Dataset Information

Metformin attenuates transforming growth factor beta (TGF-?) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer.

ABSTRACT: Mesenchymal stem-like/claudin-low (MSL/CL) breast cancers are highly aggressive, express low cell-cell adhesion cluster containing claudins (CLDN3/CLDN4/CLDN7) with enrichment of epithelial-to-mesenchymal transition (EMT), immunomodulatory, and transforming growth factor-? (TGF-?) genes. We examined the biological, molecular and prognostic impact of TGF-? upregulation and/or inhibition using in vivo and in vitro methods. Using publically available breast cancer gene expression databases, we show that upregulation and enrichment of a TGF-? gene signature is most frequent in MSL/CL breast cancers and is associated with a worse outcome. Using several MSL/CL breast cancer cell lines, we show that TGF-? elicits significant increases in cellular proliferation, migration, invasion, and motility, whereas these effects can be abrogated by a specific inhibitor against TGF-? receptor I and the anti-diabetic agent metformin, alone or in combination. Prior reports from our lab show that TNBC is exquisitely sensitive to metformin treatment. Mechanistically, metformin blocks endogenous activation of Smad2 and Smad3 and dampens TGF-?-mediated activation of Smad2, Smad3, and ID1 both at the transcriptional and translational level. We report the use of ID1 and ID3 as clinical surrogate markers, where high expression of these TGF-? target genes was correlated to poor prognosis in claudin-low patients. Given TGF-?'s role in tumorigenesis and immunomodulation, blockade of this pathway using direct kinase inhibitors or more broadly acting inhibitors may dampen or abolish pro-carcinogenic and metastatic signaling in patients with MCL/CL TNBC. Metformin therapy (with or without other agents) may be a heretofore unrecognized approach to reduce the oncogenic activities associated with TGF-? mediated oncogenesis.

SUBMITTER: Wahdan-Alaswad R

PROVIDER: S-EPMC4889288 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Metformin attenuates transforming growth factor beta (TGF-β) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer.

Wahdan-Alaswad Reema R Harrell J Chuck JC Fan Zeying Z Edgerton Susan M SM Liu Bolin B Thor Ann D AD

Cell cycle (Georgetown, Tex.) 20160101 8

Mesenchymal stem-like/claudin-low (MSL/CL) breast cancers are highly aggressive, express low cell-cell adhesion cluster containing claudins (CLDN3/CLDN4/CLDN7) with enrichment of epithelial-to-mesenchymal transition (EMT), immunomodulatory, and transforming growth factor-β (TGF-β) genes. We examined the biological, molecular and prognostic impact of TGF-β upregulation and/or inhibition using in vivo and in vitro methods. Using publically available breast cancer gene expression databases, we show ...[more]

PMID: 26919310

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Project description:There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-?) pathway-associated genes relative to other subtypes, including the TGF-? receptor type III (T?RIII). We hypothesize that T?RIII is tumor promoter in mesenchymal-stem like TNBC cells.Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of T?RIII in the MSL subtype. We stably expressed short hairpin RNAs specific to T?RIII (T?RIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine T?RIII expression patterns across all TNBC subtypes.T?RIII was the most differentially expressed TGF-? signaling gene in the MSL subtype. Silencing T?RIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when T?RIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed T?RIII-dependent phenotypes, we discovered that integrin-?2 was expressed at higher level in MSL cells after T?RIII-KD. Stable knockdown of integrin-?2 in T?RIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells.We have found that T?RIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that T?RIII-KD elevates expression of integrin-?2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both T?RIII and integrin-?2. Overall, our results indicate a potential mechanism in which T?RIII modulates integrin-?2 expression to effect MSL cell migration, invasion, and tumorigenicity.

Project description:TGFβ signaling plays crucial role during development and cancer, however the role for TGFβ signaling in regulating the noncoding part of the human genome in triple negative breast cancer (TNBC) is still being unraveled. Herein, we provide the transcriptional landscape of TNBC in response to TGFβ activation and subsequent inhibition employing SB431542, selective TGFβ1 Receptor ALK5 Inhibitor. Our data revealed 72 commonly upregulated [fold change (FC) ≥ 2.0], including PLAU, TPM1, TAGLN, COL1A1, TGFBI, and SNAI1, and 53 downregulated (FC ≤ 2.0) protein coding genes in BT-549 and MDA-MB-231 models in response to TGFβ1 activation. Alignment to the geocode (V33) identified 41 upregulated (FC ≥ 2.0) and 22 downregulated (FC ≤ 2.0) long non-coding RNA (lncRNA) in response to TGFβ1 activation, which were inhibited by concurrent treatment with SB431542. To place our data from the in vitro models into their clinical context, we identified AC015909.1, AC013451.1, CYP1B1-AS1, AC004862.1, LINC01824, AL138828.1, B4GALT1-AS1, AL353751.1, AC090826.3, AC104695.4, ADORA2A-AS1, PTPRG-AS1, LINC01943, AC026954.3, TPM1-AS, ZFPM2-AS1, AC007362.1, AC112721.2, MALAT1, AL513314.2, AC112721.1, AC010343.3, LINC01711, and MAP3K2-DT lncRNA expression to positively correlate with TGFβ1 expression in a cohort of 360 TNBC patients. To provide mechanistic insight into lncRNA regulation by TGFβ signaling, SMAD2/3 ChIp-Seq data from BT-549 TNBC model retrieved from Gene Expression Omnibus (GEO) revealed direct binding of SMAD2/SMAD3 to the promoter of AC112721.1, AC112721.2, MALAT1, HHIP-AS1, LINC00472, and SLC7A11, suggesting their direct regulation by TGFβ1/SMAD2/SMAD3 pathway. Interestingly, AC112721.1, AC112721.2 exhibited higher expression in TNBC compared to normal breast tissue suggesting a possible role for those lncRNA in TNBC biology. Our miRNA analysis in the BT-549 model in response to exogenous TGFB1 revealed several affected miRNAs (2.0 ≤ FC ≤ 2.0), whose expression pattern was reversed in the presence of SB431542, suggesting those miRNA as plausible targets for TGFβ regulation. In particular, we observed hsa-miR-1275 to be downregulated in response to TGFB1 which was highly predicted to regulate PCDH1, FIBCD1, FXYD7, GDNF, STC1, EDN1, ZSWIM4, FGF1, PPP1R9B, NUAK1, PALM2AKAP2, IGFL3, and SPOCK1 whose expression were upregulated in response to TGFβ1 stimulus. On the other hand, hsa-miR-181b-5p was among the top upregulated miRNAs in response to TGFB1, which is also predicted to regulate CDKN1B, TNFRSF11B, SIM1, and ARSJ in the BT-549 model. Taken together, our data is the first to provide such in depth analysis of lncRNA and miRNA epigenetic changes in response to TGFβ signaling in TNBC.

Dataset Information

Metformin attenuates transforming growth factor beta (TGF-?) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer.

Publications

Metformin attenuates transforming growth factor beta (TGF-β) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer.

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