Autophagy is a critical survival pathway for cancer cells under conditions of stress. Thus, induction of autophagy has emerged as a drug resistance mechanism. This study is to determine whether autophagy is activated by a novel multikinase inhibitor linifanib, thereby impairing the sensitivity of hepatocellular carcinoma (HCC) cells to this targeted therapy. Here, we found that linifanib induced a high level of autophagy in HCC cells, which was accompanied by suppression of phosphorylation of PDGFR-β and its downstream Akt/mTOR and Mek/Erk signaling pathways. Cell death induced by linifanib was greatly enhanced after autophagy inhibition by the pharmacological inhibitors or siRNAs against autophagy related genes, ATG5 and ATG7, in vitro. Moreover, HCQ, an FDA-approved drug used to inhibit autophagy, could significantly augment the anti-HCC effect of linifanib in a mouse xenograft model. In conclusion, linifanib can induce cytoprotective autophagy by suppression of PDGFR-β activities in HCC cells. Thus, autophagy inhibition represents a promising approach to improve the efficacy of linifanib in the treatment of HCC patients.
Hair loss (alopecia) is a common problem for people. The dermal papilla is the key signaling center that regulates hair growth and it engage in crosstalk with the microenvironment, including Wnt signaling and stem cells. In this study, we explored the effects of bone marrow mesenchymal stem cell overexpression of Wnt1a on mouse hair follicle regeneration. Wnt-CM accelerated hair follicle progression from telogen to anagen and enhanced the ALP expression in the DP area. Moreover, the hair induction-related genes were upregulated, as demonstrated by qRT-PCR. Wnt-CM treatment restored and increased DP cell expression of genes downregulated by dihydrotestosterone treatment, as demonstrated by qRT-PCR assays. Our study reveals that BM-MSC-generated Wnt1a promotes the DP's ability to induce hair cycling and regeneration.
Colorectal cancer (CRC) is still the third most common cancer and the second most common causes of cancer-related death around the world. Metformin, a biguanide, which is widely used for treating diabetes mellitus, has recently been shown to have a suppressive effect on CRC risk and mortality, but not all laboratory studies suggest that metformin has antineoplastic activity. Here, we investigated the effect of metformin and AMPK activator AICAR on CRC cells proliferation. As a result, metformin did not inhibit cell proliferation or induce apoptosis for CRC cell lines in vitro and in vivo. Different from metformin, AICAR emerged antitumor activity and sensitized anticancer effect of 5-FU on CRC cells in vitro and in vivo. In further analysis, we show that AMPK activation may be a key molecular mechanism for the additive effect of AICAR. Taken together, our results suggest that metformin has not antineoplastic activity for CRC cells as a single agent but AMPK activator AICAR can induce apoptosis and enhance the cytotoxic effect of 5-FU through AMPK activation.
Aberrant DNA methylation is one of the main drivers of tumor initiation and progression. The reversibility of methylation modulation makes it an attractive target for novel anticancer therapies. Clinical studies have demonstrated that high-dose decitabine, a hypomethylating agent, results in some clinical benefits in patients with refractory advanced tumors; however, they are extremely toxic. Low doses of decitabine minimize toxicity while potentially improving the targeted effects of DNA hypomethylation. Based on these mechanisms, low-dose decitabine combined with chemoimmunotherapy may be a new treatment option for patients with refractory advanced tumors. We proposed the regimen of low-dose decitabine-based chemoimmunotherapy for patients with refractory advanced solid tumors. A favorable adverse event profile was observed in our trial that was highlighted by the finding that most of these adverse events were grades 1-2. Besides, the activity of our cohort was optimistic and the clinical benefit rate was up to 60%, and the median PFS was prolonged compared with PFS to previous treatment. We also identified a significant correlation between the PFS to previous treatment and clinical response. The low-dose DAC decitabine-based chemoimmunotherapy might be a promising protocol for improving the specificity and efficiency of patients with refractory advanced solid tumors. This trial is registered in the ClinicalTrials.gov database (identifier NCT01799083).
Deficiency or mutation in the p53 tumor suppressor gene commonly occurs in human cancer and can contribute to disease progression and chemotherapy resistance. Currently, although the pro-survival or pro-death effect of autophagy remains a controversial issue, increasing data seem to support the idea that autophagy facilitates cancer cell resistance to chemotherapy treatment. Here we report that 5-FU treatment causes aberrant autophagosome accumulation in HCT116 p53−/− and HT-29 cancer cells. Specific inhibition of autophagy by 3-MA, CQ or small interfering RNA treatment targeting Atg5 or Beclin 1 can potentiate the re-sensitization of these resistant cancer cells to 5-FU. In further analysis, we show that JNK activation and phosphorylation of Bcl-2 are key determinants in 5-FU-induced autophagy. Inhibition of JNK by the compound SP600125 or JNK siRNA suppressed autophagy and phosphorylation of c-Jun and Bcl-2 but increased 5-FU-induced apoptosis in both HCT116 p53−/− and HT29 cells. Taken together, our results suggest that JNK activation confers 5-FU resistance in HCT116 p53−/− and HT29 cells by promoting autophagy as a pro-survival effect, likely via inducing Bcl-2 phosphorylation. These results provide a promising strategy to improve the efficacy of 5-FU-based chemotherapy for colorectal cancer patients harboring a p53 gene mutation.
