Human Cervical Squamous Cell Carcinoma Research Articles

Trichosanthin inhibits DNA methyltransferase and restores methylation-silenced gene expression in human cervical cancer cells

Epigenetic silencing of tumor suppressor genes is a well-established oncogenic process and the reactivation of tumor suppressor genes that have been silenced by promoter methylation is an attractive molecular target for cancer therapy. In this study, we investigated the demethylation activity of trichosanthin (TCS, the main bioactive component isolated from a Chinese medicinal herb) and its possible mechanism of action in cervical cancer cell lines. HeLa human cervical adenocarcinoma and CaSki human cervical squamous carcinoma cells were treated with various concentrations (0, 20, 40 and 80 µg/ml) of TCS for 48 h and the mRNA and protein expression levels of the tumor suppressor genes adenomatous polyposis coli (APC) and tumor suppressor in lung cancer 1 (TSLC1) were detected using reverse transcription (RT)-PCR and western blotting, respectively. We analyzed the methylation status of APC and TSLC1 using methylation-specific PCR (MSP). The expression levels and enzyme activity of DNA methyltransferase 1 (DNMT1) were also examined. The mRNA and protein expression levels of APC and TSLC1 were increased following treatment with various concentrations (0, 20, 40 and 80 µg/ml) of TCS for 48 h. The expression of the APC gene increased 2.55±0.29-, 3.44±0.31- and 4.36±0.14-fold, respectively. The expression of the TSLC1 gene increased 2.28±0.15-, 4.23±0.88- and 6.09±0.23-fold, respectively. MSP detection showed that TCS induced demethylation in HeLa and CaSki cells and that this demethylation activity was accompanied by the decreased expression of DNMT1 and reduced DNMT1 enzyme activity. Our experimental results demonstrate for the first time that TCS is capable of restoring the expression of methylation-silenced tumor suppressor genes and is potentially useful as a demethylation agent for the clinical treatment of human cervical cancer.

Study on effects of microRNA-21 antisense oligonucleotide in vivo and in vitro on bionomics of human cervical squamous carcinoma cell lines SiHa

To explore the effect of microRNA-21 (miR-21) antisense oligonucleotide on the biological characteristics of human cervical squamous carcinoma cell lines SiHa in vivo and in vitro. Specific phosphorothioate antisense oligodeoxynucleotides targeting miR-21 were synthesized and transfected into cervical cancer cells in vitro. Expression of miR-21 in SiHa after transfection was detected by real-time RT-PCR. The cell proliferation was evaluated by MTT assay and colony formation experiment. The cell apoptosis was analyzed by annexin V-FITC/PI analysis. The inhibitory effect of miR-21 antisense oligonucleotide on tumor growth was evaluated by tumor growth curves and immunohistochemistry (MaxVision method). H-E staining was used to document morphological changes and fluorometric TUNEL assay was to detect the apoptotic activity. After the transfection of antisense miR-21, the expression of miR-21 decreased along with an obvious growth inhibition, compared with that of the control groups (P < 0.05). Colony formation of both cell lines was markedly inhibited with antisense miR-21 (55.6% ± 1.4%), as compared with that in the negative group (98.3% ± 2.0%, P < 0.05). Flow cytometry assay showed that antisense miR-21 expression significantly enhanced the cell apoptosis (6.7% ± 1.3% and 29.4% ± 1.7%, P < 0.05). The tumor-forming rates of miR-21 transfected group, and negative control groups were 3/8 and 6/8, respectively (P < 0.05). Ki-67 proliferative marker staining decreased significantly (42% vs 90%) in the transfected group compared with negative control groups. Extensive dead tumor cells were seen in the miR-21 transfected cells along with a marked increase of apoptosis (P < 0.05). Targeted antisense oligonucleotide miR-21 effectively suppresses the growth of cervical carcinoma SiHa cells both in vitro and in vivo through an induction of apoptosis.

