consist of an array of complex gene dysregulation events. Although these processes are still not fully understood, the dysregulation of a number of key proteins must take place if the tumor cells are to disseminate and metastasize. It is now widely accepted that future effective and innovative treatments of cancer metastasis will have to encompass all the major components of malignant transformation. For this reason, much research is now being carried out into the mechanisms that govern the malignant transformation processes. Recent research has identified key genes involved in the development of metastases, as well as their mechanisms of action. A detailed understanding of the encoded proteins and their interrelationship generates the possibility of developing novel therapeutic approaches. This review will focus on a select group of proteins, often deregulated in breast cancer metastasis, which have shown therapeutic promise, notably, EMT, E-cadherin, Osteopontin, PEA3, Transforming Growth Factor Beta (TGF-β) and Ran.

Breast cancer is a leading cause of cancer-related deaths. Anemia is common in breast cancer patients and can be treated with blood transfusions or with recombinant erythropoietin (EPO) to stimulate red blood cell production. Clinical studies have indicated decreased survival in some groups of cancer patients treated with EPO. Numerous tumor cells express the EPO receptor (EPOR), posing a risk that EPO treatment would enhance tumor growth, but the mechanisms involved in breast tumor progression are poorly understood. Here, we have examined the functional role of the EPO-EPOR axis in pre-clinical models of breast cancer. EPO induced the activation of PI3K/AKT and MAPK pathways in human breast cancer cell lines. EPOR knockdown abrogated human tumor cell growth, induced apoptosis through Bim, reduced invasiveness, and caused downregulation of MYC expression. EPO-induced MYC expression is mediated through the PI3K/AKT and MAPK pathways, and overexpression of MYC partially rescued loss of cell proliferation caused by EPOR downregulation. In a xenotransplantation model, designed to simulate recombinant EPO therapy in breast cancer patients, knockdown of EPOR markedly reduced tumor growth. Thus, our experiments in vitro and in vivo demonstrate that functional EPOR signaling is essential for the tumor-promoting effects of EPO and underline the importance of the EPO-EPOR axis in breast tumor progression.

Emerging evidence has indicated a role of the bone morphogenetic proteins (BMP) in the pathogenesis of certain cancers. The signaling of BMP family members is tightly regulated by their antagonists, including noggin and SOST, which are, in turn, positively regulated by BMP, thereby forming a negative feedback loop. Consequently, the expression of these antagonists should be taken into account in studies on the prognostic significance of BMP. In the present paper, we correlated protein and mRNA expression levels of BMP6, noggin and SOST, alone or in combination, with patient survival in various types of cancer. We found that BMP6 alone was not significantly correlated with esophageal squamous cell carcinoma patient survival. Instead, a high level of inhibitor of differentiation 1, a downstream factor of BMP6, was associated with shorter survival in patients whose tumors stained strongly for BMP6. Knockdown of noggin in esophageal cancer cell line EC109, which expresses BMP6 strongly and SOST weakly, enhanced the non‐adherent growth of the cells. Noggin and SOST expression levels, when analyzed alone, were not significantly correlated with patient survival. However, high BMP6 activity, defined by strong BMP6 expression coupled with weak noggin or SOST expression, was significantly associated with shorter survival in esophageal squamous cell carcinoma patients. We further confirmed that BMP6 activity could be used as a prognostic indicator in prostate, bladder and colorectal cancers, using publicly available data on BMP6, noggin and SOST mRNA expression and patient survival. Our results strongly suggest that BMP6, noggin and SOST could be used in combination as a prognostic indicator in cancer progression. (Cancer Sci 2012; 103: 1145–1154)