Autophagy is a highly conserved intracellular degradation process and plays an important role in hepatocarcinogenesis. Available data show that autophagy is involved in anti-hepatocarcinoma (HCC) therapies. Autophagy regulation involves a novel target for overcoming therapeutic resistance and sensitizing HCC to currently therapeutic methods. This is a systematic review on the interface of autophagy and the development of HCC and outlining the role of autophagy in current anti-HCC approaches. Understanding the significance of autophagy in anti-HCC therapy may offer a novel therapeutic target for improving anti-cancer efficacy and prolong survival for HCC patients.
autophagy; hepatocarcinoma; anti-hepatocarcinoma therapy
Epidermal growth factor receptor (EGFR) somatic mutations are found in the majority of non-small-cell lung cancers (NSCLCs) and patients with NSCLC who harbor EGFR mutations have been shown to exhibit increased sensitivity to the small-molecule EGFR-tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. However, the majority of tumors develop acquired resistance to EGFR-TKIs after a median of 10–16 months, which limits the clinical efficacy of these drugs. Autophagy, an important homeostatic cellular recycling mechanism, has emerged as a potential target for the acquired resistance phenotype. Recently, several studies demonstrated that autophagy may be induced in a dose-dependent manner by treatment of multiple cancer cell lines with EGFR-TKIs in vitro. Furthermore, it was recently reported that autophagy, as a cytoprotective response, may be activated by EGFR-TKIs in lung cancer cells and that the inhibition of autophagy enhanced the cytotoxic effect of EGFR-TKIs. In this review, we aimed to focus on the association between resistance to EGFR-TKIs and autophagy, and assess whether autophagy inhibition represents a promising approach to improve the efficacy of EGFR-TKIs in the treatment of NSCLC patients.
epidermal growth factor receptor; autophagy; non-small-cell lung cancer; resistance
Infusion of mesenchymal stem cells (MSCs) has been shown to effectively lower blood glucose in diabetic individuals, but the mechanism involved could not be adequately explained by their potential role in promoting islet regeneration. We therefore hypothesized that infused MSCs might also contribute to amelioration of the insulin resistance of peripheral insulin target tissues. To test the hypothesis, we induced a diabetic rat model by high-fat diet/streptozotocin (STZ) administration, performed MSC infusion during the early phase (7 days) or late phase (21 days) after STZ injection, and then evaluated the therapeutic effects of MSC infusion and explored the possible mechanisms involved. MSC infusion ameliorated hyperglycemia in rats with type 2 diabetes (T2D). Infusion of MSCs during the early phase not only promoted β-cell function but also ameliorated insulin resistance, whereas infusion in the late phase merely ameliorated insulin resistance. Infusion of MSCs resulted in an increase of GLUT4 expression and an elevation of phosphorylated insulin receptor substrate 1 (IRS-1) and Akt (protein kinase B) in insulin target tissues. This is the first report of MSC treatment improving insulin sensitivity in T2D. These data indicate that multiple roles and mechanisms are involved in the efficacy of MSCs in ameliorating hyperglycemia in T2D.
Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Wharton's jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Wharton's jelly would be beneficial for in vitro expansion of MSCs. Wharton's jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.
Transforming growth factor (TGF)-β/Smad signaling plays an important role in colon cancer development, progression and metastasis. In this study we demonstrated that the microRNA-130a/301a/454 family is up-regulated in colon cancer tissues compared to paired adjacent normal mucosa, which share the same 3′-untranslational region (3′-UTR) binding seed sequence and are predicated to target Smad4. In colorectal cancer HCT116 and SW480 cells, overexpression of miRNA-130a/301a/454 mimics enhances cell proliferation and migration, while inhibitors of these miRNAs affect cell survival. The biological function of miRNA-130a/301a/454 on colon cancer cells is likely mediated by suppression of Smad4, and the up-regulation of the miRNAs is correlated with Smad4 down-regulation in human colon cancers. Collectively, these results suggest that miRNA-130a/301a/454 are novel oncogenic miRNAs contributing to colon tumorigenesis by regulating TGF-β/Smad signaling, which may have potential application in cancer therapy.
