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Photodynamic therapy (PDT) is an effective and low‑invasive tumour therapy. However, it can induce tumour angiogenesis, which is a main factor leading to tumour recurrence and metastasis. Activin receptor‑like kinase‑1 (ALK1) is a key factor regulating angiogenesis. However, it remains unclear whether ALK1 plays an unusual role in low‑dose PDT‑induced tumour angiogenesis. In the present study, human umbilical vein endothelial cells (HUVECs) co‑cultured with breast cancer MDA‑MB‑231 cells (termed HU‑231 cells) were used to construct an experimental model of tumour angiogenesis induced by low‑dose PDT. The viability, and the proliferative, invasive, migratory, as well as the tube‑forming ability of the HU‑231 cells were evaluated following low‑dose PDT. In particular, ALK1 inhibitor and and an adenovirus against ALK1 were used to further verify the role of ALK1 in low‑dose PDT‑induced tumour angiogenesis. Moreover, the expression of ALK1, inhibitor of DNA binding 1 (ID1), Smad 1, p‑Smad1/5, AKT and PI3K were detected in order to verify the underlying mechanisms. The findings indicated that low‑dose PDT enhanced the proliferative ability of the HU‑231 cells and reinforced their migratory, invasive and tube formation capacity. However, these effects were reversed with the addition of an ALK1 inhibitor or by the knockdown of ALK1 using adenovirus. These results indicated that ALK1 was involved and played a critical role in tumour angiogenesis induced by low‑dose PDT. Furthermore, ALK1 was found to participate in PDT‑induced tumour angiogenesis by activating the Smad1/5‑ID1 pathway, as opposed to the PI3K/AKT pathway. On the whole, the present study, for the first time, to the best of our knowledge, demonstrates that ALK1 is involved in PDT‑induced tumour angiogenesis. The inhibition of ALK1 can suppress PDT‑induced tumour angiogenesis, which can enhance the effects of PDT and may thus provide a novel treatment strategy for PDT.

Hepatocellular carcinoma (HCC) is the fourth most common cancer type worldwide, with a poor prognosis and high mortality rate. The aim of the present study was to investigate the mechanisms underlying HCC progression to potentially benefit the development of effective therapies for advanced HCC. The present study reported a novel mechanism that promotes the HCC malignant phenotype, in which the inhibitor of differentiation 1 (ID1) activates the aurora kinase A (AURKA)/Myc signaling pathway. Specifically, a high expression of ID1 promoted a highly malignant phenotype of HCC cells that exhibit enhanced metastatic ability and drug resistance. However, the effects of ID1 were markedly reversed by the AURKA inhibitor VX689 and the Myc inhibitor 10058‑F4. Further studies demonstrated that ID1 competitively binds with anaphase‑promoting complex/cyclosome Cdh1 (APC/CCdh1), which is responsible for ubiquitination‑mediated AURKA protein degradation, resulting in upregulation of AURKA. Increased AURKA expression subsequently enhanced Myc expression at both transcriptional and post‑transcriptional level, leading to amplification of the Myc oncogenic signaling pathway. This novel ID1/AURKA/Myc axis could be a promising therapeutic target for the development of effective therapeutic strategies for advanced HCC.

Although inhibitor of differentiation 1 (Id‑1) and NF‑κB have been shown have play a role in tumorigenesis, their mechanisms and exact roles in cervical cancer have not yet been addressed. This study aimed to investigate the clinical significance of Id‑1 and NF‑κB p65 in cervical cancer and to elucidate their roles in malignant properties. Paraffin‑embedded cervical tissue specimens (n=85) were collected for immunohistochemistry and survival analysis. Targeting Id‑1 with lentivirus in vitro allowed the examination of the NF‑κB signaling pathway and cell survival. The results demonstrated the elevated co‑expression of Id‑1 and nuclear NF‑κB p65 was more frequently associated with aggressive cervical cancer behavior and poorer clinical outcomes. Targeting Id‑1 with short hairpin RNA or Id‑1 overexpression lentivirus in SiHa cells demonstrated that Id‑1 is associated with nuclear NF‑κB p65 expression and cell survival capacity. The physical interaction between Id‑1 and NF‑κB p65 was validated in SiHa cells. Moreover, the survival‑promoting or chemoresistant effects of Id‑1 may be attributed to the subsequent activation of NF‑κB signaling. On the whole, the synchronous co‑expression of Id‑1 and nuclear NF‑κB p65 has a prominent role in cervical cancer, suggesting a combined analysis of Id‑1 and NF‑κB may help to predict malignant properties and prognosis. Aside from NF‑κB, Id‑1 may also be developed as a promising therapeutic strategy for cervical cancer.