Using inverse polymerase chain reaction, we identified CD44, located on chromosome 11p13, as a novel translocation partner of IGH in 9 of 114 cases of gastric, nongastric extranodal, follicular, and nodal diffuse large B-cell lymphoma (DLBCL). Notably, these translocations involving IGHSμ were detected in follicular lymphomas and exclusively in germinal center B cell-ike (GCB)–DLBCLs. CD44 is not expressed in reactive GC B cells. The IGHSμ/CD44 translocations substitute Sμ for the CD44 promoter and remove exon 1 of CD44, resulting in the overexpression of Iμ-CD44 hybrid mRNA transcripts activated from derivative 11 that encode a new CD44 variant lacking the leader peptide and with a unique C-terminus (CD44ΔEx1). When overexpressed in vitro in the CD44− GCB-DLBCL cell line BJAB, CD44ΔEx1–green fluorescent protein localized to the cytoplasm and nucleus, whereas CD44s–green fluorescent protein (standard form) localized to the plasma membrane. The ectopic expression of CD44ΔEx1 in BJAB cells enhanced their proliferation rate and clonogenic ability, indicating a possible pathogenic role of the translocation.

Deregulation of nuclear factor (NF)-kappaB signalling is common in cancers and is essential for tumourigenesis. Constitutive NF-kappaB activation in extranodal natural killer (NK)-cell lymphoma, nasal type (ENKL) is known to be associated with aberrant nuclear translocation of BCL10. Here we investigated the mechanisms leading to NF-kappaB activation and BCL10 nuclear localization in ENKLs. Given that ENKLs are dependent on T-cell-derived interleukin-2 (IL2) for cytotoxicity and proliferation, we investigated whether IL2 modulates NF-kappaB activation and BCL10 subcellular localization in ENKLs. In the present study, IL2-activated NK lymphoma cells were found to induce NF-kappaB activation via the PI3K/Akt pathway, leading to an increase in the entry of G(2)/M phase and concomitant transcription of NF-kappaB-responsive genes. We also found that BCL10, a key mediator of NF-kappaB signalling, participates in the cytokine receptor-induced activation of NF-kappaB. Knockdown of BCL10 expression resulted in deficient NF-kappaB signalling, whereas Akt activation was unaffected. Our results suggest that BCL10 plays a role downstream of Akt in the IL2-triggered NF-kappaB signalling pathway. Moreover, the addition of IL2 to NK cells led to aberrant nuclear translocation of BCL10, which is a pathological feature of ENKLs. We further show that BCL10 can bind to BCL3, a transcriptional co-activator and nuclear protein. Up-regulation of BCL3 expression was observed in response to IL2. Similar to BCL10, the expression and nuclear translocation of BCL3 were induced by IL2 in an Akt-dependent manner. The nuclear translocation of BCL10 was also dependent on BCL3 because silencing BCL3 by RNA interference abrogated this translocation. We identified a critical role for BCL10 in the cytokine receptor-induced NF-kappaB signalling pathway, which is essential for NK cell activation. We also revealed the underlying mechanism that controls BCL10 nuclear translocation in NK cells. Our findings provide insight into a molecular network within the NF-kappaB signalling pathway that promotes the pathogenesis of NK cell lymphomas.

Background: Id-1 is overexpressed in and correlated with metastatic potential of prostate cancer. The role of Id-1 in this metastatic process was further analysed. Methods: Conditioned media from prostate cancer cells, expressing various levels of Id-1, were used to stimulate pre-osteoclast differentiation and osteoblast mineralisation. Downstream effectors of Id-1 were identified. Expressions of Id-1 and its downstream effectors in prostate cancers were studied using immunohistochemistry in a prostate cancer patient cohort (N=110). Results: We found that conditioned media from LNCaP prostate cancer cells overexpressing Id-1 had a higher ability to drive osteoclast differentiation and a lower ability to stimulate osteoblast mineralisation than control, whereas conditioned media from PC3 prostate cancer cells with Id-1 knockdown were less able to stimulate osteoclast differentiation. Id-1 was found to negatively regulate TNF-β and this correlation was confirmed in human prostate cancer specimens (P=0.03). Furthermore, addition of recombinant TNF-β to LNCaP Id-1 cell-derived media blocked the effect of Id-1 overexpression on osteoblast mineralisation. Conclusion: In prostate cancer cells, the ability of Id-1 to modulate bone cell differentiation favouring metastatic bone disease is partially mediated by TNF-β, and Id-1 could be a potential therapeutic target for prostate cancer to bone metastasis.

TWIST, a helix-loop-helix transcription factor, is highly expressed in many types of human cancer. We have previously found that TWIST confers prostate cancer cells with an enhanced metastatic potential through promoting epithelial-mesenchymal transition (EMT) and a high TWIST expression in human prostate cancer is associated with an increased metastatic potential. The predilection of prostate cancer cells to metastasize to bone may be due to two interplaying mechanisms (i) by increasing the rate of bone remodeling and (ii) by undergoing osteomimicry. We further studied the role of TWIST in promoting prostate cancer to bone metastasis. TWIST expression in PC3, a metastatic prostate cancer cell line, was silenced by small interfering RNA and we found that conditioned medium from PC3 with lower TWIST expression had a lower activity on stimulating osteoclast differentiation and higher activity on stimulating osteoblast mineralization. In addition, we found that these effects were, at least partly, associated with TWIST-induced expression of dickkopf homolog 1 (DKK-1), a factor that promotes osteolytic metastasis. We also examined TWIST and RUNX2 expressions during osteogenic induction of an organ-confined prostate cancer cell, 22Rv1. We observed increased TWIST and RUNX2 expressions upon osteogenic induction and downregulation of TWIST through short hairpin RNA reduced the induction level of RUNX2. In summary, our results suggest that, in addition to EMT, TWIST may also promote prostate cancer to bone metastasis by modulating prostate cancer cell-mediated bone remodeling via regulating the expression of a secretory factor, DKK-1, and enhancing osteomimicry of prostate cancer cells, probably, via RUNX2.

Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Kruppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo . IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1 , FAS , and CASP8 ). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo , nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts.

Background/Aim: Cyclooxygenase-2 (COX-2), an inducible enzyme that catalyzes prostaglandin synthesis, has been implicated in a number of hepatic stellate cell (HSC) functions. In the current study, we assessed the in vivo effect of celecoxib, a COX-2-selective inhibitor, in experimental liver fibrosis in rats. Methods: Male Sprague–Dawley rats received experimental treatments for 5 weeks. Serum alanine transminase at the time of sacrifice was measured. Quantitative assessment of liver fibrosis was performed by computerized morphometry. Expression of COX-2, α smooth muscle actin and connective tissue growth factor (CTGF) was evaluated by immunohistochemistry. Real-time quantitative PCR was used to determine the expression of genes associated with fibrogenesis and extracellular matrix degradation. Results: Liver fibrosis was significantly worse in rats that received both carbon tetrachloride (CCl4) and celecoxib, compared with rats that received CCl4 and gavage of water (P=0.037). There was also more HSC activation, and upregulation of collagen α1(I), heat-shock protein 47, αB crystallin, matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of MMP (TIMP)-2. The expression of TIMP-1 and CTGF was not significantly different between the two groups. The pro-fibrogenic effect of celecoxib in toxin-induced liver fibrosis in rats was further confirmed in thioacetamide model of liver injury. Conclusions: Celecoxib potentiates experimental liver fibrosis; further studies are warranted to investigate the potential pro-fibrogenic effect of celecoxib in other animal models of liver fibrosis and in patients with chronic hepatitis.

We characterized the effects of selective Cox-2 inhibition on the angiogenesis gastric cancer cell line SGC7901 by stable transfection of antisense Cox-2 cDNA (Cox-2-AS) or pharmacological inhibition by selective cox-2 inhibitor (NS398). The conditioned media obtained from SGC7901 treated with Cox-2-AS or NS398 suppressed the proliferation, migration and tube formation of human umbilical vein endothelial cells. Moreover, both treatments inhibited in vivo angiogenesis. These inhibitions could be partially reversed by the addition of PGE2. Our findings support that selective inhibition of Cox-2 alone plays an instrumental role on gastric cancer associated angiogenesis.

Dear Sir, Clear cell carcinoma (CCC) of the ovary is a subtype of epithelial ovarian cancer (EOC) known for its relative resistance to standard platinum-based chemotherapy and poor prognosis. CCC account for up to 25% of ovarian carcinomas in Japan while make up only about 10% in North America. Analysis of archived pathology records on cases of ovarian cancer managed by Queen Mary Hospital, Hong Kong, from January 1993 to June 2012 revealed that CCC constituted 22.4% of EOC locally. Moreover, its yearly incidence showed a significant increase (unpublished results). Studies on ovarian CCC are therefore important and of regional significance. Genetic and molecular biological studies of ovarian CCC often involve in vitro experiments using several widely recognised ovarian CCC cell lines including ES2, TOV-21G, OVISE, OVTOKO and OVMANA.1 However, we found that ES2, a widely used ovarian CCC cell line established in the late 1980s, displayed unusual features distinct from other ovarian CCC cell lines. Such observations led us to question its true histological identity. Verification of hepatocyte nuclear factor 1β (HNF-1β) expression in our ovarian cancer cell lines has been carried out. HNF-1β is a transcription factor found to be considerably upregulated in almost all ovarian CCC through CpG island hypomethylation but not in other histological subtypes …

Aims - To study the prevalence of high risk oncogenic human papillomaviruses (HPV) in inverted papilloma and papillary transitional cell carcinoma of the bladder. Methods - Ten cases of inverted papilloma and 20 cases of papillary transitional cell carcinoma of the bladder from Chinese patients in Hong Kong were examined for the presence of HPV type 6, 11, 16, 18, 31, and 33 genomes using the polymerase chain reaction and HPV type specific primer probe combinations on paraffin wax embedded biopsy specimens. Results - Of the 10 cases of inverted papilloma, cases 1 and 6 showed the presence of HPV types 16 and 18, respectively. Six of the 20 papillary transitional cell carcinomas were positive for HPV type 18. The other HPV types were not detected. Conclusions - HPV type 18 was found in 60% and 30% of cases of inverted papilloma and papillary transitional cell carcinoma of the bladder, respectively. These tumours were rarely associated with HPV types 6, 11, 16, 31, and 33. The role of HPV type 18 in oncogenesis of inverted papilloma and transitional cell carcinoma of the bladder requires further studies., published_or_final_version

Inhibition of the PI3K (phosphoinositide 3-kinase)/Akt/mTORC1 (mammalian target of rapamycin complex 1) and Ras/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathways for cancer therapy has been pursued for over a decade with limited success. Emerging data have indicated that only discrete subsets of cancer patients have favourable responses to these inhibitors. This is due to genetic mutations that confer drug insensitivity and compensatory mechanisms. Therefore understanding of the feedback mechanisms that occur with respect to specific genetic mutations may aid identification of novel biomarkers that predict patient response. In the present paper, we show that feedback between the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways is cell-line-specific and highly dependent on the activating mutation of K-Ras or overexpression c-Met. We found that cell lines exhibited differential signalling and apoptotic responses to PD184352, a specific MEK inhibitor, and PI103, a second-generation class I PI3K inhibitor. We reveal that feedback from the PI3K/Akt/mTORC1 to the Ras/MEK/ERK pathway is present in cancer cells harbouring either K-Ras activating mutations or amplification of c-Met but not the wild-type counterparts. Moreover, we demonstrate that inhibition of protein phosphatase activity by OA (okadaic acid) restored PI103-mediated feedback in wild-type cells. Together, our results demonstrate a novel mechanism for feedback between the PI3K/Akt/mTORC1 and the Ras/MEK/ERK pathways that only occurs in K-Ras mutant and c-Met amplified cells but not the isogenic wild-type cells through a mechanism that may involve inhibition of a specific endogenous phosphatase(s) activity. We conclude that monitoring K-Ras and c-Met status are important biomarkers for determining the efficacy of PI103 and other PI3K/Akt inhibitors in cancer therapy.

