Kaiji Hu

Phosphorylated Insulin-Like Growth Factor-I/Insulin Receptor Is Present in All Breast Cancer Subtypes and Is Related to Poor Survival

Drugs that target the insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (IR) are currently under investigation for a variety of malignancies including breast cancer. Although we have previously reported that IGF-IR expression in primary breast tumors is common, the activation status of this receptor has not been examined in relation to survival. Phosphorylated IGF-IR/IR (P-IGF-IR/IR) and its downstream signaling partner phospho-S6 (P-S6) were evaluated immunohistochemically in tumor tissue microarrays representing 438 cases of invasive breast cancer. P-IGF-IR/IR (n = 114; P = 0.046) and total levels of IR (n = 122; P = 0.009) were indicative of poor survival, whereas total IGF-IR (n = 112; P = 0.304) was not. P-IGF-IR/IR and P-S6 were coordinately expressed in primary breast tumors (likelihood ratio, 11.57; P = 6.70 x 10(-4)). Importantly, P-IGF-IR/IR was detected in all breast cancer subtypes (luminal, 48.1%; triple negative, 41.9%; and HER2, 64.3%). In vitro, the IGF-IR/IR inhibitor BMS-536924 decreased phospho-RSK and P-S6, and significantly suppressed the growth of breast cancer cell lines MCF-7, SUM149, and AU565 representing the luminal, triple negative, and HER2 subtypes, respectively, in monolayer and soft agar. BMS-536924 also inhibited growth in tamoxifen resistant MCF-7 Tam-R cells while having little effect on immortalized normal breast epithelial cells. Thus, we can determine which patients have the activated receptor and provide evidence that P-IGF-IR/IR is a prognostic factor for breast cancer. Beyond this, P-IGF-IR/IR could be a predictive marker for response to IGF-IR and/or IR-targeted therapies, as these inhibitors may be of benefit in all breast cancer subtypes including those with acquired resistance to tamoxifen.

Y-box binding protein-1 induces the expression of CD44 and CD49f leading to enhanced self-renewal, mammosphere growth, and drug resistance.

Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in >40% of breast cancers, where it is associated with poor prognosis, disease recurrence, and drug resistance. We questioned whether this may be linked to the ability of YB-1 to induce the expression of genes linked to cancer stem cells such as CD44 and CD49f. Herein, we report that YB-1 binds the CD44 and CD49f promoters to transcriptionally upregulate their expressions. The introduction of wild-type (WT) YB-1 or activated P-YB-1(S102) stimulated the production of CD44 and CD49f in MDA-MB-231 and SUM 149 breast cancer cell lines. YB-1-transfected cells also bound to the CD44 ligand hyaluronan more than the control cells. Similarly, YB-1 was induced in immortalized breast epithelial cells and upregulated CD44. Conversely, silencing YB-1 decreased CD44 expression as well as reporter activity in SUM 149 cells. In mice, expression of YB-1 in the mammary gland induces CD44 and CD49f with associated hyperplasia. Further, activated mutant YB-1(S102D) enhances self-renewal, primary and secondary mammosphere growth, and soft-agar colony growth, which were reversible via loss of CD44 or CD49f. We next addressed the consequence of this system on therapeutic responsiveness. Here, we show that paclitaxel induces P-YB-1(S102) expression, nuclear localization of activated YB-1, and CD44 expression. The overexpression of WT YB-1 promotes mammosphere growth in the presence of paclitaxel. Importantly, targeting YB-1 sensitized the CD44(High)/CD24(Low) cells to paclitaxel. In conclusion, YB-1 promotes cancer cell growth and drug resistance through its induction of CD44 and CD49f.

Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a potential target for therapy

IntroductionBasal-like breast cancers (BLBCs) are very aggressive, and present serious clinical challenges as there are currently no targeted therapies available. We determined the regulatory role of Y-box binding protein-1 (YB-1) on epidermal growth factor receptor (EGFR) overexpression in BLBC, and the therapeutic potential of inhibiting EGFR. We pursued this in light of our recent work showing that YB-1 induces the expression of EGFR, a new BLBC marker.MethodsPrimary tumour tissues were evaluated for YB1 protein expression by immunostaining tissue microarrays, while copy number changes were assessed by comparative genomic hybridization (CGH). The ability of YB-1 to regulate EGFR was evaluated using luciferase reporter, chromatin immunoprecipitation (ChIP) and gel shift assays. The impact of Iressa on monolayer cell growth was measured using an ArrayScan VTI high-throughput analyser to count cell number, and colony formation in soft agar was used to measure anchorage-independent growth.ResultsYB-1 (27/37 or 73% of cases, P = 3.899 × 10-4) and EGFR (20/37 or 57.1% of cases, P = 9.206 × 10-12) are expressed in most cases of BLBC. However, they are not typically amplified in primary BLBC, suggesting overexpression owing to transcriptional activation. In support of this, we demonstrate that YB-1 promotes EGFR reporter activity. YB-1 specifically binds the EGFR promoter at two different YB-1-responsive elements (YREs) located at -940 and -968 using ChIP and gel shift assays in a manner that is dependent on the phosphorylation of S102 on YB-1. Inhibiting EGFR with Iressa suppressed the growth of SUM149 cells by ~40% in monolayer, independent of mutations in the receptor. More importantly anchorage-independent growth of BLBC cell lines was inhibited with combinations of Iressa and YB-1 suppression.ConclusionWe have identified for the first time a causal link for the expression of EGFR in BLBC through the induction by YB-1 where it binds specifically to two distinguished YREs. Finally, inhibition of EGFR in combination with suppression of YB-1 presents a potential opportunity for therapy in BLBC.