Establishment and characterization of a new carcinoma cell line from uterine cervix of Uyghur women

To establish a uterine cervical carcinoma cell line of Uyghur ethnical background and to evaluate the related biological characteristics for future biomedical investigations of diseases in the Uyghur population. Poorly-differentiated squamous cell carcinoma specimens of Uyghur patients were obtained and cultured in vitro by enzymatic digestion method, followed by continuous passaging to reach a stable growth determined by cell viability and growth curve. Morphological study, cell cycling and chromosomal analysis were performed. Tumorigenesis study was conducted by inoculation of nude mice. Biomarker (CK17, CD44, Ki-67, CK14 and vimentin) expression was detected by immunofluorescence and immunocytochemical techniques. A cervical carcinoma cell line was successfully established and maintained for 12 months through 70 passages. The cell line had a stable growth with a population doubling time of 51.9 h. Flask method and double agar-agar assay showed that the cell line had colony-forming rates of 32.5% and 15.6%, respectively. Ultrastructural evaluation demonstrated numerous cell surface protrusions or microvilli, a large number of rod-shape structures in cytoplasm, typical desmosomes and nuclear atypia. Chromosomal analysis revealed human karyotype with the number of chromosomes per cell varying from 32 - 97 with a majority of 54 - 86 (60.3%). Xenogeneic tumors formed in nude mice showed histological structures identical to those of the primary tumor. The cells had high expression of CK17, CD44, Ki-67 and vimentin but no CK14 expression. A cervical carcinoma cell line from a female Uyghur patient is successfully established. The cell line has the characteristics of human cervical squamous cell carcinoma, and it is stable with maintaining the characteristic biological and morphological features in vitro for more than 12 months, therefore, qualified as a stable cell line for further biomedical research.

Demethylation restores SN38 sensitivity in cells with acquired resistance to SN38 derived from human cervical squamous cancer cells

Using seven monoclonal SN38-resistant subclones established from ME180 human cervical squamous cell carcinoma cells, we examined the demethylation effects of 5-aza-2′-deoxycytidine (5-aza-CdR) on the SN38-sensitivity of the cells as well as the expression of death-associated protein kinase (DAPK) in the SN38-resistant cells. The DAPK expression levels were evaluated among parent ME180 cells, SN38-resistant ME180 cells and cisplatin-resistant ME180 cells by methylation-specific DAPK-PCR, quantitative RT-PCR and western blot analysis. The SN38-resistant cells co-treated with SN38 and 5-aza-CdR strongly exhibited enhanced SN38-sensitivities resembling those found in the parent cells. In the SN38-resistant subclones, no relationships were found between the restored SN38 sensitivity and hypermethylation of the DAPK promoter, DAPK mRNA expression, DAPK protein expression and induction of DAPK protein after 5-aza-CdR treatment, unlike the strong suppression of 5-aza-CdR-induced DAPK protein expression in the cisplatin-resistant subclones. These findings indicate that reversibly methylated molecules, but not DAPK, may regulate SN38 resistance, and that demethylating agents can be strong sensitizing anticancer chemotherapeutic drugs for SN38-resistant cancers.

The Role of the Globular Heads of C1q Receptor (gC1qR) Gene in Regulating Apoptosis of Human Cervical Squamous Cell Carcinoma

Background: The globular heads of the human C1q receptor (gC1qR) are multi-compartmental and multi-functional cellular proteins. The list of biological responses mediated by the gC1qR includes growth perturbation and morphological abnormalities, along with the initiation of apoptosis. However, the effects of the gC1qR on the apoptosis of cervical squamous carcinoma cells (C33a and SiHa) have not been demonstrated. Methods: Here, human cervical tissues were examined for the expression of the gC1qR using real-time PCR and Western blot analysis. Apoptotic death of C33a and SiHa cells was assessed by flow cytometric analysis to detect the subG1 population. Viability, migration and proliferation of C33a and SiHa cells were detected via the water-soluble tetrazolium salt (WST-1) assay, the Transwell assay and the ³H-thymidine incorporation into DNA assay (³H-TdR), respectively. Results: These data showed that expression of the gC1qR protein was significantly decreased in human cervical squamous cell carcinoma tissues relative to normal cervix tissues. C33a and SiHa cells transfected with a GFP-gC1qR vector resulted in the up-regulation of cellular apoptosis and an apparent increase in the expression of the p38 mitogen-activated protein kinase (p38 MAPK). Further, the changes in C33a and SiHa cells viability, migration and proliferation observed upon overexpression of gC1qR could be abrogated using the p38 MAPK inhibitor SB202190. Conclusion: These data indicate that gC1qR inhibits viability, migration and proliferation of cervical squamous cells carcinoma via the p38 MAPK signalling pathway.