The transcription factors Pea3, Erm, and Er81 can promote cancer initiation and progression in various types of solid tumors. However, their role in esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we found that the expression levels of Pea3 and Erm, but not that of Er81, were significantly higher in ESCC compared with nontumor esophageal epithelium. A high level of Pea3 expression was significantly correlated with a shorter overall survival in a cohort of 81 patients with ESCC and the subgroup with N1 stage tumor (Wilcoxon-Gehan test, P = 0.016 and P = 0.001, respectively). Pea3 was overexpressed in seven ESCC cell lines compared with two immortalized esophageal cell lines. Pea3 knockdown reduced cell proliferation and suppressed nonadherent growth, migration, and invasion in ESCC cells in vitro. In addition, Pea3 knockdown in ESCC cells resulted in a down-regulation of phospho-Akt and matrix metalloproteinase 13, whereas a significant positive correlation in the expression levels was observed between Pea3 and phospho-Akt (r = 0.281, P < 0.013) and between Pea3 and matrix metalloproteinase 13 in the human specimens (r = 0.462, P < 0.001). Moreover, Pea3 modulated the sensitivity of EC109 cells to doxorubicin, probably via reduced activity of the phosphatidylinositol 3-kinaseAktmammalian target of Rapamycin complex 1 pathway on Pea3 knockdown. In conclusion, our results suggest that Pea3 plays an important role in the progression of ESCC. © 2011 American Society for Investigative Pathology., link_to_subscribed_fulltext

Deregulation of nuclear factor (NF)-kappaB signalling is common in cancers and is essential for tumourigenesis. Constitutive NF-kappaB activation in extranodal natural killer (NK)-cell lymphoma, nasal type (ENKL) is known to be associated with aberrant nuclear translocation of BCL10. Here we investigated the mechanisms leading to NF-kappaB activation and BCL10 nuclear localization in ENKLs. Given that ENKLs are dependent on T-cell-derived interleukin-2 (IL2) for cytotoxicity and proliferation, we investigated whether IL2 modulates NF-kappaB activation and BCL10 subcellular localization in ENKLs. In the present study, IL2-activated NK lymphoma cells were found to induce NF-kappaB activation via the PI3K/Akt pathway, leading to an increase in the entry of G(2)/M phase and concomitant transcription of NF-kappaB-responsive genes. We also found that BCL10, a key mediator of NF-kappaB signalling, participates in the cytokine receptor-induced activation of NF-kappaB. Knockdown of BCL10 expression resulted in deficient NF-kappaB signalling, whereas Akt activation was unaffected. Our results suggest that BCL10 plays a role downstream of Akt in the IL2-triggered NF-kappaB signalling pathway. Moreover, the addition of IL2 to NK cells led to aberrant nuclear translocation of BCL10, which is a pathological feature of ENKLs. We further show that BCL10 can bind to BCL3, a transcriptional co-activator and nuclear protein. Up-regulation of BCL3 expression was observed in response to IL2. Similar to BCL10, the expression and nuclear translocation of BCL3 were induced by IL2 in an Akt-dependent manner. The nuclear translocation of BCL10 was also dependent on BCL3 because silencing BCL3 by RNA interference abrogated this translocation. We identified a critical role for BCL10 in the cytokine receptor-induced NF-kappaB signalling pathway, which is essential for NK cell activation. We also revealed the underlying mechanism that controls BCL10 nuclear translocation in NK cells. Our findings provide insight into a molecular network within the NF-kappaB signalling pathway that promotes the pathogenesis of NK cell lymphomas.

