YM155 sensitizes triple‐negative breast cancer to membrane‐bound TRAIL through p38 MAPK‐ and CHOP‐mediated DR5 upregulation

Because available treatments have limited efficacy in triple‐negative breast cancer (TNBC), the identification of new therapeutic strategies to improve patients' outcome is urgently needed. In our study, we investigated the effects of the administration of the small molecule selective survivin suppressant YM155, alone or in association with CD34+ cells transduced with a replication‐deficient adenovirus encoding the human tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) gene (CD34‐TRAIL+ cells), in three TNBC cell models. YM155 exposure significantly impaired TNBC cell growth and selectively modulated survivin expression at both mRNA and protein level. In addition, co‐culturing YM155‐treated TNBC cells with CD34‐TRAIL+ cells resulted in markedly increased cytotoxic effect and apoptotic response in comparison with single treatments. Such a chemosensitizing effect was observed only in TNBC cells inherently expressing DR5 and relied on the ability of YM155 to upregulate DR5 expression through a p38 MAPK‐ and CHOP‐dependent mechanism. YM155/CD34‐TRAIL+ combination also showed a significant inhibitory effect on the growth of DR5‐expressing TNBC cells following xenotransplantation into NOD/SCID mice, in the absence of toxicity. Overall, our data (i) provide, for the first time, evidence that YM155 sensitizes TNBC cells to CD34‐TRAIL+ cells‐induced apoptosis by a mechanism involving the downregulation of survivin and the simultaneous p38 MAPK‐ and CHOP‐mediated upregulation of DR5, and (ii) suggest the combination of YM155 with TRAIL‐armed CD34+ progenitor cells as a promising therapeutic option for patients with TNBC expressing DR5.     

Gene-viro-therapy targeting liver cancer by a dual-regulated oncolytic adenoviral vector harboring IL-24 and TRAIL

Cancer-targeting gene-viro-therapy is a promising cancer therapeutic strategy that strengthens the antitumor effect of oncolytic viruses by expressing an inserted foreign antitumor gene. To achieve liver cancer targeting and to improve the safety of the ZD55 vector (a widely-used E1B55KD gene-deleted oncolytic adenoviral vector (OV), we previously constructed), we designed a novel OV named Ad·AFP·D55 that selectively replicates in hepatocellular carcinoma (HCC) cells by replacing the E1A promoter with the liver-cancer specific α-Fetoprotein (AFP) promoter based on the ZD55 vector. We found that the oncolytic adenoviruses Ad·AFP·D55-IL-24 and Ad·AFP·D55-TRAIL express tumor-suppressor gene interleukin-24 (IL-24) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), respectively, significantly suppressed the HCC cell growth in vitro by inducing apoptosis by the caspase-8 and mitochondria-dependent caspase-9 signaling pathways. Furthermore, the combined treatment of Ad·AFP·D55-IL-24 and Ad·AFP·D55-TRAIL showed strong antitumor effects in vivo by significantly inhibiting the tumor growth in HCC HuH-7 cell xenograft mice, and markedly increasing animal survival rate. Therefore, this novel HCC cell-targeting OV carrying tumor-suppressor genes may provide a promising approach for liver cancer gene therapy.     

Oncolytic adenovirus armed with IL-24 Inhibits the growth of breast cancer in vitro and in vivo

ABSTRACT: BACKGROUND: Interleukin-24 (IL-24) is a cytokine that belongs to the IL-10 family. It can selectively induce cancer cell apoptosis which has been utilized as a cancer gene therapy strategy. METHODS: A recombinant type five adenovirus containing IL-24 gene (designated CNHK600-IL24) was constructed, whose replication is activated only in tumor cells. The replication of CNHK600-IL24 in breast tumor cells and fibroblasts were assessed by TCID50 and MTT assay; the secretion of IL-24 was measured by ELISA and western blotting. The in vivo anti-tumor effect of CNHK600-IL24 was investigated in in nude mice carrying orthotopic or metastatic breast tumor. RESULTS: We observed that CNHK600-IL24 could replicate efficiently and resulted in high level IL-24 expression and massive cell death in human breast cancer cell MDA-MB-231 but not in normal fibroblast cell MRC-5. In addition, orthotopic breast tumor growth in the nude mice model was significantly suppressed when CNHK600-IL24 was administered. In the metastatic model generated by tail vein injection, CNHK600-IL24 virotherapy significantly improved survival compared with the same virus expressing EGFP (median survival CNHK600-IL24, 55 days vs. CNHK600-EGFP, 41 day, p<0.05 Mantal-Cox test). A similar phenomenon was observed in the metastatic model achieved by left ventricular injection as suggested by in vivo luminescence imaging of tumor growth. CONCLUSION: The oncolytic adenovirus armed with IL-24, which exhibited enhanced anti-tumor activity and improved survival, is a promising candidate for virotherapy of breast cancer.     

Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.     

E1B-55kD-deleted oncolytic adenovirus armed with canstatin gene yields an enhanced anti-tumor efficacy on pancreatic cancer

Conditionally-replicating adenovirus (CRAd) therapy is currently being tested against pancreatic cancer and has shown some promise. To improve the efficacy, a novel virus CRAd-Cans was designed by deletion of E1B-55 kDa gene for selective replication in tumor cells, as well as carrying a new angiogenesis inhibitor gene, canstatin. CRAd-Cans mediated higher expression of canstatin in BxPC-3 pancreatic cancer cell line compared to the replication-deficient adenovirus Ad5-Cans. The modified CRAd-Cans manifested the same selective replication and cytocidal effects in pancreatic cancer cells as ONYX-015 in vitro, yet showed greater reduction of tumor growth in nude mice with markedly prolonged survival rate in vivo (P < 0.05), compared to that of either ONYX-015 or Ad5-Cans. Pathological examination revealed viral replication, decreased microvessel density and increased cancer cell apoptosis in CRAd-Cans-treated xenografts. The results suggest that the novel oncolytic virus CRAd-Cans, showing synergistic effects of oncolytic therapy and anti-angiogenesis therapy, is a new promising therapeutics for pancreatic cancer.     

Assessment of a combined, adenovirus-mediated oncolytic and immunostimulatory tumor therapy

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.t.) injection of adenovirus vectors into mice increases plasma levels of proinflammatory cytokines and chemokines, including TNF-alpha, INF-gamma, and MCP-1, which are potent inducers of dendritic cell maturation. Furthermore, we showed that in vivo expression of Flt3L from an adenovirus vector increases the number of CD11b+ and CD11c+ cells (populations that include dendritic cells) in the blood circulation. Based on these findings, we tested whether Ad.IR-E1A/TRAIL induced killing of tumor cells in combination with dendritic cell mobilization by Ad.Flt3L or, for comparison, Ad.GM-CSF would have an additive antitumor effect. As a model, we used immunocompetent C3H mice with syngeneic s.c. tumors derived from C3L5 cells. We found that vaccination of mice with C3L5 cells that underwent viral oncolysis in combination with Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression induces a systemic antitumor immune response. I.t. injection of the oncolytic and Flt3L expressing vectors into established tumors delayed tumor growth but did not cause efficient tumor elimination. This study shows the effectiveness of a combined oncolytic/immunostimulatory tumor therapy approach.     

Potentiation of tumor radiotherapy by a radiation-inducible oncolytic and oncoapoptotic adenovirus in cervical cancer xenografts

The p53 tumor suppressor pathway is impaired in more than 90% of cervical cancers and cancer-derived cell lines as a result of infection by human papillomavirus (HPV). The HPV E6 oncoprotein forms complexes with p53 and promotes its degradation via ubiquitin-dependent mechanism. In our study, we attempted to improve the clinical outcomes of this combined therapy by modifying the p53-targeted adenovirus to become radiation-responsive. The antitumor adenovirus was constructed by inserting a radiation-responsive expression cassette composed of the promoter of early growth response-1 (Egr-1) and the proapoptotic protein TRAIL. We showed that the addition of adenovirus containing Egr-1/TRAIL significantly increased cell death and apoptosis caused by radiotherapy. In mice bearing xenograft tumors, intratumoral administration of the Egr-1/TRAIL adenovirus followed by radiation significantly reduced tumor growth and enhanced tumor survival. Our Egr-1/TRAIL adenoviral gene product may offer a novel "one-two punch" tumor therapy for cervical cancers not only by potentiating radiation treatment but also by preserving p53 defect-specific tumor killing of the oncolytic adenovirus.     