Finding a New Prognostic Biomarker for Metastatic Colorectal Cancer

See Article on Page 315-320 For detecting new biomarkers in colorectal cancer, whether they are prognostic or predictive, we should understand the mechanism of the metastatic process at the gene expression level. This article presents a new prognostic biomarker, 'placental growth factor (PlGF)' to the clinical field. PlGF, which is a member of the vascular endothelial growth factor (VEGF) family in conjunction with VEGF-A and VEGF-B, is a ligand for VEGF receptor-1, which is expressed on endothelial cells, monocytes/macrophages, and some tumor cells. PlGF was isolated in 1991 from the placenta and was found only in very low levels under physiological conditions, but it was up-regulated in pathological circumstances such as wound healing, ischemia and tumor growth [1]. PlGF up-regulation has been found in human meningiomas, hemangioblastomas, melanomas, and cervical squamous cell carcinomas and is associated with angiogenesis in renal cell carcinomas. On the contrary, PlGF is down-regulated in thyroid carcinomas, germ cell tumors and cervical adenocarcinomas. Chen et al. [2] reported that PlGF was significantly up-regulated in gastric cancer tissue and was significantly correlated with microvessel density-evaluated angiogenesis and tumor stage. In this research, the authors found that the PlGF protein expression level in colorectal cancer tissue was significantly correlated with microvessel density, overall patient survival, and clinicopathological factors such as lymph-node metastasis, tumor stage, and lymphovascular invasion. According to the authors, PlGF seems to be an independent surrogate prognostic factor for colorectal cancer progression. However, for these results to be adopted in the clinical filed, it is very important that a large-sample-size study be brought conducted in a validation setting. The findings of this research finding suggest that PlGF may constitute a novel approach to colorectal cancer treatment. Several agents against PlGF have already developed or are currently under development. Among these agents, two novel agents against PlGF will be briefly discussed. Firstly, aflibercept (ZALTRAP) is a recombinant fusion protein that acts as a soluble receptor that binds to VEGF-A, VEGF-B and PIGF. In April 2011, Van Cutsem et al. [3] reported that aflibercept improved the primary endpoint of overall survival in the Velour phase III clinical trial for second-line treatment for metastatic colorectal cancer. Secondly, the humanized anti-PlGF mAb (TB-403) is directed against PlGF. A trial with TB-403 has only completed its first phase. Antitumor activity has been demonstrated with TB-403 in human tumor xenograft models of renal cell carcinomas and hepatocellular carcinomas. The mechanism of action for TB-403 is currently under investigation; preclinical studies suggest its effects may include blocking tumor angiogenesis and primary tumor growth, as well as inhibition of metastasis. In addition, PIGF inhibition may complement and potentiate the antitumor effects of VEGF inhibition, in part by inhibiting macrophage recruitment [4].

Expression of RAGE protein in human cervical squamous cell carcinoma

Expression of RAGE protein in human cervical squamous cell carcinoma

Thymoquinone from Nigella sativa was more potent than cisplatin in eliminating of SiHa cells via apoptosis with down-regulation of Bcl-2 protein

Thymoquinone (TQ), the active constituent of Nigella sativa or black cumin exhibited cytotoxic effects in several cancer cell lines. In this study, the cytotoxicity of TQ in human cervical squamous carcinoma cells (SiHa) was investigated. TQ was cytotoxic towards SiHa cells with IC50 values of 10.67±0.12 and 9.33±0.19μg/mL as determined by MTT assay and trypan blue dye exclusion test, respectively, after 72h of incubation. TQ was more cytotoxic towards SiHa cells compared to cisplatin. Interestingly, TQ was less cytotoxic towards the normal cells (3T3-L1 and Vero). Cell cycle analysis performed by flowcytometer showed a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Apoptosis induction by TQ was further confirmed by Annexin V/PI and AO/PI staining. Significant elevation of p53 and down-regulation of the anti-apoptotic Bcl-2 protein was found in the treated cells, without any changes in the expression of the pro-apoptotic Bax protein. In conclusion, thymoquinone from N. sativa was more potent than cisplatin in elimination of SiHa cells via apoptosis with down-regulation of Bcl-2 protein.