Targeting YB-1 in HER-2 Overexpressing Breast Cancer Cells Induces Apoptosis via the mTOR/STAT3 Pathway and Suppresses Tumor Growth in Mice

The Y-box binding protein-1 (YB-1) is a transcription/translation factor that is highly expressed in primary breast tumors where it is consistently associated with poor survival. It induces human epidermal growth factor receptor (her-2) along with its dimerization partner egfr by directly binding to their promoters. In addition to promoting growth by inducing receptor tyrosine kinases, YB-1 also protects cells against apoptosis through mechanisms that have not been fully revealed. Given this, we addressed whether YB-1 might be an eventual therapeutic target for breast cancer by inhibiting it with small interfering RNAs in vitro and in vivo. Inhibiting YB-1 suppressed the growth of six of seven breast cancer cell lines that had amplified her-2 or were triple negative. Importantly, targeting YB-1 induced apoptosis in BT474-m1 and Au565 breast cancer cells known to have her-2 amplifications. The potential role of signal transducers and activators of transcription 3 (STAT3) was pursued to address the underlying mechanism for YB-1-mediated survival. Inhibition of YB-1 decreased P-STAT3(S727) but not P-STAT3(Y705) or total STAT3. This was accompanied by decreased P-ERK1/2(T202/Y204), P-mTOR(S2448), and total mammalian target of rapamycin mTOR. Furthering the role of STAT3 in these cells, we show that knocking it down recapitulated the induction of apoptosis. Alternatively, constitutively active P-STAT3 rescued YB-1-induced apoptosis. Finally, targeting YB-1 with 2 different siRNAs remarkably suppressed tumor cell growth in soft agar by >90% and delayed tumorigenesis in nude mice. We conclude that HER-2 overexpressing as well as triple-negative breast cancer cells are YB-1 dependent, suggesting it may be a good therapeutic target for these exceptionally aggressive tumors.

Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells

IntroductionTriple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.MethodsA genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44high/CD24-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.ResultsOf the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER+/HER2+, a metastatic variant), and HR5 (ER+/HER2+, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse.ConclusionInhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.

Targeting p90 Ribosomal S6 Kinase Eliminates Tumor-Initiating Cells by Inactivating Y-Box Binding Protein-1 in Triple-Negative Breast Cancers†‡§

Y‐box binding protein‐1 (YB‐1) is the first reported oncogenic transcription factor to induce the tumor‐initiating cell (TIC) surface marker CD44 in triple‐negative breast cancer (TNBC) cells. In order for CD44 to be induced, YB‐1 must be phosphorylated at S102 by p90 ribosomal S6 kinase (RSK). We therefore questioned whether RSK might be a tractable molecular target to eliminate TICs. In support of this idea, injection of MDA‐MB‐231 cells expressing Flag‐YB‐1 into mice increased tumor growth as well as enhanced CD44 expression. Despite enrichment for TICs, these cells were sensitive to RSK inhibition when treated ex vivo with BI‐D1870. Targeting RSK2 with small interfering RNA (siRNA) or small molecule RSK kinase inhibitors (SL0101 and BI‐D1870) blocked TNBC monolayer cell growth by ∼100%. In a diverse panel of breast tumor cell line models RSK2 siRNA predominantly targeted models of TNBC. RSK2 inhibition decreased CD44 promoter activity, CD44 mRNA, protein expression, and mammosphere formation. CD44+ cells had higher P‐RSKS221/227, P‐YB‐1S102, and mitotic activity relative to CD44− cells. Importantly, RSK2 inhibition specifically suppressed the growth of TICs and triggered cell death. Moreover, silencing RSK2 delayed tumor initiation in mice. In patients, RSK2 mRNA was associated with poor disease‐free survival in a cohort of 244 women with breast cancer that had not received adjuvant treatment, and its expression was highest in the basal‐like breast cancer subtype. Taking this further, we report that P‐RSKS221/227 is present in primary TNBCs and correlates with P‐YB‐1S102 as well as CD44. In conclusion, RSK2 inhibition provides a novel therapeutic avenue for TNBC and holds the promise of eliminating TICs. STEM CELLS2012;30:1338–1348