Penile squamous cell carcinoma (PSCC) occurs more frequently in developing countries, and is commonly diagnosed at an advanced stage with an unfavorable prognosis. At present, few biomarkers for PSCC have been identified and used in clinical practice. Aberrant expression of inhibitor of DNA binding 1 (ID1) has been suggested as a potential regulator of tumor progression in various types cancer. Herein, we evaluated ID1 expression in PSCC and analyzed its association with the clinicopathological parameters of PSCC. Our findings indicated that ID1 overexpression is associated with histological subtype and lymph node metastasis. Kaplan‑Meier survival analysis showed that the overexpression of ID1 is associated with unfavorable cancer‑specific survival. In Cox proportional hazard models, ID1 overexpression was found to be an independent predictor of cancer‑specific survival. Furthermore, we investigated the function of ID1 in PSCC using a PSCC cell line Penl1. Silencing of ID1 expression retarded cell growth, inhibited clonogenesis, and attenuated cell migration and invasion in Penl1 cells. ID1 may regulate key oncogenic and metastasis‑related molecules, as depletion of ID1 expression affected the levels of p‑AKT, p16, PTEN and cleaved caspase‑3, and reduced MMP2/9 secretion in Penl1 cells. Nevertheless, the mRNA expression of p16 and PTEN increased following ID1 knockdown, suggesting that ID1 may repress p16 and PTEN expression in Penl1 cells. Therefore, overexpression of ID1 could serve as a potential prognostic biomarker for the clinical management of PSCC. Strategies targeting ID1‑regulated signaling pathways may have therapeutic benefit in PSCC.

The inhibitor of DNA‑binding (ID) proteins are dominant‑negative modulators of transcription factors with basic helix‑loop‑helix (bHLH) structures, which control a variety of genes in cell cycle regulation. An increasing volume of evidence has demonstrated that the deregulated expression of IDs in several types of malignancy, including breast carcinoma, has been proven to serve crucial regulatory functions in tumorigenesis and the development of breast cancer (BC). The present study evaluated the prognostic values of the ID family members by investigating a set of publicly accessible databases, including Oncomine, bc‑GenExMiner, Kaplan‑Meier plotter and the Human Protein Atlas. The results demonstrated that mRNA levels of distinct IDs exhibited diverse profiles between BC and normal counterparts. The mRNA expression level of ID2 was significantly higher in breast cancer than normal tissues, while the mRNA expression levels of ID1, ID3 and ID4 were significantly lower in breast cancer tissues than in normal tissues. Furthermore, higher mRNA expression levels of ID1 and ID4 were associated with subgroups with lower pathological grades and fewer lymph node metastases. Survival analysis revealed that elevated mRNA levels of ID1 and ID4 predicted an improved survival in all patients with BC. Increased ID1 mRNA levels were associated with higher relapse‑free survival rates in all patients with BC, particularly in those with ER positive and Luminal A subtype tumors. Increased ID4 mRNA expression predicted longer survival times in all patients with BC, particularly in those with hormone receptor‑positive tumors or those treated with endocrine therapy. These results indicated that IDs are essential prognostic indicators in BC. Future studies on the effect of IDs on the pathogenesis and development of BC are warranted.

Ribosomal protein L9 (RPL9), a component of the 60S subunit for protein synthesis, is upregulated in human colorectal cancer. In the present study, we investigated whether RPL9 gained extraribosomal function during tumorigenesis and whether targeting of RPL9 with small interfering (si) RNA could alter the course of colorectal cancer progression. Our results showed that siRNA knockdown of RPL9 suppresses colorectal cancer (CRC) cell growth and long-term colony formation through an increase in sub-G1 cell population and a strong induction of apoptotic cell death. To obtain insights into the molecular changes in response to RPL9 knockdown, global changes in gene expression were examined using RNA sequencing. It revealed that RPL9-specific knockdown led to dysregulation of 918 genes in HCT116 and 3178 genes in HT29 cells. Among these, 296 genes showed same directional regulation (128 upregulated and 168 downregulated genes) and were considered as a common RPL9 knockdown signature. Particularly, we found through a network analysis that Id-1, which is functionally associated with activation of NF-κB and cell survival, was commonly downregulated. Subsequent western blot analysis affirmed that RPL9 silencing induced the decrease in the levels of Id-1 and phosphorylated IκBα in both HCT116 and HT29 cells. Also, the same condition decreased the levels of PARP-1 and pro-caspase-3, accelerating apoptosis. Furthermore, inhibition of RPL9 expression significantly suppressed the growth of human CRC xenografts in nude mice. These findings indicate that the function of RPL9 is correlated with Id-1/NF-κB signaling axis and suggest that targeting RPL9 could be an attractive option for molecular therapy of colorectal cancer.

Vascular endothelial damage is the major contributing factor to cardiovascular diseases. Recently, the therapeutic significance of endothelial progenitor cells (EPCs) has drawn increasing attention due to their roles in re-endothelialization following injury. The inhibitor of DNA-binding 1 (ID1) has been proven to promote EPC proliferation and migration, suggesting a critical function of ID1 in re-endothelialization. However, the underlying mechanisms remain undefined. In this study, ID1 was found to interact with E2-2 using immunoprecipitation analysis. Moreover, ID1 overexpression suppressed E2-2 expression and luciferase reporter activity; however, these effects were not observed in cells transfected with ID1 lacking the helix-loop-helix (HLH) domain (ID1ΔHLH). Further functional analysis corroborated that the upregulation of E2-2 markedly attenuated the ID1-mediated increase in EPC proliferation and migration. Furthermore, the HLH domain plays an important role in ID1-induced EPC proliferation and migration, as its deletion suppressed the positive regulatory effects of ID1 on EPC proliferation and migration. Taken together, the findings of our study confirm that ID1 promotes EPC proliferation and migration by suppressing E2-2 through the HLH domain in ID1. Therefore, ID1 may represent a potential therapeutic target for EPC-mediated re-endothelialization following vascular injury.

The underlying mechanisms of inhibitory effects induced by tetramethylpyrazine (TMP) on angiogenesis and tumor growth of lung cancer were investigated. In vitro cell proliferation, migration, and tube formation of human microvascular endothelial cells (HMEC-1) were evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide (MTT), wound healing, Transwell, and Matrigel assays. The expression of BMP/Smad/Id-1 signals was detected by RT-PCR and western blotting. In an A549 xenograft tumor model, TMP (40 and 80 mg/kg/day) was intraperitoneally injected into mice. The expressions of CD31, phosphorylated Smad1/5/8, and Id-1 were measured by immunohistochemistry. We demonstrated that TMP inhibited proliferation, migration, and capillary tube formation of HMEC-1 in a dose- and time-dependent manner. Furthermore, treatment of HMEC-1 cells with TMP (0.4 mg/ml) significantly upregulated BMP2 expression and downregulated BMPRIA, BMPRII, phosphorylated Smad1/5/8, and Id-1 expression. In addition, administrations of TMP remarkably inhibited tumor growth of A549 xenograft in nude mice. The CD31, phosphorylated Smad1/5/8, and Id-1 expression were significantly inhibited in TMP-treated xenograft tumors compared with the vehicle. In conclusion, our results indicated that TMP suppressed angiogenesis and tumor growth of lung cancer via blocking the BMP/Smad/Id-1 signaling.

Inhibitor of DNA-binding protein 1 (ID1) is commonly abnormally overexpressed in colorectal cancer (CRC); yet, the functional significance of ID1 in the growth and invasive properties of CRC cells remains largely unclear. The present study investigated the effects of ID1 downregulation on the cell growth and metastatic features of CRC. Using lentiviral shRNA infection, stable ID1-knockdown (KD) HCT116 and SW620 cells, human metastatic CRC cell lines, were created. In vitro, the migration/invasion capacity of the ID1-KD CRC cells was assessed by a wound healing assay. The activities of MMP2 and MMP-9 were measured by gelatin zymography. The expression of CXC chemokine receptor 4 (CXCR4), PCNA and survivin were determined by immunoblot analysis and qRT-PCR. The effects of ID1 knockdown on tumor growth and hepatic metastasis were demonstrated by a xenograft study in mice. The results showed evident decreases in proliferation, migration and invasion and an increased apoptosis rate in the ID1-KD CRC cells. Similarly, ID1 knockdown significantly decreased mRNA and protein levels of PCNA, survivin, CXCR4, MMP2 and MMP9. Overexpression of CXCR4 antagonized the negative effect on the migration and invasion abilities of the ID1-KD cells. As compared with the control, ID1 knockdown prevented tumor growth and profoundly suppressed hepatic metastasis in vivo. The present study demonstrated the significance of ID1 in colon cancer progression, and its effect on tumor invasiveness and metastatic properties may be partly dependent on CXCR4.