Comparative proteomic profiling in squamous cell carcinoma of the uterine cervix

Proteomic profiling of human uterine cervical squamous cell carcinoma (SCC) tissues was performed to find new biomarkers for the early diagnosis and molecular target therapies. Proteins extracted from five of normal tissues and five of the SCC tissues were subjected to 2-DE followed by LC-MS/MS. Twenty-seven of the protein spots were increased, and five of them were identified by LC-MS/MS to be cytokeratin-19, HSP70, HSP27, glyceraldehyde-3-phosphate dehydrogenase, and transgellin-2. The upregulation of HSP70, HSP27, and transgelin-2 was examined by Western blot of cultured SCC cell lines, ten additional normal tissues, ten additional SCC tissues, and three paired samples of the SCC tissue and its corresponding noncancerous tissue. The positive rate of HSP70 expression was higher in the SCC tissues than in normal tissues. Transgelin-2 was observed only in the SCC tissues, and also those in the early stage. The present study successfully identified three upregulated proteins in the uterine cervical SCC by proteomic analyses, and suggested that these proteins could be the candidates for new biomarkers for early diagnosis of SCC and molecular target therapies.

MDM2 SNP 309 human papillomavirus infection in cervical carcinogenesis

Objective To investigate the biological significance of single nucleotide polymorphism (SNP) at murine double-minute 2 homolog (MDM2) promoter 309 in cervical carcinogenesis. Methods SNP at MDM2 promoter 309 (T/G) together with human papillomavirus (HPV) types was examined in a total of 195 cervical smear samples and 8 human cervical squamous carcinoma cell lines using two independent PCR assays and PCR-RFLP techniques. Results Forty-one patients with high-grade squamous intraepithelial lesion (HSIL) had higher frequency of high-risk HPV than 102 with low-grade SIL (LSIL) and 52 controls. There was an increased OR (8.88; CI = 2.34–33.63; P = 0.003) for TG + GG genotype in HSIL cases compared to controls among 68 patients with high-risk HPV. Twenty-one cases with HPV types 16 and/or 18 had significantly higher frequency of the TG + GG genotype and G allele than 47 with other types of high-risk HPV. Seven of 8 cervical carcinoma cell lines also showed TG or GG genotype. Conclusion MDM2-SNP309 (T/G) and high-risk HPV infection may be closely associated with cervical carcinogenesis in a Japanese population.

In Vitro Release of a Tumor-Antigen (TA-4) by Human Cervical Squamous Cell Carcinoma

The release of a human squamous cell carcinoma antigen (TA-4) was studied by in vitro incubation of squamous cell carcinoma tissues and normal squamous epithelium of the uterine cervix. TA-4 activity was determined by a radioimmunoassay method using a monospecific antiserum to TA-4. TA-4 activity was found in both malignant and non-malignant tissues. However, the malignant tissue released significantly larger amounts of TA-4 than the non-malignant tissue during 72 hours incubation.

S100A8/A9 Induces Apoptosis and Inhibits Metastasis of CasKi Human Cervical Cancer Cells

S100 proteins, a family of Ca(2+)-binding proteins, have been linked to several human diseases in recent years. Deregulated expression of S100 proteins, including S100A9 and its partner S100A8, was reported to be associated with neoplastic disorders. In our previous study using serial analysis of gene expression, we identified decreased expressions of S100A9 and S100A8 in human cervical squamous cell carcinoma. To investigate the functions of S100A8 and S100A9 in cervical cancer, we purified recombinant S100A8 and S100A9 proteins and treated CaSki human cervical cancer cells with these proteins. We found that S100A8/A9 induced apoptosis and inhibited migration of CaSki cells; S100A8/A9 also reduced the expression of matrix metalloproteinase (MMP)-2 in CaSki cells. In summary, this study suggests that S100A8 and S100A9 have inhibitory effects on the proliferation of CaSki carcinoma cells by inducing cell apoptosis and on the invasiveness of CaSki cells.

