Topological Data Analysis: A New Method to Identify Genetic Alterations in Cancer.

Cancer is the largest health problem worldwide. A number of targeted therapies are currently employed for the treatment of different cancers. Determining the molecular mechanisms that are necessary for cancer development and progression is the most critical step in targeted therapies. Currently, many studies have identified a large number of frequently mutated cancer-associated genes using recurrence-based methods. However, only the cancer-associated mutations with a mutation frequency >15% can be identified by these methods. In other words, they cannot be used to identify driver genes that have low mutation frequency but play a major role in tumorigenesis and development. Thus, there is an urgent need for a method for identifying cancer-associated genes that are not based on recurrence. In a study, recently published in Nature Communications, research team led by Prof. Raúl Rabadán from the Columbia University successfully devised a novel topological data analysis approach to identify low-prevalence cancer-associated gene mutations using expression data from multiple cancers.

Transcriptional coregualtor NUPR1 maintains tamoxifen resistance in breast cancer cells.

To support cellular homeostasis and mitigate chemotherapeutic stress, cancer cells must gain a series of adaptive intracellular processes. Here we identify that NUPR1, a tamoxifen (Tam)-induced transcriptional coregulator, is necessary for the maintenance of Tam resistance through physical interaction with ESR1 in breast cancers. Mechanistically, NUPR1 binds to the promoter regions of several genes involved in autophagy process and drug resistance such as BECN1, GREB1, RAB31, PGR, CYP1B1, and regulates their transcription. In Tam-resistant ESR1 breast cancer cells, NUPR1 depletion results in premature senescence in vitro and tumor suppression in vivo. Moreover, enforced-autophagic flux augments cytoplasmic vacuolization in NUPR1-depleted Tam resistant cells, which facilitates the transition from autophagic survival to premature senescence. Collectively, these findings suggest a critical role for NUPR1 as a transcriptional coregulator in enabling endocrine persistence of breast cancers, thus providing a vulnerable diagnostic and/or therapeutic target for endocrine resistance.

SPIB promotes anoikis resistance via elevated autolysosomal process in lung cancer cells.

Anoikis (detachment-induced cell death) is a specific type of programmed cell death which occurs in response to the loss of the correct extracellular matrix connections. Anoikis resistance is an important mechanism in cancer invasiveness and metastatic behavior. Autophagy, on the other hand, involves the degradation of damaged organelles and the recycling of misfolded proteins and intracellular components. However, the intersection of these two cellular responses in lung cancer cells has not been extensively studied. Here, we identified that upon matrix deprivation, the lymphocyte lineage-specific Ets transcription factor SPIB was activated and directly enhanced SNAP47 transcription in certain lung cancer cells. Loss of attachment-induced autophagy significantly increased anoikis resistance by SPIB activation. Consistent with this function, SPIB depletion by short hairpin RNA abrogated SNAP47 transcriptional activation upon matrix deprivation. Therefore, these data delineate an important role of SPIB in autophagy-mediated anoikis resistance in lung cancer cells. Accordingly, these findings suggest that manipulating SPIB-regulated pathways in vivo and evaluating the impact of anoikis resistance warrant further investigation. DATABASE: RNA sequencing and ChIP sequencing data are available in Gene Expression Omnibus database under the accession numbers GSE106592 and GSE125561, respectively.

Transcriptional regulation of autophagy-lysosomal pathway in cancer.

The transcriptional regulation of autophagy-lysosomal pathway adapts to cellular stress and enables advanced cancer cells survive. This pathway plays an oncopromoting or oncosuppressing role, depending on context-dependent stresses and treatment resistance. It remains controversial whether this pathway represents a target for drugs, although autophagy-lysosomal inducers and inhibitors have been tested in clinical trials for cancer treatment. Therefore, identifying the transcriptional regulators of autophagy-lysosomal pathway may lead to the development of effective cancer treatment and the improvement of the existing targeted cancer therapies. In this review, we summarize findings from several published studies on transcriptional regulation of autophagy-lysosomal pathway in cancer biology, and evaluate its functional role as a therapeutic target.

Oroxylin A Suppresses the Cell Proliferation, Migration, and EMT via NF-κB Signaling Pathway in Human Breast Cancer Cells.

Oroxylin A is a natural extract and has been reported to have a remarkable anticancer function. However, the mechanism of its anticancer activity remains not quite clear. In this study, we examined the inhibiting effects of Oroxylin A on breast cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT) and its possible molecular mechanism. The cytoactive and inflammatory factors were analyzed via Cell Counting Kit-8 assay and ELISA assay, respectively. Flow cytometry and western blotting were used to assess the cell proliferation. In addition, a wound healing assay and transwell assay were used to detect cell invasion and migration. qRT-PCR and western blot were employed to determine the effect of Oroxylin A on the EMT formation. Moreover, expression level of protein related to NF-κB signaling pathway was determined by western blot. The results revealed that Oroxylin A attenuated the cytoactivity of MDA-MB-231 cells in a dose- and a time-dependent manner. Moreover, cell proliferation, invasion, and migration of breast cancer cells were inhibited by Oroxylin A compared to the control. The mRNA and protein expression levels of E-cadherin were remarkably increased while N-cadherin and Vimentin remarkably decreased. Besides, Oroxylin A suppressed the expression of inflammatory factors and NF-κB activation. Furthermore, we also found that supplement of TNF-α reversed the effects of Oroxylin A on the cell proliferation, invasion, migration, and EMT in breast cancer cells. Taken together, our results suggested that Oroxylin A inhibited the cell proliferation, invasion, migration, and EMT through inactivating NF-κB signaling pathway in human breast cancer cells. These findings strongly suggest that Oroxylin A could be a therapeutic potential candidate for the treatment of breast cancer.

Spleen tyrosine kinase SYK(L) interacts with YY1 and coordinately suppresses SNAI2 transcription in lung cancer cells.

Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase with dual properties of an oncoprotein and an oncosuppressor in distinctive cell types. In solid cancers, two isoforms SYK(L) and SYK(S) of SYK were recently identified due to its alternative mRNA splicing. However, the cellular activity and the biological significance of the long isoform of SYK, SYK(L), is still not well defined in human lung cancers. Here, we describe an interaction between SYK(L) and the ubiquitously expressed transcription regulator Yin Yang 1 (YY1) in the nucleus, which suppresses the epithelial-to-mesenchymal transition (EMT) by inactivating SNAI2 (coding transcription factor SLUG) transcription. ChIP indicated that endogenous SYK(L) interacts directly with a YY1 binding cis-regulatory element in the SNAI2 promoter. Importantly, knockdown of YY1 activates SYK(L)-dependent EMT suppression in human lung cancer H1155 cells. We also found that the protein level of SYK(L) is markedly upregulated in various types of human lung cancers, and its nuclear localization is strongly correlated with clinical benefits of lung adenocarcinomas. Collectively, our data reveal a SYK(L)-dependent transcriptional regulation of EMT through SLUG as a potential biomarker for lung cancer aggressiveness.

Extent of breast cancer type 1 promoter methylation correlates with clinicopathological features in breast cancers.

AIM OF STUDY: The current meta-analysis investigated the correlation between breast cancer type 1 (BRCA1) promoter methylation and the clinicopathological features of breast cancer (BC). MATERIALS AND METHODS: An electronic literature search was performed to identify and select cohort studies, by employing stringent inclusion and exclusion criteria, for data relevant to promoter methylation of BRCA1 and BC. Statistical analysis of the extracted data was performed using comprehensive meta-analysis 2.0 software (CMA 2.0) (Biostat Inc., Englewood, New Jersey, USA). RESULTS: A total of 125 published studies were retrieved from the literature search, and finally, 18 cohort studies meeting our inclusion criteria were incorporated into our meta-analysis. The 18 studies contained a total of 3213 BC patients. Meta-analysis results revealed that BRCA1 promoter methylation in BC patients with high and moderately differentiated tumors (I-II) was significantly lower than patients with poorly-differentiation tumors (III) (odds ratio [OR] =0.450, 95% confidence interval [95% CI] =0.241-0.838, P = 0.012). BRCA1 promoter methylation in BC patients with lymph node (LN) metastasis was significantly higher than patients without LN metastasis (OR = 2.244, 95% CI = 1.278-3.940, P = 0.005). The results of ethnicity-based subgroup analysis showed a significant difference in histological grade of BC on Asians, LN metastasis of BC in Asians and Caucasians, subtypes of BC in Caucasians, and age at diagnosis of BC patients in Caucasians (all P < 0.05). CONCLUSIONS: Our meta-analysis revealed that BRCA1 promoter methylation status is linked to tumor grade and LN metastasis of BC.

Correction to: Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer.