Recent evidence suggests that experimental induction of hepatocytes into pancreatic cells provides new cell transplantation therapy prospects for type 1 diabetes mellitus. Stepwise differentiation from rat liver epithelial stem-like WB-F344 cells (WB cells) into functional insulin-secreting cells will identify key steps in β-cell development and may yet prove useful for transplantation therapy for diabetic patients. An essential step in this protocol was the generation of pancreatic precursor cell that express Pdx1 based on induction by a combination of 5-aza-2′-deoxycytidine, trichostatin A, retinoic acid, and a mix of insulin, transferrin and selenite. The Pdx1-expressing cells express other pancreatic markers and contribute to endocrine cells in vitro and in vivo. This study indicates an efficient chemical protocol for differentiating WB cells into functional insulin-producing cells using small molecules, and represents a promising hepatocyte-based treatment for diabetes mellitus.
Hemoglobin (Hgb) is the main oxygen and carbon dioxide carrier in cells of erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. However, Hgb is also expressed in nonerythroid cells. In the present study, the expression of Hgb in human uterine cervix carcinoma cells and its role in cervical cancer were investigated.
The expression level of Hgb in cervical cancer tissues was assessed by quantitative reverse transcriptase-PCR (qRT-PCR). We applied multiple methods, such as RT-PCR, immunoblotting, and immunohistochemical analysis, to confirm Hgb expression in cervical cancer cells. The effects of ectopic expression of Hgb and Hgb mutants on oxidative stress and cell viability were investigated by cellular reactive oxygen species (ROS) analysis and lactate dehydrogenase (LDH) array, respectively. Both Annexin V staining assay by flow cytometry and caspase-3 activity assay were used, respectively, to evaluate cell apoptosis.
qRT-PCR analysis showed that Hgb-α- (HBA1) and Hgb-β-globin (HBB) gene expression was significantly higher in cervical carcinoma than in normal cervical tissues, whereas the expression of hematopoietic transcription factors and erythrocyte specific marker genes was not increased. Immunostaining experiments confirmed the expression of Hgb in cancer cells of the uterine cervix. Hgb mRNA and protein were also detected in the human cervical carcinoma cell lines SiHa and CaSki, and Hgb expression was up-regulated by hydrogen peroxide-induced oxidative stress. Importantly, ectopic expression of wild type HBA1/HBB or HBA1, rather than mutants HBA1H88R/HBBH93R unable to bind hemo, suppressed oxidative stress and improved cell viability.
The present findings show for the first time that Hgb is expressed in cervical carcinoma cells and may act as an antioxidant, attenuating oxidative stress-induced damage in cervical cancer cells. These data provide a significant impact not only in globin biology but also in understanding of cervical cancer pathogenesis associated with oxidative stress.
The interplay between tumor cells and their microenvironment plays a pivotal role in tumor development and progression. Although a growing body of evidence has established the importance of the tumor microenvironment, an understanding of the crosstalk between its components and cancer cells remains elusive. The pathways triggered by microenvironmental factors could modulate cancer-related gene transcription, also affecting small noncoding RNAs, microRNAs, which have emerged as key posttranscriptional regulators of gene expression, directly involved in human cancers. Although microRNAs regulate most biological mechanisms, their role in the tumor microenvironment has only recently become the focus of intense research. In this paper, we focus on the intertwined connection between the tumor microenvironment and aberrant expression of microRNAs involved in carcinogenesis. We also discuss the emerging roles of microRNAs in the tumor microenvironment as it relates to cancer progression. We conclude that microRNAs are critical for our understanding of the development of cancer, and that targeting microRNA signaling pathways in the microenvironment as well as in tumor cells opens new therapeutic avenues to the global control of cancer.
The spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively.
We have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run.
The cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.
As a nature phytoalexin found in grapes, resveratrol has been proposed as a potential drug for cancer chemoprevention and treatment. However, its poor bioavailability limits its potential clinical application. Pterostilbene, the natural dimethylated analog of resveratrol with greater bioavailability, was confirmed to inhibit tumor growth both in vivo and in vitro, demonstrating its potential for further clinical application. In the current study, we found that pterostilbene could markedly inhibit the growth of two independent breast cancer cell lines. Both apoptosis and cell cycle arrest as well as the inhibition of wnt singling was induced by pterostilbene. The dominant-active mutant of β-catenin could reverse the growth inhibitory effect of pterostilbene, indicating that the inhibition of wnt signaling is important to the growth inhibitory effect of pterostilbene. Interestingly, pterostilbene induced autophagy and blockage of autophagy augmented pterostilbene-induced growth inhibition, suggesting that the combination of autophagy inhibitors with pterostilbene and other therapeutics such as endocrine drugs could serve as a new and promising strategy for the treatment of breast cancer cells.