Distinct DNA Methylation Profiles Between Adenocarcinoma and Squamous Cell Carcinoma of Human Uterine Cervix

Alterations in DNA methylation offer unique prospects as tumor markers. The big limitation in cervical cancer research is that it is too hard to obtain the pure normal tissue from a cervical cancer mass. So, we first profile type-specific DNA methylation of major two types of human uterine cervical cancer, adenocarcinoma (ACA) and squamous cell carcinoma (SCC), to establish a precise source of marker research. To assess the DNA methylation status of promoter regions in human uterine cervical ACAs and SCCs, fresh frozen tissues were obtained from bulky tumor masses to minimize the contamination from normal tissues and two array platforms using digestion with methylation-sensitive restriction-enzyme HpaII, ligation, and PCR were performed: an array of 11,994 (approximately 1.5 kb) PCR products from 10,445 promoter regions, and an array of 355,264 oligonucleotides for 18,212 HpaII fragments in 12,617 promoter regions. Loci near 21 genes showed significant differences between six ACA and four SCC from the analysis of two array data. Real-time PCR-based validation was performed on 13 loci using other nearby candidate methylation targets in the same promoter. Methylation patterns of 11 of 13 linked loci concurred with the microarray results. Four loci were further studied using tissues from additional patients (23 ACA and 24 SCC). Hypermethylation of loci in PAK6 and NOGOR most strongly correlated with ACA. Therefore, we have identified the 21 genes with differential methylation pattern between ACA and SCC and, furthermore, we found that PAK6 and NOGOR could be useful markers of ACA to be distinct from SCC.

Symmetric Division and Expression of Its Regulatory Gene Numb in Human Cervical Squamous Carcinoma Cells

Objective: To investigate the mode of cell division and Numb expression in cervical squamous cells. Method: We measured the cleavage angles of cervical squamous epithelia basal cells and Numb protein expression in 50 cases of cervical squamous cell carcinomas (CSCC), 59 cervical intraepithelial neoplasias (CIN) grade II–III, 21 CIN grade I and 48 controls. Results: We found an increase in horizontal divisions in CIN grade I and a decrease in horizontal divisions in CIN grade II–III. Numb expression had a tendency to be stronger from control to CSCC. In CSCC there was a significantly higher expression of mRNA for the Numb-PRR^L isoform but not for the Numb-PRR^S isoform. Conclusion: Our findings suggest that in the initial stage of CSCC, carcinomatous cells may acquire an increase of symmetric division by altering cleavage orientation and proliferation. Expression of Numb protein in Cervical squamous epithelia and an increased expression of Numb-PRR^L isoforms may be among the mechanisms involved in the genesis of CSCC.

Epidermal Growth Factor-activated Aryl Hydrocarbon Receptor Nuclear Translocator/HIF-1β Signal Pathway Up-regulates Cyclooxygenase-2 Gene Expression Associated with Squamous Cell Carcinoma

Hypoxia-inducible factor (HIF) accumulates when tumors grow under hypoxic conditions. The genesis of tumors, however, usually involves normoxic conditions. In this study, we were interested in examining the potential role of aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta in tumor growth under normoxic conditions, specifically when cells are treated with epidermal growth factor (EGF), which is known to affect the gene expression of tumor growth-related protein COX-2 (cyclooxygenase-2). The results showed that EGF receptor inhibitor, AG1478, abolished EGF-induced nuclear accumulation of ARNT as well as the expression of COX-2. ARNT small interfering RNA inhibited the promoter activity, mRNA level, and protein expression of COX-2 in cells treated with EGF. In contrast, CoCl(2)-induced HIF-1alpha exhibited no effect on COX-2 expression. EGF also stimulated the formation of the ARNT.c-Jun complex as well as the complex binding to the COX-2 promoter. ARNT small interfering RNAs blocked EGF-activated cell migration. Moreover, COX-2 and ARNT were cohorts present distinctively in clinical specimens of human cervical squamous cell carcinoma and were almost nondetectable in adjacent normal or noncancerous cervical tissues. Our results revealed that ARNT plays an important role in EGF-regulated COX-2 gene expression and may thus be related to either a cause or a consequence of tumorigenesis in cervical cancer.