Id1 and NF-κB are highly expressed in oral squamous cell carcinoma (OSCC). Whether they have a synergistic role in the carcinogenesis of OSCC is unclear. The current study was designed to demonstrate the synergy of both Id1 and NF-κB in the underlying disease mechanisms of OSCC using in vitro and in vivo animal models. Id1 and NF-κB strengthened the expression of both CD133 and BMI-1 in OSCC cell cultures. CD133(+) and BMI-1(+) keratinocytes from OSCC tissues and cell cultures initiated the growth of xenograft tumors in SCID/Beige mice. Id1 and NF-κB regulate the expression of CD133 and BMI-1 in an additive or synergistic manner in OSCC, which is associated with the generation of naïve and self-renewable keratinocytes and initiate the growth of xenograft tumors in vivo.

Repulsive guidance molecule (RGM) family members RGMA, RGMB and RGMC are GPI-linked membrane proteins recently identified as co-receptor of bone morphogenetic proteins (BMPs). BMPs are a group of proteins enriched in bone and play important roles in prostate cancer. The current study aimed to investigate roles played by RGMs in prostate cancer. Expression of RGMs was examined in prostate cancer cell lines and prostate cancer tissues using RT-PCR and immunohistochemical staining. Knockdown of each RGM in prostate cancer cells was performed using the respective anti-RGMA, RGMB and RGMC transgenes. A variety of in vitro function tests were employed to analyze the influence on cancer cell functions by RGM knockdown. The implications of RGM knockdown in BMP signalling were also examined using both Western blot and real-time quantitative PCR. Knockdown of RGMA had no effect on cell growth, migration and invasion, but promoted cell-matrix adhesion. Knockdown of RGMB and RGMC increased growth and adhesion, but only RGMB knockdown increased capacities of migration and invasion in PC-3 cells. Further investigations showed an increase in Smad-3 activation and reduced levels of Smad-1 in PC-3 cells by RGMB and RGMC knockdown, and also an up-regulation of ID1, a BMP target gene particularly in exposure to BMP7. RGMs play inhibitory roles in prostate cancer by suppressing cell growth, adhesion, migration and invasion. RGMs can coordinate Smad-dependent signalling of BMPs in prostate cancer cells.

The inhibitor of differentiation/DNA binding 1 (Id-1), a negative regulator of basic helix-loop-helix transcription factors, plays an important role in the regulation of cell proliferation and differentiation. We examined the Id-1 expression by immunohistochemistry in 9 adenomas, 79 primary colorectal adenocarcinomas matched with 40 adjacent normal mucosa specimens and its relationship with clinicopathological factors. The Id-1 expression was increased in the carcinoma compared to the adjacent normal mucosa either in the unmatched and matched samples or to the adenoma. There was no significant difference in the Id-1 expression between normal mucosa and adenoma. The Id-1 expression of carcinoma was increased from Dukes' stages A to B, to C and to D. The cases with lymph node metastasis had a higher rate of a stronger Id-1 expression than those without lymph node metastasis. In conclusion, Id-1 overexpression plays an important role in colorectal cancer progression.

Transforming growth factor beta 1 (TGF-beta1) is a proposed regulator of Ids (inhibitors of DNA binding/differentiation) gene expression in epithelial cells. We previously reported that Id proteins are variously expressed in human hepatocellular carcinomas (HCC). However, the mechanism of regulation of Ids in HCC remains obscure. Here, we examined the relationship between Id1 and TGF-beta1 in four HCC cell lines, and studied the changes in cell proliferation, cell cycle and differentiation. The four HCC cell lines expressed Id1, TGF-beta1 and their receptors at various levels. TGF-beta1 strongly inhibited the growth of HuH7 cells, while the growth inhibition was moderate in PLC/PRF/5, and was not observed in HLE and HLF cell lines. TGF-beta1-induced growth inhibition in HuH7 cells was associated with cell accumulation in the G1 phase and partial induction of differentiation (with reduction of AFP and AFP-L3). Induction by TGF-beta1 dose-dependently suppressed Id1 expression in HuH7 cells; 1 ng/ml TGF-beta1 inhibited Id1 by 84.0 and 78.6% that of the untreated control at transcriptional and protein levels, respectively. HLE and HLF cells, which did not exhibit a TGF-beta1 growth inhibitory effect, lacked TGF-beta receptors and Id1 expression was not altered. In PLC/PRF/5 cells, Id1 augmentation was not observed in response to TGF-beta1, indicating that TGF-beta1-induced growth inhibition was not related to Id1 in this cell line. Our results suggest that, in some HCC cells, the pathway of suppression of Id1 by TGF-beta1 may be important in TGF-beta1-induced growth inhibition and partial differentiation.