携带人端粒酶反转录酶启动子的溶瘤腺病毒RCA-TERT-Ad35对乳腺癌干细胞的靶向作用

目的 探讨携带人端粒酶反转录酶启动子的溶瘤腺病毒RCA-TERT-Ad35在靶向乳腺癌干细胞中的作用。方法 乳腺癌细胞系MCF-7以球体形式在加入生长因子的无血清培养基中生长。同时构建携带TERT启动子的溶瘤腺病毒RCA-TERT-Ad35,并通过溶瘤、MTT及体内抗肿瘤实验观察RCA-TERT-Ad35对干细胞样癌细胞的杀伤效果。结果 与亲代细胞相比,MCF-7球体细胞显示出干细胞特性,且MCF-7球体细胞中hTERT基因过表达,其对细胞毒性剂紫杉醇具有耐药性。溶瘤腺病毒RCA-TERT-Ad35在TERT过表达的细胞中进行复制并抑制细胞生长。在裸鼠移植瘤模型中,与RCA-Ad35相比,RCA-TERT-Ad35显著抑制肿瘤生长并改善小鼠的存活状态。结论 溶瘤腺病毒RCA-TERT-Ad35可优先杀伤TERT过表达的乳腺癌干细胞。     

ARID1B在三阴性乳腺癌中的表达及其临床意义

目的 探讨AT富集结构域蛋白1B（ARID1B）在三阴性乳腺癌（TNBC）中的表达情况及其临床意义。方法 通过免疫组化法检测ARID1B在TNBC、非TNBC及癌旁正常组织中的表达差异,并分析其表达与TNBC临床病理特征及预后的关系。结果 ARID1B在120 例乳腺癌组织和30例癌旁正常乳腺组织中的阳性表达率分别为57.5％（69／120）、13.3％（4／30）,差异有统计学意义（P＜0.001）。ARID1B在90例TNBC组织中的阳性表达率为61.1％（55／90）,在30例非TNBC组织中的阳性表达率为33.3％（10／30）,差异亦有统计学意义（P=0.007）。TNBC患者ARID1B的表达与年龄、肿瘤大小、组织学分级及Ki-67增殖指数密切相关（P＜0.05）,而与淋巴结转移、TNM分期、p53表达均无关（P＞0.05）。生存分析显示ARID1B高表达TNBC患者的中位生存时间（OS）为27.9个月（95％CI：25.1～30.8个月）,显著低于低表达者的49.1个月（95％CI：40.2～57.9个月）,差异有统计学意义（P=0.009）。结论 ARID1B在TNBC中高表达且与年龄、肿瘤大小、组织学分级、Ki-67指数及生存时间密切相关,其有可能是TNBC预后评估和治疗的有效生物标志物。     

Systemic efficacy of oncolytic adenoviruses in imagable orthotopic models of hormone refractory metastatic breast cancer

Conditionally replicating oncolytic adenoviruses represent a promising developmental strategy for the treatment of cancer refractory to current treatments, such as hormone refractory metastatic breast cancer. In clinical cancer trials, adenoviral agents have been well tolerated, but gene transfer has been insufficient for clinical benefit. One of the main reasons may be the deficiency of the primary adenovirus receptor, and therefore viral capsid modifications have been employed. Another obstacle to systemic delivery is rapid clearance of virus by hepatic Kupffer cells and subsequent inadequate bioavailability. In this study, we compared several capsid-modified oncolytic adenoviruses for the treatment of breast cancer with and without Kupffer cell inactivation. Replication deficient capsid-modified viruses were analyzed for their gene transfer efficacy in vitro in breast cancer cell lines and clinical samples and in vivo in orthotopic models of breast cancer. The effect of Kupffer cell depleting agents on gene transfer efficacy in vivo was evaluated. An aggressive lung metastatic model was developed to study the effect of capsid-modified oncolytic adenoviruses on survival. Capsid-modified viruses displayed increased gene transfer and cancer cell killing in vitro and resulted in increased survival in an orthotopic model of lung metastatic breast cancer in mice. Biodistribution of viruses was favorable, tumor burden and treatment response could be monitored repeatedly. Kuppfer cell inactivation led to enhanced systemic gene delivery, but did not increase the survival of mice. These results facilitate clinical translation of oncolytic adenoviruses for the treatment of hormone refractory metastatic breast cancer.     

Oncolytic adenovirus mediated Survivin knockdown by RNA interference suppresses human colorectal carcinoma growth in vitro and in vivo

Background

Colorectal cancer is a one of the most common alimentary malignancies. Survivin has been proved by many studies to be an ideal target for cancer gene therapy because of its strong anti-apoptotic effect. The reduction of Survivin expression by means of chemically synthesized small interfering RNA or small hairpin RNA expressed from plasmid and resulted growth inhibition of cancer cells had been proved by many studies including ours, but the transfection efficiency was not encouraging. So for the first time we constructed the Survivin shRNA into an oncolytic adenovirus, tested its effects on colorectal cancer cell lines and nude mice xenograft model.

Methods

In this study, we constructed an oncolytic adenovirus with a Survivin targeted small hairpin RNA and a reporter gene (ZD55-Sur-EGFP). The expression of Survivin mRNA and protein were analyzed by RT-PCR and western blot. The cell growth and apoptosis were tested by in vitro cytopathic assay, MTT assay and flow cytometry respectively. The effect of the constructed virus on xenograft model was evaluated by tumor volume and western blot analysis.

Results

ZD55-Sur-EGFP replicated in cancer cells specifically, reduced the expression of Survivin mRNA and protein expression effectively (P < 0.0001), induced cancer cell apoptosis and inhibited SW480 cell growth both in vitro and in vivo significantly.

Conclusion

We conclude Survivin RNA interference combining with oncolytic adenovirus virotherapy to be a promising treatment for colorectal cancer.     

A novel Golgi protein (GOLPH2)-regulated oncolytic adenovirus exhibits potent antitumor efficacy in hepatocellular carcinoma

Golgi apparatus is the organelle mainly functioning as protein processing and secretion. GOLPH2 is a resident Golgi glycoprotein, usually called GP73. Recent data displayed that GOLPH2 is a superb hepatocellular carcinoma (HCC) marker candidate, and even its specificity is better than liver cancer marker AFP. Oncolytic adenoviruses are broadly used for targeting cancer therapy due to their selective tumor-killing effect. However, it was reported that traditionally oncolytic adenovirus lack the HCC specificity. In this study, a novel dual-regulated oncolytic adenovirus GD55 targeting HCC was first constructed based on our cancer targeted gene-viral therapeutic strategy. To verify the targeting and effectiveness of GOLPH2-regulated oncolytic adenovirus GD55 in HCC, the anticancer capacity was investigated in HCC cell lines and animal model. The results proved that the novel GOLPH2-regulated GD55 conferred higher adenovirus replication and infectivity for liver cancer cells than oncolytic adenovirus ZD55. The GOLPH2-regulated GD55 exerted a significant grow-suppressing effect on HCC cells in vitro but little damage to normal liver cells. In animal experiment, antitumor effect of GD55 was more effective in HCC xenograft of nude mice than that of ZD55. Thus GOLPH2-regulated GD55 may be a promising oncolytic virus agent for future liver cancer treatment.     

Synergistic antitumor activity of XIAP-shRNA and TRAIL expressed by oncolytic adenoviruses in experimental HCC

RNA interference (RNAi) induced by small interfering RNA (siRNA) can trigger sequence-specific gene silencing in mammalian cells. It has been proposed that siRNA can be developed as a novel strategy for cancer therapy. However effective delivery of therapeutically active siRNAs into the target tissue/cells in vivo is still a major obstacle for successful application. Oncolytic adenoviral vector mediated RNAi provides the potential advantages of minimizing the harm of normal cells, regenerating siRNAs within the tumor microenvironment and inspiring an additive antitumor outcome through viral oncolysis. Hepatocellular carcinoma (HCC) displays a high resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death, partially due to high expression levels of the X-linked Inhibitor-of-Apoptosis protein (XIAP). Here, we utilized an oncolytic adenovirus (ZD55) for expressing short hairpin RNA (shRNA), a precursor of siRNA, to knockdown XIAP. To increase sensitivity of HCC cells to TRAIL, we have used ZD55 to deliver both XIAP-shRNA and TRAIL into HCC cells. The results showed taht the combination of ZD55-XIAP-shRNA and ZD55-TRAIL resulted in significant reduction of XIAP expression and potent antitumor activity both in HCC cells and in animal model with tumor. This pilot study offers a promise of using oncolytic adenovirus to deliver siRNA targeting overexpressed oncogenes and a novel strategy for cancer therapy by regulating the equilibrium between the proapoptotic and antiapoptotic factors.     

Induction of proapoptotic antibodies to triple-negative breast cancer by vaccination with TRAIL death receptor DR5 DNA

TNF-related apoptosis-inducing ligand receptor 2 [TRAIL-R2 or death receptor 5 (DR5)] is expressed at elevated levels in a broad range of solid tumors to mediate apoptotic signals from TRAIL or agonist antibodies. We tested the hypothesis that DR5 DNA vaccination will induce proapoptotic antibody to trigger apoptosis of tumor cells. BALB/c mice were electrovaccinated with DNA-encoding wild-type human DR5 (phDR5) or its derivatives. Resulting immune serum or purified immune IgG induced apoptosis in triple-negative breast cancer (TNBC) cells, which were also TRAIL sensitive. The proapoptotic activity of immune serum at dilutions of 0.5-2% was comparable to that of 1-2 μg/ml of TRAIL. Apoptotic activity of immune serum was enhanced by antibody crosslinking. Apoptotic cell death induced by anti-DR5 antibody was shown by the cleavage of PARP and caspase-3. In contrast, immune serum had no effect on the proliferation of activated human T cells, which expressed low levels of DR5. In vivo, hDR5 reactive immune serum prevented growth of SUM159 TNBC cells in severe combined immune-deficient mice. DR5-specific IFN-γ-secreting T cells were also induced by DNA vaccination. Furthermore, the feasibility to overcome immune tolerance to self DR5 was shown by the induction of mouse DR5-binding antibody after electrovaccination of BALB/c mice with pmDR5ectm-Td1 encoding a fusion protein of mouse DR5 and an immunogenic fragment of tetanus toxin. These findings support DR5 as a promising vaccine target for controlling TNBC and other DR5-positive cancers.     

A capsid-modified, conditionally replicating oncolytic adenovirus vector expressing TRAIL Leads to enhanced cancer cell killing in human glioblastoma models

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, and patients rarely survive for more than 2 years. Gene therapy may offer new treatment options and improve the prognosis for patients with GBM. Adenovirus-mediated gene therapy strategies for brain tumors have been limited by inefficient gene transfer due to low expression of the adenovirus serotype 5 (Ad5) receptor. We have used an adenovirus vector that specifically replicates in tumor cells and uses an Ad5 capsid and the adenovirus serotype (Ad35) fiber for efficient infection of malignant tumor cells. This vector also expresses adenovirus E1A and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a tumor-specific manner. Here, we show that this oncolytic vector (Ad5/Ad35.IR-E1A/TRAIL) efficiently infects the GBM tumor cell lines SF767, T98G, and U-87 MG. Tumor cell killing was markedly enhanced with Ad5/Ad35.IR-E1A/TRAIL compared with wild-type Ad5 and Ad35 virus or Ad5/Ad35.IR-E1A- vectors without TRAIL expression in vitro. In vivo experiments using s.c. xenografted U-87 MG cells in NOD/SCID mice showed a significant growth delay of tumors after i.t. injection of Ad5/Ad35.IR-E1A/TRAIL, whereas adenovirus wild-type injections showed only marginal or no effect. Our findings indicate that the use of a capsid-modified adenoviral vector, in combination with TRAIL expression, is a promising novel approach for gene therapy of glioblastoma.     

Progesterone receptor membrane component 1 promotes survival of human breast cancer cells and the growth of xenograft tumors

Triple negative breast cancers (TNBCs) are highly aggressive and grow in response to sex steroid hormones despite lacking expression of the classical estrogen (E2) and progesterone (P4) receptors. Since P4 receptor membrane component 1 (PGRMC1) is expressed in breast cancer tumors and is known to mediate P4-induced cell survival, this study was designed to determine the expression of PGRMC1 in TNBC tumors and the involvement of PGRMC1 in regulating proliferation and survival of TNBC cells in vitro and the growth of TNBC tumors in vivo. For the latter studies, the MDA-MB-231 (MDA) cell line derived from TNBC was used. These cells express PGRMC1 but lack expression of the classical P4 receptor. A lentiviral-based shRNA approach was used to generate a stably transfected PGRMC1-deplete MDA line for comparison to the PGRMC1-intact MDA line. The present studies demonstrate that PGRMC1: 1) is expressed in TNBC cells; 2) mediates the ability of P4 to suppress TNBC cell mitosis in vitro; 3) is required for P4 to reduce the apoptotic effects of doxorubicin in vitro; and 4) facilitates TNBC tumor formation and growth in vivo. Taken together, these findings indicate that PGRMC1 plays an important role in regulating the growth and survival of TNBC cells in vitro and ultimately in the formation and development of these tumors in vivo. Thus, PGRMC1 may be a therapeutic target for TNBCs.     

选择性增殖腺病毒CNHK500治疗乳腺癌的实验研究

目的: 观察选择性增殖腺病毒CNHK500对乳腺癌的特异性杀伤作用.方法: 行病毒增殖实验和细胞生长抑制实验,验证CNHK500选择性复制和杀伤能力; Western blot检测腺病毒E1A和E1B在细胞中的表达.结果: CNHK500在乳腺癌细胞中复制能力与野生型腺病毒wtAd5相似,较ONYX-015增殖能力强.在正常成纤维细胞中CNHK500病毒增殖能力明显减弱, 较wtAd5增殖能力弱1 000倍左右.CNHK500可有效杀伤乳腺癌细胞株;而CNHK500对正常成纤维细胞的杀伤力较wtAd5减弱约100倍.CNHK500病毒的E1A可以选择性在端粒酶阳性的乳腺癌细胞株中表达,在端粒酶阴性的正常成纤维细胞株BJ中不表达, CNHK500可以选择性地在缺氧条件下表达E1B.动物实验结果显示,静脉注射CNHK500可以显著抑制MCF-7乳腺癌细胞裸鼠移植瘤的生长,治疗效果与给药剂量相关.结论: 肿瘤选择性增殖腺病毒CNHK500可选择性在端粒酶阳性的乳腺癌细胞中复制,并产生体内外杀伤作用.     

Integrin α9 depletion promotes β-catenin degradation to suppress triple-negative breast cancer tumor growth and metastasis

Although integrin α9 (ITGA9) is known to be involved in cell adhesion and motility, its expression in cancer and its role in tumor growth and metastasis remain largely unknown. Our study was designed to investigate the role of ITGA9 in triple-negative breast cancer (TNBC). ITGA9 expression in TNBC cells was knocked out (KO) using CRISPR/Cas9 technology. Four orthotopic mouse mammary xenograft tumor models coupled with cell culture studies were performed to determine the effect of ITGA9 depletion on TNBC tumor growth and metastasis and the underlying mechanism. Bioinformatics analysis showed that ITGA9 level is significantly higher in TNBC than other breast cancer subtypes, and higher ITGA9 level is associated with significantly worse distant metastasis-free survival and recurrence-free survival in TNBC patients. Experimentally, ITGA9 KO significantly reduced TNBC cell cancer stem cell (CSC)-like property, tumor angiogenesis, tumor growth and metastasis by promoting β-catenin degradation. Further mechanistic studies revealed that ITGA9 KO causes integrin-linked kinase (ILK) relocation from the membrane region to the cytoplasm, where it interacts with protein kinase A (PKA) and inhibits PKA activity leading to increased activity of glycogen synthase kinase 3 (GSK3) and subsequent β-catenin degradation. Overexpressing β-catenin in ITGA9 KO cells reversed the inhibitory effect of ITGA9 KO on tumor growth and metastasis. Furthermore, ITGA9 downregulation in TNBC tumors by nanoparticle-mediated delivery of ITGA9 siRNA drastically decreased tumor angiogenesis, tumor growth and metastasis. These findings indicate that ITGA9 depletion suppresses TNBC tumor growth and metastasis by promoting β-catenin degradation through the ILK/PKA/GSK3 pathway.