Differential radiation sensitization of human cervical cancer cell lines by the proteasome inhibitor velcade (bortezomib, PS-341)

Cervical cancer is one of the deadliest cancers in women with a death toll of 230,000 worldwide each year, nearly 80% in developing countries. Radiotherapy (RT) is a major treatment modality for advanced cervical cancer but the local relapse rate is 30-44% in patients treated with RT alone and 19-25% in patients treated with concurrent chemoradiotherapy. Previous studies have shown that the transcription factor NF-kappaB is constitutively expressed in human cervical squamous cell carcinomas. NF-kappaB activation also contributes to the resistance of cervical cancer cells to apoptosis induced by chemotherapeutic agents and radiation. Therefore, inhibition of NF-kappaB in tumor cells may render them more sensitive to chemo/radiation therapies. The objective of this study is to investigate the potential of radiosensitization of NF-kappaB inhibition by Velcade in human cervical cancer cell lines. We used the human cervical cancer cell lines HeLa and SiHa. Both are highly radioresistant and chemoresistant as compared to other cervical cancer cell lines. These cells had been treated with Velcade before they were irradiated with different doses of ionizing radiation. MTT metabolic assays and clonogenic cell survival assays were performed to evaluate the effects of Velcade on radiation resistance. Inhibition of NF-kappaB by Velcade alone decreased metabolic potential (MTT) and clonogenic survival in SiHa, but not in HeLa cells. Furthermore, pre-treatment of SiHa, but not HeLa cells with Velcade enhanced radiation sensitivity. Inhibition of NF-kappaB by the proteasome inhibitor Velcade enhances radiosensitivity of certain human cervical carcinoma cancer cells in vitro. These results raise the possibility that inhibition of NF-kappaB will result in radiosensitization only in those tumor cells which are more dependent on NF-kappaB for their metabolism and survival, however, the radiosensitivity of "NF-kappaB independent" cells are not likely influenced by it.

Human papillomavirus detection and utility of testing

Abstract Screening for cervical cancer remains an important health and economic concern in the United States and throughout the world. Molecular and epidemiologic studies have solidified the association between infection with high-risk strains of human papilloma virus (HPV) and cervical squamous cell carcinoma. Cervical cancer has a well-demonstrated pre-malignant phase of many years, which allows early detection and intervention. Pap smear cytology has been the basis of cervical cancer screening programs, but it suffers from low sensitivity. Tests have been developed that detect high-risk HPV DNA in cervical samples with high sensitivity. Large-scale studies have shown the utility of HPV DNA testing in the management of women with Pap smear results showing atypical squamous cells of undetermined significance. New screening options include testing for high-risk HPV DNA in combination with cytology. Clinical trials and cost-modeling studies are also under way to evaluate the use of HPV detection alone as the primary screening test. The recent introduction of an HPV vaccine may also eventually impact screening algorithms.

A new fatty acid ester of triterpenoid from Celastrus rosthornianus with anti-tumor activitives

The methanol extract of the stalks of Celastrus rosthornianus Loes. afforded a new fatty acid ester of triterpene: 1β,3β-dihydroxy-olean-9(11),12-dienyl-3-palmitate (1), and three known compounds β-amyrin palmitate (2), 3β-hydroxy-11-oxo-olean-12-enyl-3-palmitate (3) and lupeol palmitate (4). Their structures were established by HRMS, 1D and 2D NMR experiments, as well as, by chemical degradation. The new compound showed cytotoxicity against human cervical squamous carcinoma (Hela) cells.

Novel Triterpenoids from Microtropis triflora with Antitumor Activities

AbstractTwo unprecedented norfriedelane‐type triterpenoids, triflora lactone (1) and triptocalline B (2), were isolated from the MeOH extract of the stalks of Microtropis triflora Merr. et Freem., together with five known compounds (3–7). Their structures were elucidated on the basis of spectroscopic and mass‐spectrometric data (Tables 1 and 2). The new compounds were found to be cytotoxic against human cervical squamous carcinoma (Hela) cells, with IC50 values of 21.6 and 17.6 μg/ml, respectively.