YB-1 evokes susceptibility to cancer through cytokinesis failure, mitotic dysfunction, and HER2 amplification

Y-box binding protein-1 (YB-1) expression in the mammary gland promotes breast carcinoma that demonstrates a high degree of genomic instability. In the present study, we developed a model of pre-malignancy to characterize the role of this gene during breast cancer initiation and early progression. Antibody microarray technology was used to ascertain global changes in signal transduction following the conditional expression of YB-1 in human mammary epithelial cells (HMEC). Cell cycle-associated proteins were frequently altered with the most dramatic being LIM kinase 1/2 (LIMK1/2). Consequently, the misexpression of LIMK1/2 was associated with cytokinesis failure that acted as a precursor to centrosome amplification. Detailed investigation revealed that YB-1 localized to the centrosome in a phosphorylation-dependent manner, where it complexed with pericentrin and γ-tubulin. This was found to be essential in maintaining the structural integrity and microtubule nucleation capacity of the organelle. Prolonged exposure to YB-1 led to rampant acceleration toward tumorigenesis, with the majority of cells acquiring numerical and structural chromosomal abnormalities. Slippage through the G1/S checkpoint due to overexpression of cyclin E promoted continued proliferation of these genomically compromised cells. As malignancy further progressed, we identified a subset of cells harboring HER2 amplification. Our results recognize YB-1 as a cancer susceptibility gene, with the capacity to prime cells for tumorigenesis.

The expression of activated Y-box binding protein-1 serine 102 mediates trastuzumab resistance in breast cancer cells by increasing CD44 + cells

The development of acquired resistance to trastuzumab remains a prevalent challenge in the treatment of patients whose tumors express human epidermal growth factor 2 (HER2). We previously reported that HER2 overexpressing breast cancers are dependent on Y-box binding protein-1 (YB-1) for growth and survival. As YB-1 is also linked to drug resistance in other types of cancer, we address its possible role in trastuzumab insensitivity. Employing an in vivo model of acquired resistance, we demonstrate that resistant cell lines have elevated levels of P-YB-1S102 and its activating kinase P-RSK and these levels are sustained following trastuzumab treatment. Further, to demonstrate the importance of YB-1 in mediating drug resistance, the expression of the active mutant YB-1S102D rendered the BT474 cell line insensitive to trastuzumab. Questioning the role of tumor-initiating cells (TIC) and their ability to escape cancer therapies, we investigate YB-1s role in inducing the cancer stem cell marker CD44. Notably, the resistant cells express more CD44 mRNA and protein compared with BT474 cells, which correlated with increased mammosphere formation. Expression of YB-1S102D in the BT474 cells increase CD44 protein levels, resulting in enhanced mammosphere formation. Further, exposing BT474 cells to trastuzumab selected for a resistant sub-population enriched for CD44. Conversely, small intefering RNA inhibition of CD44 restored trastuzumab sensitivity in the resistant cell lines. Our findings provide insight on a novel mechanism employed by tumor cells to acquire the ability to escape the effects of trastuzumab and suggest that targeting YB-1 may overcome resistance by eliminating the unresponsive TIC population, rendering the cancer sensitive to therapy.

YB-1 transforms human mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer

There is growing evidence that cancer‐initiation could result from epigenetic changes. Y‐box binding protein‐1 (YB‐1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the underlying molecular events are not understood. To explore this in a human model system, YB‐1 was expressed in mammary epithelial cells under the control of a tetracycline‐inducible promoter. The induction of YB‐1 promoted phenotypes associated with malignancy in three‐dimensional breast acini cultures. This was attributed to YB‐1 enhancing the expression and activity of the histone acetyltransferase p300 leading to chromatin remodeling. Specifically, this relaxation of chromatin allowed YB‐1 to bind to the BMI1 promoter. The induction of BMI1 engaged the Polycomb complex resulting in histone H2A ubiquitylation and repression of the CDKN2A locus. These events manifested functionally as enhanced self‐renewal capacity that occurred in a BMI1‐dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB‐1 from binding to the BMI1 promoter and thereby subverted self‐renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 became overexpressed concomitant with elevated human telomerase reverse transcriptase (hTERT) activity. The YB‐1/RSK2/hTERT expressing cells formed tumors in mice that were molecularly subtyped as basal‐like breast cancer. We conclude that YB‐1 cooperates with p300 to allow BMI1 to over‐ride p16INK4a‐mediated cell cycle arrest enabling self‐renewal and the development of aggressive breast tumors. Stem Cells 2014;32:1437–1450

Overcoming resistance to Sonic Hedgehog inhibition by targeting p90 ribosomal S6 kinase in pediatric medulloblastoma.

Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However, resistance inevitably emerges against these therapies, particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets.