Role of NuMA1 in breast cancer stem cells with implications for combination therapy of PIM1 and autophagy inhibition in triple negative breast cancer.

Background: Nuclear mitotic apparatus protein 1 (NuMA1) is a cell cycle protein and upregulated in breast cancer. However, the role of NuMA1 in TNBC and its regulation in heterogenous populations remains elusive. Methods: We performed CRISPR mediated deletion of NuMA1 in mouse TNBC cells, BF3M. FACS was utilized to isolate BCSCs, and bulk cells based on CD29 and CD61 markers. Cell viability, migration, and invasion ability of BCSCs and bulk cells was evaluated using MTT, wound healing and transwell invasion assays, respectively. In vivo mouse breast cancer and lung metastatic models were generated to evaluate the combination treatment of SMI-4a and Lys-o5 inhibitors. Results: We identified that high expression of NuMA1 associated with poor survival of breast cancer patients. Further, human tissue microarray results depicted high expression of NuMA1 in TNBC relative to non-adjacent normal tissues. Therefore, we performed CRISPR mediated deletion of NuMA1 in a mouse mammary tumor cell line, BF3M and revealed that NuMA1 deletion reduced mammary tumorigenesis. We also showed that NuMA1 deletion reduced ALDH+ and CD29hiCD61+ breast cancer stem cells (BCSCs), indicating a role of NuMA1 in BCSCs. Further, sorted and characterized BCSCs from BF3M depicted reduced metastasis with NuMA1 KO cells. Moreover, we found that PIM1, an upstream kinase of NuMA1 plays a preferential role in maintenance of BCSCs associated phenotypes, but not in bulk cells. In contrast, PIM1 kinase inhibition in bulk cells depicted increased autophagy (FIP200). Therefore, we applied a combination treatment strategy of PIM1 and autophagy inhibition using SMI-4a and Lys05 respectively, showed higher efficacy against cell viability of both these populations and further reduced breast tumor formation and metastasis. Together, our study demonstrated NuMA1 as a potential therapeutic target and combination treatment using inhibitors for an upstream kinase PIM1 and autophagy inhibitors could be a potentially new therapeutic approach for TNBC. Conclusions: Our study demonstrated that combination treatment of PIM1 inhibitor and autophagy inhibitor depicted reduced mammary tumorigenesis and metastasis by targeting NuMA1 in BCSCs and bulk cells of TNBC, demonstrating this combination treatment approach could be a potentially effective therapy for TNBC patients.

In vivo CRISPR knockout screen identifies p47 as a suppressor of HER2+ breast cancer metastasis by regulating NEMO trafficking and autophagy flux.

Autophagy is a conserved cellular process, and its dysfunction is implicated in cancer and other diseases. Here, we employ an in vivo CRISPR screen targeting genes implicated in the regulation of autophagy to identify the Nsfl1c gene encoding p47 as a suppressor of human epidermal growth factor receptor 2 (HER2)+ breast cancer metastasis. p47 ablation specifically increases metastasis without promoting primary mammary tumor growth. Analysis of human breast cancer patient databases and tissue samples indicates a correlation of lower p47 expression levels with metastasis and decreased survival. Mechanistic studies show that p47 functions in the repair of lysosomal damage for autophagy flux and in the endosomal trafficking of nuclear factor κB essential modulator for lysosomal degradation to promote metastasis. Our results demonstrate a role and mechanisms of p47 in the regulation of breast cancer metastasis. They highlight the potential to exploit p47 as a suppressor of metastasis through multiple pathways in HER2+ breast cancer cells.

AZI2 mediates TBK1 activation at unresolved selective autophagy cargo receptor complexes with implications for CD8 T-cell infiltration in breast cancer.

Most breast cancers do not respond to immune checkpoint inhibitors and there is an urgent need to identify novel sensitization strategies. Herein, we uncovered that activation of the TBK-IFN pathway that is mediated by the TBK1 adapter protein AZI2 is a potent strategy for this purpose. Our initial observations showed that RB1CC1 depletion leads to accumulation of AZI2, in puncta along with selective macroautophagy/autophagy cargo receptors, which are both required for TBK1 activation. Specifically, disrupting the selective autophagy function of RB1CC1 was sufficient to sustain AZI2 puncta accumulation and TBK1 activation. AZI2 then mediates downstream activation of DDX3X, increasing its interaction with IRF3 for transcription of pro-inflammatory chemokines. Consequently, we performed a screen to identify inhibitors that can induce the AZI2-TBK1 pathway, and this revealed Lys05 as a pharmacological agent that induced pro-inflammatory chemokine expression and CD8+ T cell infiltration into tumors. Overall, we have identified a distinct AZI2-TBK1-IFN signaling pathway that is responsive to selective autophagy blockade and can be activated to make breast cancers more immunogenic.Abbreviations: AZI2/NAP1: 5-azacytidine induced 2; CALCOCO2: calcium binding and coiled-coil domain 2; DDX3X: DEAD-box helicase 3 X-linked; FCCP: carbonyl cyanide p-triflouromethoxyphenylhydrazone; a protonophore that depolarizes the mitochondrial inner membrane; ICI: immune checkpoint inhibitor; IFN: interferon; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1.

Regulation of RB1CC1/FIP200 stability and autophagy function by CREBBP-mediated acetylation in an intrinsically disordered region.

RB1CC1/FIP200 is an essential macroautophagy/autophagy protein that plays an important role in a variety of biological and disease processes through its canonical autophagy-dependent and -independent functions. However, it remains largely unknown whether post-translational modifications could regulate RB1CC1 and its associated autophagy functions. Here, we report acetylation of several lysine residues of RB1CC1 by acetyltransferase CREBBP (CREB binding protein), with K276 as the major CREBBP acetylation site. K276 is also identified as a ubiquitination site by mass spectrometry, and acetylation at this site reduces ubiquitination of RB1CC1 to inhibit its ubiquitin-dependent degradation. We also find that RB1CC1 contains an N-terminal intrinsically disordered region (IDR) capable of forming liquid-liquid phase separation (LLPS) in vitro, which may drive formation of RB1CC1 puncta with LLPS properties in cells independent of SQSTM1/p62 and other autophagy receptors CALCOCO2/NDP52, NBR1, TAX1BP1 and OPTN. Mutational analysis shows that both K276 acetylation and the N-terminal IDR containing it are important for maintaining canonical autophagy function of RB1CC1 in breast cancer cells. Our findings demonstrate regulation of RB1CC1 by a new post-translational mechanism and suggest potential therapeutic application of inducing RB1CC1 degradation through blocking K276 acetylation in the treatment of cancer and other diseases.Abbreviations: Baf-A1: bafilomycin A1; CREBBP/CBP: CREB binding protein; CHX: cycloheximide; EP300/p300: E1A binding protein p300; FRAP: fluorescence recovery after photobleaching; HADCs: histone deacetylases; IDR: intrinsically disordered region; LLPS: liquid-liquid phase separation; KAT2A/GCN5: lysine acetyltransferase 2A; KAT2B/PCAF: lysine acetyltransferase 2B; KAT5/TIP60: lysine acetyltransferase 5; KAT8/MOF: lysine acetyltransferase 8; NAM: nicotinamide; PAS: phagophore assembly site; PEG-8000: polyethylene glycol 8000; RB1CC1/FIP200: RB1 inducible coiled-coil 1; TSA: trichostatin A.

Targeting HIC1/TGF-ß axis-shaped prostate cancer microenvironment restrains its progression.

Prostate cancer (PCa) is a malignant tumor that seriously threatens men's health worldwide. Recently, stromal cells in the tumor microenvironment (TME) have been reported to contribute to the progression of PCa. However, the role and mechanism of how PCa cells interact with stromal cells to reshape the TME remain largely unknown. Here, using a spontaneous prostate adenocarcinoma (PRAD) model driven by the loss of Pten and Hic1, we found that M2 macrophages markedly infiltrated the stroma of Pten and Hic1 double conditional knockout (dCKO) mice compared with those in control (Ctrl) mice due to higher TGF-ß levels secreted by HIC1-deleted PCa cells. Mechanistically, TGF-ß in TME promoted the polarization of macrophages into "M2" status by activating the STAT3 pathway and modulating c-Myc to upregulate CXCR4 expression. Meanwhile, TGF-ß activated the fibroblasts to form cancer-associated fibroblasts (CAFs) that secrete higher CXCL12 levels, which bound to its cognate receptor CXCR4 on M2 macrophages. Upon interaction with CAFs, M2 macrophages secreted more CXCL5, which promoted the epithelial-mesenchymal transition (EMT) of PCa via CXCR2. Moreover, using the TGF-ß receptor I antagonist, galunisertib, significantly inhibited the tumor growth and progression of the TRAMP-C1 cell line-derived subcutaneous tumor model. Finally, we confirmed that the stromal microenvironment was shaped by TGF-ß in HIC1-deficient PCa and was associated with the progression of PCa.

Biglycan Promotes Cancer Stem Cell Properties, NFκB Signaling and Metastatic Potential in Breast Cancer Cells.

It is a major challenge to treat metastasis due to the presence of heterogenous BCSCs. Therefore, it is important to identify new molecular targets and their underlying molecular mechanisms in various BCSCs to improve treatment of breast cancer metastasis. Here, we performed RNA sequencing on two distinct co-existing BCSC populations, ALDH+ and CD29hi CD61+ from PyMT mammary tumor cells and detected upregulation of biglycan (BGN) in these BCSCs. Genetic depletion of BGN reduced BCSC proportions and tumorsphere formation. Furthermore, BCSC associated aggressive traits such as migration and invasion were significantly reduced by depletion of BGN. Glycolytic and mitochondrial metabolic assays also revealed that BCSCs exhibited decreased metabolism upon loss of BGN. BCSCs showed decreased activation of the NFκB transcription factor, p65, and phospho-IκB levels upon BGN ablation, indicating regulation of NFκB pathway by BGN. To further support our data, we also characterized CD24-/CD44+ BCSCs from human luminal MCF-7 breast cancer cells. These CD24-/CD44+ BCSCs similarly exhibited reduced tumorigenic phenotypes, metabolism and attenuation of NFκB pathway after knockdown of BGN. Finally, loss of BGN in ALDH+ and CD29hi CD61+ BCSCs showed decreased metastatic potential, suggesting BGN serves as an important therapeutic target in BCSCs for treating metastasis of breast cancer.

Prefused lysosomes cluster on autophagosomes regulated by VAMP8.

Lysosome-autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes-vesicle-associated membrane protein 8 (VAMP8)-plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

Autophagy inhibition perturbs ERBB2 trafficking and abolishes tumorigenesis in ERBB2-driven breast cancer.

Macroautophagy/autophagy modulation is increasingly recognized as a potential strategy for cancer therapy. Using a recently developed Rb1cc1 mutant knockin mice model, we have taken a rigorous genetic approach to assess the role of both its autophagy and non-canonical functions in an ERBB2-driven BrCA model. We found that autophagy abrogation virtually abolishes mammary tumorigenesis in the ERBB2-driven model, exhibiting stronger inhibitory effects than in our previous studies using PyMT and brca1-null mouse models. Mechanistically, autophagy inhibition perturbs ERBB2 intracellular trafficking and triggers its release via small extracellular vesicles. Our results demonstrate a new mechanism for autophagy to promote tumorigenesis in ERBB2-driven BrCA and could supplement current strategies for anti-ERBB2 therapy.

Functional cooperation between co-amplified genes promotes aggressive phenotypes of HER2-positive breast cancer.

MED1 (mediator subunit 1) co-amplifies with HER2, but its role in HER2-driven mammary tumorigenesis is still unknown. Here, we generate MED1 mammary-specific overexpression mice and cross them with mouse mammary tumor virus (MMTV)-HER2 mice. We observe significantly promoted onset, growth, metastasis, and multiplicity of HER2 tumors by MED1 overexpression. Further studies reveal critical roles for MED1 in epithelial-mesenchymal transition, cancer stem cell formation, and response to anti-HER2 therapy. Mechanistically, RNA sequencing (RNA-seq) transcriptome analyses and clinical sample correlation studies identify Jab1, a component of the COP9 signalosome complex, as the key direct target gene of MED1 contributing to these processes. Further studies reveal that Jab1 can also reciprocally regulate the stability and transcriptional activity of MED1. Together, our findings support a functional cooperation between these co-amplified genes in HER2+ mammary tumorigenesis and their potential usage as therapeutic targets for the treatment of HER2+ breast cancers.

Autophagy Blockade Limits HER2+ Breast Cancer Tumorigenesis by Perturbing HER2 Trafficking and Promoting Release Via Small Extracellular Vesicles.

Autophagy modulation is an emerging strategy for cancer therapy. By deleting an essential autophagy gene or disrupting its autophagy function, we determined a mechanism of HER2+ breast cancer tumorigenesis by directly regulating the oncogenic driver. Disruption of FIP200-mediated autophagy reduced HER2 expression on the tumor cell surface and abolished mammary tumorigenesis in MMTV-Neu mice. Decreased HER2 surface expression was due to trafficking from the Golgi to the endocytic pathways instead of the plasma membrane. Autophagy inhibition led to HER2 accumulation in early and late endosomes associated with intraluminal vesicles and released from tumor cells in small extracellular vesicles (sEVs). Increased HER2 release from sEVs correlated with reduced tumor cell surface levels. Blocking sEVs secretion rescued HER2 levels in tumor cells. Our results demonstrate a role for autophagy to promote tumorigenesis in HER2+ breast cancer. This suggests that blocking autophagy could supplement current anti-HER2 agents for treating the disease.

Single-cell RNA-sequencing reveals distinct patterns of cell state heterogeneity in mouse models of breast cancer.

Breast cancer stem cells (BCSCs) contribute to intra-tumoral heterogeneity and therapeutic resistance. However, the binary concept of universal BCSCs co-existing with bulk tumor cells is over-simplified. Through single-cell RNA-sequencing, we found that Neu, PyMT and BRCA1-null mammary tumors each corresponded to a spectrum of minimally overlapping cell differentiation states without a universal BCSC population. Instead, our analyses revealed that these tumors contained distinct lineage-specific tumor propagating cells (TPCs) and this is reflective of the self-sustaining capabilities of lineage-specific stem/progenitor cells in the mammary epithelial hierarchy. By understanding the respective tumor hierarchies, we were able to identify CD14 as a TPC marker in the Neu tumor. Additionally, single-cell breast cancer subtype stratification revealed the co-existence of multiple breast cancer subtypes within tumors. Collectively, our findings emphasize the need to account for lineage-specific TPCs and the hierarchical composition within breast tumors, as these heterogenous sub-populations can have differential therapeutic susceptibilities.

FIP200 Suppresses Immune Checkpoint Therapy Responses in Breast Cancers by Limiting AZI2/TBK1/IRF Signaling Independent of Its Canonical Autophagy Function.

Immune checkpoint inhibitors (ICI) have the potential to induce durable therapeutic responses, yet response rates in breast cancer are modest and limited to particular subtypes. To expand the applicability of ICI, we examined the role of an essential autophagy gene, FIP200, which has been shown to be important for tumor progression in mammary tumors. Specific disruption of the autophagy function of FIP200 or complete ablation of FIP200 in genetic mouse models revealed that FIP200 autophagy function was required for progression of PyMT-driven mammary tumors. However, a noncanonical autophagy function of FIP200 was responsible for limiting T-cell recruitment and activation of the TBK1-IFN signaling axis. FIP200 also interacted with the TBK1 adaptor protein, AZI2, which was crucial for activation of TBK1 following FIP200 ablation. Accordingly, disrupting the noncanonical autophagy function of FIP200 in combination with ICI therapy led to superior, durable responses in immune-competent models of breast cancer. Collectively, these insights could guide future development of therapeutic agents against FIP200 for combinatorial ICI therapies in nonresponsive breast cancers. SIGNIFICANCE: These findings show that deletion of FIP200 enhances immune checkpoint inhibitor efficacy in nonresponsive breast cancer.

FAK signaling in cancer-associated fibroblasts promotes breast cancer cell migration and metastasis by exosomal miRNAs-mediated intercellular communication.

Cancer-associated fibroblasts (CAFs) are activated fibroblasts that constitute the major components of tumor microenvironment (TME) and play crucial roles in tumor development and metastasis. Here, we generated fibroblast-specific inducible focal adhesion kinase (FAK) knockout (cKO) mice in a breast cancer model to study potential role and mechanisms of FAK signaling in CAF to promote breast cancer metastasis in vivo. While not affecting primary tumor development and growth, FAK deletion significantly suppressed breast cancer metastasis in vivo. Analyses of CAFs derived from cKO mice as well as human CAFs showed that FAK is required for their activity to promote mammary tumor cell migration. We further showed that FAK ablation in CAFs decreased exosome functions to promote tumor cell migration and other activities, which could contribute to the reduced metastasis observed in cKO mice. Lastly, profiling of miRs from CAF exosomes showed alterations of several exosomal miRs in FAK-null CAFs, and further analysis suggested that miR-16 and miR-148a enriched in exosomes from FAK-null CAFs contribute to the reduced tumor cell activities and metastasis. Together, these results identify a new role for FAK signaling in CAFs that regulate their intercellular communication with tumor cells to promote breast cancer metastasis.

Tumor suppressor HIC1 is synergistically compromised by cancer-associated fibroblasts and tumor cells through the IL-6/pSTAT3 axis in breast cancer.

BACKGROUND: Interleukin-6 (IL-6) is commonly highly secreted in the breast cancer (BrCA) microenvironment and implicated in disease development. In this study, we aimed to determine the role of the IL-6/pSTAT3/HIC1 axis in the breast cancer microenvironment, including in cancer-associated fibroblasts (CAFs) and breast cancer cells. METHODS: Stromal fibroblasts from the breast cancer tissue were isolated, and the supernatants of the fibroblasts were analyzed. Recombinant human IL-6 (rhIL-6) was applied to simulate the effect of CAF-derived IL-6 to study the mechanism of HIC1 (tumor suppressor hypermethylated in cancer 1) downregulation. IL-6 was knocked down in the high IL-6-expressing BrCA cell line MDA-MB-231, which enabled the investigation of the IL-6/pSTAT3/HIC1 axis in the autocrine pathway. RESULTS: Increased IL-6 was found in the supernatant of isolated CAFs, which suppressed HIC1 expression in cancer cells and promoted BrCA cell proliferation. After stimulating the BrCA cell line SK-BR-3 (where IL-6R is highly expressed) with rhIL-6, signal transducers and activators of transcription 3 (STAT3) was found to be phosphorylated and HIC1 decreased, and a STAT3 inhibitor completely rescued HIC1 expression. Moreover, HIC1 was restored upon knocking down IL-6 expression in MDA-MB-231 cells, accompanied by a decrease in STAT3 activity. CONCLUSIONS: These findings indicate that IL-6 downregulates the tumor suppressor HIC1 and promotes BrCA development in the tumor microenvironment through paracrine or autocrine signaling.

HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk.

Breast cancer (BrCa) is the malignant tumor that most seriously threatens female health; however, the molecular mechanism underlying its progression remains unclear. Here, we found that conditional deletion of hypermethylated in cancer 1 (HIC1) in the mouse mammary gland might contribute to premalignant transformation in the early stage of tumor formation. Moreover, the chemokine (C-X-C motif) ligand 14 (CXCL14) secreted by HIC1-deleted BrCa cells bound to its cognate receptor GPR85 on mammary fibroblasts in the microenvironment and was responsible for activating these fibroblasts via the ERK1/2, Akt, and neddylation pathways, whereas the activated fibroblasts promoted BrCa progression via the induction of epithelial-mesenchymal transition (EMT) by the C-C chemokine ligand 17 (CCL17)/CC chemokine receptor 4 (CCR4) axis. Finally, we confirmed that the HIC1-CXCL14-CCL17 loop was associated with the malignant progression of BrCa. Therefore, the crosstalk between HIC1-deleted BrCa cells and mammary fibroblasts might play a critical role in BrCa development. Exploring the progression of BrCa from the perspective of microenvironment will be beneficial for identifying the potential prognostic markers of breast tumor and providing more effective treatment strategies.

Targeting CXCR7 improves the efficacy of breast cancer patients with tamoxifen therapy.

Chemokine (C-X-C motif) receptor 7 (CXCR7) has been established to be involved in breast cancer (BCa) progression. However, the role of CXCR7 in different subtype of BCa still remains unclear. Here we note that CXCR7 expression is significantly amplified in Luminal type BCa tissues as compared with Her2 and TNBC types through data-mining in TCGA datasets, and its protein level positively correlates with ERα expression by staining of human BCa tissue. Interestingly, alteration of CXCR7 expression in Luminal type BCa cells is able to modulate the expression of ERα through ubiquitination at post-translational level. Additionally, overexpression of CXCR7 in these cells greatly induces 4-OHT insensitivity in vitro and is associated with earlier recurrence in patients with tamoxifen therapy. Notably, silencing ERα expression potentially rescues the sensitivity of the above cells to 4-OHT, suggesting that elevated level of ERα is responsible for CXCR7-induced 4-OHT insensitivity in Luminal type BCa. Finally, mechanistic analyses show that the reduced BRCA1 (ubiquitin E3 ligase) and elevated OTUB1 (deubiquitinase) expression, which are regulated by CXCR7/ERK1/2 signaling pathway, are responsible for stabilizing ERα protein. In conclusion, our results suggest that targeting CXCR7 may serve as a potential therapeutic strategy for improving the efficacy of BCa patients with tamoxifen therapy.

HIC1 loss promotes prostate cancer metastasis by triggering epithelial-mesenchymal transition.

Metastatic disease is the leading cause of death due to prostate cancer (PCa). Although the hypermethylated in cancer 1 (HIC1) gene has been observed to be epigenetically modified in PCa, its intrinsic role and mechanism in PCa metastasis still remain uncertain. Here, we show that hypermethylation of the HIC1 promoter markedly reduces its suppressive function in metastatic PCa tissues as compared with primary and adjacent normal prostate tissues, and is associated with poor patient survival. PCas in cancer-prone mice homozygous for a prostate-targeted Hic1 conditional knockout showed stronger metastatic behaviour than those in heterozygous mice, as a result of epithelial-mesenchymal transition (EMT). Moreover, impairment of HIC1 expression in PCa cells induced their migration and metastasis through EMT, by enhancing expression of Slug and CXCR4, both of which are critical to PCa metastasis; the CXCL12-CXCR4 axis promotes EMT by activating the extracellular signal-regulated kinase (ERK) 1/2 pathway. Taken together, our results suggest that evaluation of HIC1-CXCR4-Slug signalling may provide a potential predictor for PCa aggressiveness. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer.

BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein that can directly regulate apoptosis and metastasis. In this study, we investigated the functional and molecular mechanisms by which TIMP-1 influences triple-negative breast cancer (TNBC). METHODS: The expression level of TIMP-1 in breast cancer tissues was analyzed using the ONCOMINE microarray database. The overall survival of patients with distinct molecular subtypes of breast cancer stratified by TIMP-1 expression levels was evaluated using Kaplan-Meier analysis. Bisulfate sequencing PCR (BSP) was used to analyze the methylation status of the TIMP-1 promoter. Real-time-PCR (RT-PCR), Western blot and ELISA assays were used to evaluate gene and protein expression in cell lines and human tissue specimens. In addition, TIMP-1 function was analyzed using a series of in vitro and in vivo assays with cells in which TIMP-1 was inhibited using RNAi or neutralizing antibodies. RESULTS: We found that serum TIMP-1 levels were strongly enhanced in patients with TNBC and that elevated TIMP-1 levels were associated with a poor prognosis in TNBC. However, TIMP-1 levels were not significantly associated with overall survival in other subtypes of breast cancer or in the overall population of breast cancer patients. We also report the first evidence that the TIMP-1 promoter is hypomethylated in TNBC cell lines compared with non-TNBC cell lines, suggesting that aberrant TIMP-1 expression in TNBC results from reduced DNA methylation. RNAi-mediated silencing of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and reduced cyclin D1 expression. In addition, mechanistic analyses revealed that the p-Akt and p-NF-κB signaling pathways, but not the GSK-3ß and MAPK1/2 pathways, are associated with TIMP-1 overexpression in TNBC cells. Moreover, neutralizing antibodies against TIMP-1 significantly decreased the rate of tumor growth in vivo. CONCLUSIONS: Our findings suggest that TIMP-1 is a biomarker indicative of a poor prognosis in TNBC patients and that targeting TIMP-1 may provide an attractive therapeutic intervention specifically for triple-negative breast cancer patients.

Hypermethylated in cancer 1(HIC1) suppresses non-small cell lung cancer progression by targeting interleukin-6/Stat3 pathway.

Non-small cell lung cancer (NSCLC), which accounts for more than 80% of lung cancers, is a leading cause of cancer mortality worldwide. However, the mechanism underlying its progression remains unclear. Here we found that HIC1 promoter was heavily methylated in NSCLC cell lines and tissues contributing to its low expression compared to normal controls. Restoring HIC1 expression inhibited migration, invasion and promoted inducible apoptosis of NSCLC cells. Notably, HIC1 is a tumor suppressor through inhibiting the transcription of IL-6 by sequence-specific binding on its promoter. Restoring IL-6 expression could partially rescue these phenotypes induced by HIC1 in vitro and in vivo. Mechanistic analyses show that autocrine secretion of IL-6 induced by loss of HIC1 activated STAT3 through IL-6/JAK pathway and was associated with NSCLC progression. The HIC1/IL-6 axis may serve as a prognostic biomarker and provide an attractive therapeutic target for NSCLC.

The feedback loop between miR-124 and TGF-ß pathway plays a significant role in non-small cell lung cancer metastasis.

Increasing evidence shows that micro RNAs (miRNAs) play a critical role in tumor development. However, the role of miRNAs in non-small cell lung cancer (NSCLC) metastasis remains largely unknown. Here, we found that miR-124 expression was significantly impaired in NSCLC tissues and associated with its metastasis. In vitro and in vivo experiments indicate that restoring miR-124 expression in NSCLC cells had a marked effect on reducing cell migration, invasion and metastasis. Mechanistic analyses show that Smad4, a cobinding protein in transforming growth factor-ß (TGF-ß) pathway, was identified as a new target gene of miR-124. Restoring Smad4 expression in miR-124-infected cells could partially rescue miR-124-induced abolition of cell migration and invasion. Notably, upon TGF-ß stimulation, phosphorylation of Smad2/3 was modulated by alteration of miR-124 or Smad4 expression, followed by inducing some special transcription of downstream genes including Snail, Slug and ZEB2, all of which may trigger epithelial-mesenchymal transition and be associated with NSCLC metastasis. Moreover, activation of TGF-ß pathway may enhance expression of DNMT3a, leading to hypermethylation on miR-124 promoter. Therefore, heavily loss of miR-124 expression further enhances Smad4 level by this feedback loop. Taken together, our data show for the first time that the feedback loop between miR-124 and TGF-ß pathway may play a significant role in NSCLC metastasis. Targeting the loop may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for NSCLC.