Background: The antiapoptotic and epithelial-mesenchymal transition activities of Twist have been implicated in the neoplastic transformation and the development of metastasis, respectively. Upregulation of Twist, described in several types of human cancer, also acts as a prognostic marker of poor outcome. Aim: To investigate Twist expression in oesophageal squamous cell carcinoma (SCC) and its prognostic value in a Chinese cohort of patients with oesophageal SCC. Methods: Twist expression in primary oesophageal SCC of 87 Chinese patients was investigated by immunohistochemical staining. Twist protein level in one immortalised normal oesophageal epithelial cell line and six oesophageal SCC cell lines was measured by western blot analysis. Twist mRNA level in 30 pairs of frozen specimens of primary oesophageal SCC and non-neoplastic oesophageal epithelium from the upper resection margin of corresponding oesophagectomy specimen was also determined by semiquantitative reverse transcription-PCR. Results: It was found that Twist was upregulated in oesophageal SCC cell lines, and its mRNA and protein levels were both increased in oesophageal SCC and the non-neoplastic oesophageal epithelium (p, published_or_final_version

A 70-year-old man presented with rash, microscopic haematuria and proteinuria. The renal biopsy showed mesangial proliferative glomerulonephritis with mesangial IgA deposition. He was subsequently found to have basaloid squamous carcinoma of the oesophagus. This possible association between IgA nephropathy and oesophageal basaloid squamous carcinoma has never been reported.

Cyclooxygenase-2 (COX-2), an inducible enzyme that catalyzes prostaglandin synthesis, has been implicated in a number of hepatic stellate cell (HSC) functions. In the current study, we assessed the in vivo effect of celecoxib, a COX-2-selective inhibitor, in experimental liver fibrosis in rats.Male Sprague-Dawley rats received experimental treatments for 5 weeks. Serum alanine transminase at the time of sacrifice was measured. Quantitative assessment of liver fibrosis was performed by computerized morphometry. Expression of COX-2, alpha smooth muscle actin and connective tissue growth factor (CTGF) was evaluated by immunohistochemistry. Real-time quantitative PCR was used to determine the expression of genes associated with fibrogenesis and extracellular matrix degradation.Liver fibrosis was significantly worse in rats that received both carbon tetrachloride (CCl4) and celecoxib, compared with rats that received CCl4 and gavage of water (P = 0.037). There was also more HSC activation, and upregulation of collagen alpha1(I), heat-shock protein 47, alphaB crystallin, matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of MMP (TIMP)-2. The expression of TIMP-1 and CTGF was not significantly different between the two groups. The pro-fibrogenic effect of celecoxib in toxin-induced liver fibrosis in rats was further confirmed in thioacetamide model of liver injury.Celecoxib potentiates experimental liver fibrosis; further studies are warranted to investigate the potential pro-fibrogenic effect of celecoxib in other animal models of liver fibrosis and in patients with chronic hepatitis.

Aims: To determine the extent of Fas expression in oesophageal squamous cell carcinomas (ESCCs) from Chinese patients and to correlate Fas expression with clinicopathological prognostic parameters. Methods: Clinicopathological data were collected from 58 patients with ESCC who underwent oesophagectomy and had no prior radiotherapy or chemotherapy. Immunostaining was performed on the primary tumours. Expression of Fas was correlated with patients' demographics, tumour characteristics and stage, R category of surgery, and patients' survival. Results: The actuarial survival rates of all patients at two and five years after surgery were 48% and 14%, respectively. Fas expression was detected in 89.7% of ESCCs. Higher Fas expression recorded on a four point scale correlated with better tumour differentiation (p < 0.01), but not with other patient or tumour variables. Importantly, higher Fas expression was associated with better survival (p = 0.0317). Conclusions: These findings suggest that Fas activated apoptosis is important in the pathogenesis of ESCC. This molecular pathway may be a potential therapeutic target for ESCC., published_or_final_version

Although gastric cancer with cyclooxygenase (COX)-2 overexpression is associated with poor prognosis, the mechanistic pathway remains unknown. We examined the associations between expressions of COX-2 and vascular endothelial growth factor (VEGF) in both gastric cancer cells and in human gastric cancer. The gastric cell line, Kato III, was transiently transfected with cox-2 expressing vector. The levels of COX-2, prostaglandin (PG) E 2 and VEGF expression were measured post-transfection. Additionally, expressions of COX-2 and VEGF in human gastric cancer were determined by immunohistochemistry in archive gastrectomy specimens. Tumor angiogenesis was assessed by the microvessel density (MVD), which was determined by anti-CD34 immunostaining. Transient transfection of Kato III with cox-2 was associated with increased COX-2 expression, higher PGE 2 production and upregulated VEGF expressions. Treatment with NS398, a specific COX-2 inhibitor, reduced VEGF expression in COX-2 expressing Kato III cells by 25%. Among the 67 gastric cancers examined, COX-2 overexpression was found in 45 (67%) cases whereas increased VEGF expression was detected in 46 (69%) cases. There was a significant association between COX-2 and VEGF expressions in gastric cancer (r=0.25, p=0.041). Additionally, tumor MVD was associated with both COX-2 (r=0.32, p=0.008) and VEGF (r=0.39, p=0.001) expressions. Our results showed that overexpression of COX-2 in both gastric cells and primary gastric cancer is associated with upregulation of VEGF and angiogenesis. Future studies should evaluate the potential anti-angiogenic effect of COX-2 inhibitors on human gastric cancer.

Although gastric cancer with cyclooxygenase (COX)-2 overexpression is associated with poor prognosis, the mechanistic pathway remains unknown. We examined the associations between expressions of COX-2 and vascular endothelial growth factor (VEGF) in both gastric cancer cells and in human gastric cancer. The gastric cell line, Kato III, was transiently transfected with cox-2 expressing vector. The levels of COX-2, prostaglandin (PG) E2 and VEGF expression were measured post-transfection. Additionally, expressions of COX-2 and VEGF in human gastric cancer were determined by immunohistochemistry in archive gastrectomy specimens. Tumor angiogenesis was assessed by the microvessel density (MVD), which was determined by anti-CD34 immunostaining. Transient transfection of Kato III with cox-2 was associated with increased COX-2 expression, higher PGE2 production and upregulated VEGF expressions. Treatment with NS398, a specific COX-2 inhibitor, reduced VEGF expression in COX-2 expressing Kato III cells by 25%. Among the 67 gastric cancers examined, COX-2 overexpression was found in 45 (67%) cases whereas increased VEGF expression was detected in 46 (69%) cases. There was a significant association between COX-2 and VEGF expressions in gastric cancer (r=0.25, p=0.041). Additionally, tumor MVD was associated with both COX-2 (r=0.32, p=0.008) and VEGF (r=0.39, p=0.001) expressions. Our results showed that overexpression of COX-2 in both gastric cells and primary gastric cancer is associated with upregulation of VEGF and angiogenesis. Future studies should evaluate the potential anti-angiogenic effect of COX-2 inhibitors on human gastric cancer.

Background: Ovarian cancer is one of the most lethal cancers among women worldwide. Despite of an initial response to chemotherapy, relapse may happen due to the chemoresistant phenotypes. However, underlying mechanism conferring chemoresistance is not well defined. p21-activated serine/threonine kinases (PAKs) are serine/threonine kinases involved in various cellular functions including cell motility, survival and angiogenesis.PAK4 is the prototypic member of Group II PAKs. In this study, we aim to delineate the crucial domains in PAK4 that may contribute to the development of chemoresistance in ovarian cancer cells by analysis of various PAK4 mutants. Materials and Methods: Ovarian cancer cell line SKOV-3 stably expressing various PAK4 mutants was used as an in vitro model. SKOV-3 was transfected with Flag-tagged wild type PAK4, constitutively active PAK4 (445N/474E), kinase-dead PAK4 (M350), or the control vector, p3XFLAG-CMV-10. To illustrate the functional impact of nuclear PAK4, PAK4 constructs with various nucleus localization signals (NLS) mutation were generated. PAK4 NLS mutants were then stably transfected into SKOV-3 and the expression and nuclear exclusion were confirmed by western blotting and immunofluorescence microscope respectively.They were also treated with cisplatin and the discrepancies in response to cisplatin were accessed by MTT assay and through the evaluation of apoptotic activity using flow cytometry and caspase-3 activation. In order to unveil the significance of PAK4 amplification and mutation status, genomic DNA of two immortalized ovarian epithelial cell lines HOSE6-3 and HOSE-11-12, and eleven ovarian cancer cell lines were extracted. Exon 3, which encodes the Cdc42-Rac interaction/binding (CRIB) domain and exons 5 - 10, which encode the kinase domain were sequenced to detect possible mutation in comparison to the wild-type sequence. Results: By MTT assay, overexpression of wild type PAK4 was demonstrated to enhance the survival of SKOV3 cells when compared with vector-transfected control cells when challenged with cisplatin. Flow Cytometry Analysis confirmed the above survival pattern. No significant differences were found between the two PAK4 kinase domain mutants and the wild type one. At genomic level, no amplification or mutation in PAK4 gene could be found in the ovarian cancer cell lines studied. Wild type PAK4 expression was found in both nucleus and cytoplasm. Mutation of NLS3, but not NLS1, could abolish the nuclear-localization of PAK4. Conclusion: In summary, aberrant expression of PAK4 contributes to resistance of ovarian cancer cells towards cisplatin.Amplification or mutation in PAK4 kinase domain cannot be detected and therefore may not be the crucial factor underlying overexpression in ovarian cancers. Further study on PAK4, including its subcellular localization, may further shed light on its potential as a novel therapeutic target of ovarian cancer. Citation Format: Ya Nan Du, Oscar GW Wong, Ka Kui Chan, Esther SY Wong, Annie NY Cheung. PAK4 mediated mechanisms contributing to chemoresistance of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2621. doi:10.1158/1538-7445.AM2014-2621

Aims - To determine retrospectively the prevalence of human papillomavirus (HPV) types 16 and 18 in penile carcinomas. Methods - Forty one surgically resected penile carcinomas from the archives at Queen Mary Hospital, Hong Kong, were reviewed and classified into verrucous carcinoma, and well, moderately, and poorly differentiated squamous cell carcinomas. Paraffin wax embedded tumour tissue was sectioned and analysed for HPV 16 and HPV 18 using the polymerase chain reaction with type specific internal probes. Results - There were seven verrucous carcinomas, and 11 well, 17 moderately, and six poorly differentiated squamous cell carcinomas. Six of the 41 (15%) patients had penile carcinoma containing HPV 16 or HPV 18 DNA, or both, with HPV 16 found in four (10%) and HPV 18 in four (10%). The mean ages of HPV positive and HPV negative groups of patients were 68.5 and 57.6 years, respectively (p < 0.05). None of the seven verrucous and 11 well differentiated squamous cell carcinomas was positive for HPV. The mean age of patients who had these carcinomas was 52.4 years. As a group, these low grade carcinomas occurred in patients younger by more than a decade than those who had carcinomas of the higher grades (mean age 64.4 years; p < 0.01). Conclusions - Penile carcinomas had much lower rates of infection by HPV 16 or HPV 18 than cervical carcinomas in this Hong Kong population. Based on our findings and on data collated from published findings, it is concluded that penile verrucous carcinomas are not associated with HPV 16 and HPV 18, The overall low prevalence of HPV 16 and HPV 18 in penile carcinomas suggests that other HPV types might be important in the pathogenesis of these tumours., published_or_final_version