After the publication of this work [1] an error in Fig. 1c was brought to our attention: the Western blots for PRDX6 and ß-actin were similar to those shown in lanes 5-6 of Fig. 4g. To verify these findings, we have repeated this experiment and the results are shown in a new Fig. 1c below. The repeated experimental results are consistent with the previously reported findings in the original study [1] and the functional role for PRDX6 in malignant progression of human cancer including breast cancer has been widely documented and recognized in numerous other studies [2]. We apologize for the error. However, this correction does not affect the conclusions of the article.

NUPR1 maintains autolysosomal efflux by activating SNAP25 transcription in cancer cells.

In the advanced stages of cancer, autophagy is thought to promote tumor progression through its ability to mitigate various cellular stresses. However, the details of how autophagy is homeostatically regulated in such tumors are unknown. Here, we report that NUPR1 (nuclear protein 1, transcriptional regulator), a transcriptional coregulator, is aberrantly expressed in a subset of cancer cells and predicts low overall survival rates for lung cancer patients. NUPR1 regulates the late stages of autolysosome processing through the induction of the SNARE protein SNAP25, which forms a complex with the lysosomal SNARE-associated protein VAMP8. NUPR1 depletion deregulates autophagic flux and impairs autolysosomal clearance, inducing massive cytoplasmic vacuolization and premature senescence in vitro and tumor suppression in vivo. Collectively, our data show that NUPR1 is a potent regulator of autolysosomal dynamics and is required for the progression of some epithelial cancers.

Association of PKCζ expression with clinicopathological characteristics of breast cancer.

The protein kinase C (PKC) family has been functionally linked to cancer. It has been suggested that atypical PKCs contribute to cell proliferation and cancer progression. With respect to breast cancer, PKCζ has been found to play a key role in intracellular transduction of mitogenic and apoptotic signals using mammary cell lines. However, little is known about its function in vivo. Here we examined the correlation between PKCζ protein levels and important clinicopathologic factors in breast cancer using patient samples. To conduct the study, 30 invasive ductal carcinoma cases and their paired normal tissues were used for tissue microarray analysis (TMA) and 16 were used for western blot analysis. In addition, the correlation between PKCζ expression levels and clinicopathologic characteristics was determined in 176 cases with relevant clinical data. Finally, the correlation between PKCζ and epithelial growth factor receptor 2 (HER2) expressions was determined using three breast cancer cell lines by western blot analysis. Both TMA and western blot results showed that PKCζ protein was highly expressed in primary tumors but not in paired normal tissue. The correlation study indicated that high PKCζ levels were associated with premenopausal patients (p = 0.019) and worse prognostic factors, such as advanced clinical stage, more lymph node involvement and larger tumor size. Both disease-free survival and overall survival rates were lower in the high PKCζ group than those in the low PKCζ group. No correlation was observed between PKCζ levels and age, histological grade, or estrogen or progesterone receptor expression status. A positive correlation between PKCζ and HER2 levels was observed in both tumor samples and cell lines. Our observations link PKCζ expression with factors pointing to worse prognosis, higher HER2 levels and a lower survival rate. This suggests that PKCζ protein levels may serve as a prognostic marker of breast cancer.

A retrospective study of different local treatments in breast cancer patients with synchronous ipsilateral supraclavicular lymph node metastasis.

AIM: To evaluate the local treatment outcome and efficacy of supraclavicular lymph node dissection and radical radiotherapy for breast cancer patients with synchronous ipsilateral supraclavicular lymph node metastasis (ISLM). MATERIALS AND METHODS: A total of 29 patients with ISLM in the absence of distant metastases were retrospectively analyzed. All patients received radical or modified radical mastectomy and systemic therapy. Thirteen patients received supraclavicular lymph node dissection surgery and the other patients were treated with radical radiotherapy. RESULTS: At the median follow-up of 47 months, 23 patients had developed distant metastases. The 3-year distant metastasis-free survival (DMFS) rates were 46.2% for the supraclavicular lymph node dissection group and 31.3% for the radical radiotherapy group. The 5-year overall survival rates were 46.2% for the supraclavicular lymph node dissection group and 37.5% for the radical radiotherapy group. CONCLUSION: Breast cancer with ISLM should be considered as a locoregional disease. Besides systemic therapy, local therapy may be helpful in enhancing local control and correspondingly reducing distant metastasis. In some individual patients, supraclavicular lymph node dissection might get a good prognosis.

ARK5 is associated with the invasive and metastatic potential of human breast cancer cells.

PURPOSE: To investigate the effects of Akt/ARK5 pathways on the metastatic potential of human breast cancer cells. MATERIALS AND METHODS: The human ARK5 gene was transfected into MDA-MB-231 cells. Effects of ARK5 on MDA-MB-231 cells were investigated in vitro. The tumorigenicity and spontaneously metastatic capability regulated by ARK5 were determined using an orthotopic xenograft tumor model. RESULTS: ARK5 enhanced the invasive and metastatic potential of MDA-MB-231 cells under regulation by Akt. The enhancement was associated with increasing MMP-2, MMP-9, and MT1-MMP expression. The results were further demonstrated by RNA interference experiment. In an in vivo study, we also demonstrated that ARK5-transfected breast cancer cells grew faster and had more pulmonary metastases than its parental counterparts. CONCLUSION: ARK5 led to a more invasive phenotype and metastatic potential in human breast cancer dependent on Akt.

Enhanced expression of trophinin promotes invasive and metastatic potential of human gallbladder cancer cells.

PURPOSE: To study effects of trophinin on the metastatic potential of human gallbladder cancer cells and its potential mechanism. MATERIALS AND METHODS: Expression of trophinin in the highly metastatic GBC-SDH(i) cells was investigated by real time RT-PCR and western blot. Recombinant expression plasmid vector of the human trophinin gene was constructed and transfected into GBC-SD cells. Effects of trophinin on the invasion of GBC-SD cells were investigated by adhesion assay and invasion assay in vitro. The siRNA was used to down-regulate the expression of trophinin. Some genes related to the invasion and metastasis of cancer were determined by real time RT-PCR and western blot. The pulmonary metastasis regulated by trophinin was determined in the nude mice. RESULTS: Overexpression of trophinin in GBC-SDH(i) cells was confirmed compared with its parental counterparts. Up-regulation of trophinin enhanced the in vitro invasion in the GBC-SD/TRO cells. The enhancement was associated with increasing integrin alpha3, MMP-7, MMP-9, and Ets-1 expression. The results were further demonstrated by RNA interference experiment in vitro. In in vivo study, we also demonstrated that trophinin-transfected gallbladder cancer cells had more pulmonary metastases than the vector-transfected one or its parental counterparts. CONCLUSION: Overexpression of trophinin leads to a more invasive phenotype and metastatic potential in human gallbladder cancer, at least in part, through regulating integrin alpha3, MMP-7, MMP-9, and Ets-1 expression.

Identification of the functional role of AF1Q in the progression of breast cancer.

A novel highly metastatic MDA-MB-231HM cells, derived from MDA-MB-231, was established in our institute. RT-PCR, real-time PCR and Western blot showed that AF1Q gene was differentially expressed between highly metastatic MDA-MB-231HM cells and its parental MDA-MB-231 cells. However, its molecular mechanisms in breast cancer metastasis remain to be characterized. To investigate the effects of AF1Q on the progression of human breast cancer cells, in the present study, recombinant expression plasmid vectors of the human AF1Q gene was transfected into MDA-MB-231 cells. We demonstrated that AF1Q overexpression enhanced the in vitro proliferation and invasive potential of breast cancer cells. Focused microarray analyses showed that 22 genes were differentially expressed between AF1Q transfected cells and its parental counterparts. Integrin alpha3, accompanied by up-regulation of Ets-1 and MMP-2, significantly enhanced the in vitro invasive potential of human breast cancer cells mediated by AF1Q. Estrogen-responsive ring finger protein gene (EFP), also played a role in the enhancement of in vitro proliferation of human breast cancer cells mediated by AF1Q, accompanied by down-regulation of 14-3-3delta. The association was ERalpha independent. These results were further demonstrated by RNA interference (RNAi) experiment in vitro. In in vivo study, we also demonstrated that AF1Q transfected breast cancer cells grew much faster and had more pulmonary metastases than vector-transfected or its parental counterparts. On the contrary, AF1Q knockdown cells grew slower and had less pulmonary metastasis. Similar effects of AF1Q on integrin alpha3, Ets-1, MMP-2, EFP, and 14-3-3delta expression observed in vitro studies were also found in the in vivo study. Taken together, these results provide functional evidences that overexpression of AF1Q leads to a more progression in human breast cancer, at least in part, through regulating the integrin alpha3, Ets-1, MMP-2, EFP, and 14-3-3delta expression.

Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer.

INTRODUCTION: The molecular mechanisms involved in breast cancer metastasis still remain unclear to date. In our previous study, differential expression of peroxiredoxin 6 was found between the highly metastatic MDA-MB-435HM cells and their parental counterparts, MDA-MB-435 cells. In this study, we investigated the effects of peroxiredoxin 6 on the proliferation and metastatic potential of human breast cancer cells and their potential mechanism. METHODS: Expression of peroxiredoxin 6 in the highly metastatic MDA-MB-231HM cells was investigated by RT-PCR, real-time PCR and western blot. A recombinant expression plasmid of the human peroxiredoxin 6 gene was constructed and transfected into MDA-MB-231 and MDA-MB-435 cells. The effects of peroxiredoxin 6 on the proliferation and invasion of MDA-MB-231 and MDA-MB-435 cells were investigated by the Cell Counting Kit-8 method, colony-formation assay, adhesion assay, flow cytometry and invasion assay in vitro. miRNA was used to downregulate the expression of peroxiredoxin 6. Genes related to the invasion and metastasis of cancer were determined by RT-PCR, real-time PCR and western blot. The tumorigenicity and spontaneously metastatic capability regulated by peroxiredoxin 6 were determined using an orthotopic xenograft tumor model in athymic mice. RESULTS: Overexpression of peroxiredoxin 6 in MDA-MB-231HM cells compared with their parental counterparts was confirmed. Upregulation of peroxiredoxin 6 enhanced the in vitro proliferation and invasion of breast cancer cells. The enhancement was associated with decreasing levels of tissue inhibitor of matrix metalloproteinase (TIMP)-2 and increasing levels of the urokinase-type plasminogen activator receptor (uPAR), Ets-1 (E26 transformation-specific-1), matrix metalloproteinase (MMP)-9 and RhoC (ras homolog gene family, member C) expression. The results were further demonstrated by RNA interference experiments in vitro. In an in vivo study, we also demonstrated that peroxiredoxin 6-transfected breast cancer cells grew much faster and had more pulmonary metastases than control cells. By contrast, peroxiredoxin 6 knockdown breast cancer cells grew more slowly and had fewer pulmonary metastases. Effects similar to those of peroxiredoxin 6 on the uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression observed in in vitro studies were found in the in vivo study. CONCLUSION: Overexpression of peroxiredoxin 6 leads to a more invasive phenotype and metastatic potential in human breast cancer, at least in part, through regulation of the levels of uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression.

Gene expression pattern in apoptotic QGY-7703 cells induced by homoharringtonine.

AIM: To classify the genes responsible for apoptosis in QGY-7703 cells induced by homoharringtonine (HHT). METHODS: Apoptosis in QGY-7703 cells induced by HHT was demonstrated by DNA fragmentation and morphological observation. cDNA microarray technology was used to detect gene transcription, and the result of microarrays for genes was confirmed by RT-PCR. RESULTS: Seventy-eight individual mRNA were identified and their transcription levels changed significantly. Those genes, of which 68% were upregulated and 32% were downregulated, were partially related to apoptosis. They were mostly oncogenes, tumor suppressors, enzymes, and kinases. CONCLUSION: HHT is a potential drug in the treatment of liver cancer. TGF-beta, TNF, FAS, p38MAPK, and p53 apoptosis signaling pathways were activated during apoptosis in QGY-7703 cells. Such inducible genes may play important roles in apoptosis and deserve to be further studied.

Isolation of a human gallbladder cancer cell clone with high invasive phenotype in vitro and metastatic potential in orthotopic model and inhibition of its invasiveness by heparanase antisense oligodeoxynucleotides.

The mechanisms involved in gallbladder cancer metastasis still remain unclear to date. The poor understanding is due, in part, to the lack of ideal cell line and animal model for study. In the present study, 21 cell clones were isolated from the human gallbladder carcinoma cells GBC-SD and the cell clone GBC-SDH(i) with high invasive phenotype was fished out. The invasive phenotype and metastatic potential of GBC-SDH(i) were confirmed in a novel surgical orthotopic implantation model of gallbladder cancer in nude mice. Heparanase, an endoglycosidase that degrades heparan sulfate, is a critical mediator of tumor metastasis and angiogenesis. RT-PCR, real time RT-PCR and western blot showed that the expression levels of heparanase were significant difference between GBC-SDH(i) and its parent cells. After treated with antisense oligodeoxynucleotides, the heparanase mRNA and protein expression in GBC-SDH(i) cells were significantly decreased and its invasive potential in vitro was inhibited in a dose-dependent manner. The study provides a useful cell clone and a clinically relevant orthotopic tumor model for the metastatic study in human gallbladder cancer. The roles of heparanase in gallbladder cancer are also evaluated.