Understanding the Presence and Function of Erythropoietin Receptors on Cancer Cells

The expression of erythropoietin (EPO) and the EPO receptor (EPOR) have been recognized in a variety of human cancers, including breast, prostate, colon, ovary, uterine, cervical, glioblastoma, and head and neck squamous cell carcinoma (HNSCC). A growing number of studies demonstrate functional EPO/EPOR signaling events in cancer cells that contribute to disease progression. These findings have been difficult to reconcile with the current clinical practice of treating or preventing anemia in cancer patients with recombinant EPO (rEPO). Anemia is a wellrecognized negative prognostic factor for cancer patients treated with radiation therapy and chemotherapy. EPO has been an appealing alternative to blood transfusion for these patients to increase hemoglobin levels and to improve quality of life by decreasing fatigue. In addition to the identification of EPO and EPOR on cancer cells, a number of recent clinical trials have heightened concerns regarding the potential effects of EPO on cancer progression. The Breast Cancer Erythropoietin Trial (BEST) examined rEPO treatment in nonanemic, metastatic breast cancer patients. This study was terminated early when a higher mortality rate was observed among the rEPO treatment group. In a previous report, Henke and colleagues performed a randomized, double-blind, multi-institutional trial comparing anemic head and neck cancer patients receiving radiation therapy with or without rEPO treatment. These authors reported that rEPO treatment corrected hemoglobin levels, but was associated with decreased locoregional cancer control and patient survival. Although there was some concern regarding study design and methodology, that study and the BEST trial raised legitimate concerns regarding current recommendations for the routine use of rEPO in cancer patients. In the study reported in this issue of the Journal of Clinical Oncology, Henke and colleagues examined the expression of EPOR in a subset of 154 patients from a single clinical center derived from their original multicenter population (351 patients). Expression of EPOR was assessed by immunohistochemistry and reviewed by two independent, blinded pathologists. Within their patient population, 104 patients (67.5%) demonstrated EPOR expression. Their findings demonstrated that decreased locoregional progression-free survival due to treatment with rEPO was significant only in patients with HNSCC that expressed EPOR. rEPO treatment did not impact outcome in patients who had tumors that did not express EPOR. Although originally identified as a cytokine that promoted RBC progenitor survival and differentiation, the role of EPO in normal cells has been dramatically redefined within the past decade. EPO/EPOR signaling regulates physiologic cyclic uterine angiogenesis. In the CNS, EPO is produced by astrocytes and EPOR is expressed by neurons. EPO appears to have a neuroprotective effect against ischemic injury. Similarly, rEPO protects cardiac myocytes against ischemic injury in preclinical studies. The ability of cancer cells to subvert the EPO/EPOR system should not be surprising. A significant number of oncogenes represent signaling molecules that have been appropriated by cancer cells to facilitate proliferation and survival. The overexpression of the epidermal growth factor receptor in HNSCC is crucial for growth, invasion, metastasis, and angiogenesis. A number of preclinical studies have demonstrated EPO-mediated activation of the mitogenactivated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)–Akt, JAK-STAT (Janus kinase-Signal Transducer and Activator of Transcription), and nuclear factor-kappa B (NF B) signaling pathways in a variety of human cancers. Activation of these signaling cascades has been associated with cellular functions that promote tumor progression. rEPO appears to induce cancer cell proliferation and inhibit apoptosis. In addition, EPO stimulates not only chemotaxis of endothelial cells, but also migration and invasion of breast cancer and HNSCC cells. Pretreatment with rEPO protects some cancer cell lines from the cytotoxic effects of the chemotherapeutic agent, cisplatin. Thus, EPO/EPOR signaling appears to contribute to a wide variety of tumor-promoting functions in different cancer types. The contribution of the EPO/EPOR signaling axis to cancer progression is not completely straightforward. The influence of EPO/EPOR on different cancer types appears to be quite variable and remains incompletely understood. A number of different tumor cell lines do not demonstrate any proliferation response to rEPO. Similarly, certain cancer types undergo increased apoptosis or are more sensitive to fluorouracil treatment. Clearly, the complex biology of EPO/EPOR signaling in normal and cancer cells requires continued research. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 24 NUMBER 29 OCTOBER 1