Pterostilbene; phytoalexins; tumor growth inhibition; wnt singling; apoptosis
Anthracycline daunorubicin (DNR) is one of the major antitumor agents widely used in the treatment of myeloid leukemia. Unfortunately, the clinical efficacy of DNR was limited because of its cytotoxity at high dosage. As a novel cytoprotective mechanism for tumor cell to survive under unfavorable conditions, autophagy has been proposed to play a role in drug resistance of tumor cells. Whether DNR can activate to impair the sensitivity of cancer cells remains unknown. Here, we first report that DNR can induce a high level of autophagy, which was associated with the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Moreover, cell death induced by DNR was greatly enhanced after autophagy inhibition by the pharmacological inhibitor chloroquine (CQ) and siRNAs targeting Atg5 and Atg7, the most important components for the formation of autophagosome. In conclusion, we found that DNR can induce cytoprotective autophagy by activation of ERK in myeloid leukemia cells. Autophagy inhibition thus represents a promising approach to improve the efficacy of DNR in the treatment of patients with myeloid leukemia.
Epidermal growth factor receptor tyrosine kinase inhibitors gefitinib and erlotinib have been widely used in patients with non-small-cell lung cancer. Unfortunately, the efficacy of EGFR-TKIs is limited because of natural and acquired resistance. As a novel cytoprotective mechanism for tumor cell to survive under unfavorable conditions, autophagy has been proposed to play a role in drug resistance of tumor cells. Whether autophagy can be activated by gefitinib or erlotinib and thereby impair the sensitivity of targeted therapy to lung cancer cells remains unknown. Here, we first report that gefitinib or erlotinib can induce a high level of autophagy, which was accompanied by the inhibition of the PI3K/Akt/mTOR signaling pathway. Moreover, cytotoxicity induced by gefitinib or erlotinib was greatly enhanced after autophagy inhibition by the pharmacological inhibitor chloroquine (CQ) and siRNAs targeting ATG5 and ATG7, the most important components for the formation of autophagosome. Interestingly, EGFR-TKIs can still induce cell autophagy even after EGFR expression was reduced by EGFR specific siRNAs. In conclusion, we found that autophagy can be activated by EGFR-TKIs in lung cancer cells and inhibition of autophagy augmented the growth inhibitory effect of EGFR-TKIs. Autophagy inhibition thus represents a promising approach to improve the efficacy of EGFR-TKIs in the treatment of patients with advanced non-small-cell lung cancer.
Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation.
GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples.
We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens.
Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.
Previous reports have suggested that treatment with
cytokine-induced killer (CIK) cells may benefit patients with
various types of tumor. The aim of this study was to evaluate the
antitumor effects of CIK cells against the colorectal cancer line
SW1116 in vitro and in vivo. CIK
cells were generated routinely from peripheral blood mononuclear
cells of healthy human donors, and the number of CD3+CD56+ cells
was expanded more than 1300-fold after 14-day culture. At an
effector : target cell ratio of
50 : 1, the percentage lysis of SW1116 cells
reached 68% in the presence of CIK cells, Experimental mice
injected with SW1116 cells subcutaneously were divided randomly
into four groups: untreated, 5-fluorouracil (5-FU)-treated,
CIK-consecutive treated (injected once/day) and CIK-interval
treated (injected once every 5 days). CIK cells were injected
abdominally five times in total. Compared with the untreated
group, xenograft growth was inhibited greatly by CIK treatment, to
nearly the same extent as with 5-FU treatment. We demonstrated
that the necrotic area in the tumor xenograft was markedly larger
in the CIK-treated groups than in the other groups. These findings
suggest that CIK-based immunotherapy may represent an effective
choice for patients with colorectal cancer.
Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.
Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERα-mediated signaling and promoted cell cycle progression.
K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERα and promotes the proliferation of ERα-positive breast tumor cells. K18 plays an important functional role in regulating the ERα signaling pathway.
Inhibitor of differentiation 2 (Id2) is a natural inhibitor of the basic helix–loop–helix transcription factors. Although Id2 is well known to prevent differentiation and promote cell-cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Here, we identified that Four-and-a-half LIM-only protein 2 (FHL2) is a novel functional repressor of Id2. Moreover, we demonstrated that FHL2 can directly interact with all members of the Id family (Id1–4) via an N-terminal loop–helix structure conserved in Id proteins. FHL2 antagonizes the inhibitory effect of Id proteins on basic helix–loop–helix protein E47-mediated transcription, which was abrogated by the deletion mutation of Ids that disrupted their interaction with FHL2. We also showed a competitive nature between FHL2 and E47 for binding Id2, whereby FHL2 prevents the formation of the Id2–E47 heterodimer, thus releasing E47 to DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 was opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Altogether, these results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.
Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor α (ERα)-positive MCF-7 and SKOV-3 cancer cells.
MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.
Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.
Overexpression of Id2 in ERα-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.