MKNK1 is a YB-1 target gene responsible for imparting trastuzumab resistance and can be blocked by RSK inhibition.

Trastuzumab (Herceptin) resistance is a major obstacle in the treatment of patients with HER2-positive breast cancers. We recently reported that the transcription factor Y-box binding protein-1 (YB-1) leads to acquisition of resistance to trastuzumab in a phosphorylation-dependent manner that relies on p90 ribosomal S6 kinase (RSK). To explore how this may occur we compared YB-1 target genes between trastuzumab-sensitive cells (BT474) and those with acquired resistance (HR5 and HR6) using genome-wide chromatin immunoprecipitation sequencing (ChIP-sequencing), which identified 1391 genes uniquely bound by YB-1 in the resistant cell lines. We then examined differences in protein expression and phosphorylation between these cell lines using the Kinexus Kinex antibody microarrays. Cross-referencing these two data sets identified the mitogen-activated protein kinase-interacting kinase (MNK) family as potentially being involved in acquired resistance downstream from YB-1. MNK1 and MNK2 were subsequently shown to be overexpressed in the resistant cell lines; however, only the former was a YB-1 target based on ChIP-PCR and small interfering RNA (siRNA) studies. Importantly, loss of MNK1 expression using siRNA enhanced sensitivity to trastuzumab. Further, MNK1 overexpression was sufficient to confer resistance to trastuzumab in cells that were previously sensitive. We then developed a de novo model of acquired resistance by exposing BT474 cells to trastuzumab for 60 days (BT474LT). Similar to the HR5/HR6 cells, the BT474LT cells had elevated MNK1 levels and were dependent on it for survival. In addition, we demonstrated that RSK phosphorylated MNK1, and that this phosphorylation was required for ability of MNK1 to mediate resistance to trastuzumab. Furthermore, inhibition of RSK with the small molecule BI-D1870 repressed the MNK1-mediated trastuzumab resistance. In conclusion, this unbiased integrated approach identified MNK1 as a player in mediating trastuzumab resistance as a consequence of YB-1 activation, and demonstrated RSK inhibition as a means to overcome recalcitrance to trastuzumab.

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 G(1)/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 transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1.

PIK3CA, which codes for the p110alpha catalytic subunit of phosphatidylinositol-3-kinase (PI3K), is implicated as an oncogene. Despite importance of PIK3CA in cancer, little is known about what drives up its expression in tumor cells. We recently characterized the PIK3CA promoter and reported that it is transcriptionally silenced by the tumor suppressor protein p53. In the present study, we demonstrate that PIK3CA can be induced by the oncogenic transcription factor Y-box binding protein-1 (YB-1). Three YB-1-responsive elements were identified on the PIK3CA promoter using chromatin immunoprecipitation and electrophoretic mobility shift assays. Interestingly, silencing YB-1 with siRNA in models of basal-like breast cancer decreased p110alpha protein levels regardless of whether PIK3CA was wild type, amplified or mutated. This decrease in p110alpha led to a reduction in PI3K activity and the downstream signaling primarily through p90 ribosomal S6 kinase and S6 ribosomal protein. Disruption in PIK3CA-dependent signaling suppressed cellular invasion correlative with loss of urokinase plasminogen activator (uPA). Similarly, silencing YB-1 suppressed invasion and uPA production however this was reversible through the introduction of constitutively active PIK3CA. In conclusion, YB-1 is the first reported oncogene to induce the expression of PIK3CA through transcriptional control of its promoter.

Peptidylprolyl isomerase A (PPIA) as a preferred internal control over GAPDH and beta-actin in quantitative RNA analyses.

A good internal control is critical in all quantitative analyses of gene expression. Levels of bet-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and peptidylprolyl isomerase A (PPIA) were analyzed in 78 samples (data obtained from our laboratory and from a publicly available database at http://www.ncbi.nlm.nih.gov/SAGE/). These libraries included cell lines and tissues from brain, breast, colon, kidney, ovary, pancreas, prostate, skin, and vascular origin. The level of PPIA mRNA is the most constant among the three genes. Hence, our study suggests that PPIA is a better internal control than beta-actin or GAPDH, the two most commonly used internal controls.

A functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin.

To gain better understanding of the molecular alterations responsible for the aggressive growth potential of epidermal growth factor receptor (EGFR)-positive breast cancers, we utilized an expression cloning strategy to seek gene products that mediate the EGF-independent growth of human breast cancer cells. A retroviral cDNA expression library was constructed from the EGFR-positive SUM-149PT cell line, and transduced into growth factor-dependent human mammary epithelial (HME) cells. Recipient cells were functionally selected for their ability to proliferate in serum-free, EGF-free medium. Library cDNAs were recovered from EGF-independent colonies by PCR amplification or by biological rescue. Clone H55a#1 contained a library insert encoding amphiregulin. This EGFR ligand was able to confer EGF independence when transduced into HME cells. SUM-149PT and H55a#1 cells overexpressed amphiregulin transcripts, and secreted moderate EGF-like activity in conditioned media, indicating a possible autocrine loop. EGFR membrane levels and constitutive phosphorylation were consistent with this hypothesis, as well as the sensitivity of the cells to an ErbB-specific kinase inhibitor. Expression of the WT1 Wilms' tumor suppressor gene, a transcriptional activator of amphiregulin, did not parallel amphiregulin transcript levels, suggesting that another factor regulates amphiregulin in SUM-149PT. Our data confirm the importance of amphiregulin in the etiology of breast cancer.

Transcription of human cathepsin B is mediated by Sp1 and Ets family factors in glioma.

Cathepsin B expression is increased at both the mRNA and protein levels in a wide variety of tumors. The mechanisms responsible for this regulation are not well elucidated. We have isolated a 2.2-kb cathepsin B genomic fragment that contains the 5'-flanking region of the cathepsin B gene. Using reporter gene analysis in human glioblastoma U87MG cells, we have mapped a 228-bp fragment (-172 to +56) having high promoter activity. This promoter region has a high G+C content; contains potential Spl, Ets, and USF binding motifs; and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site. Cotransfection experiments demonstrated that Spl and Ets1 could trans-activate cathepsin B transcription, whereas Ets2 could not. Electrophoretic mobility shift assays and supershift assays revealed that three of the four putative Sp1 sites in this promoter region form a specific complex containing the Sp1 transcription factor. Mutating all four of the Spl binding sites individually markedly reduced the promoter activity of transfected reporter genes in U87 cells. Cotransfection of this cathepsin B promoter construct with Spl family expression vectors in Schneider's Drosophila line 2 (SL2) cells demonstrated that Spl and Sp3, but not Sp4, activated cathepsin B transcription. Taken together, these results suggest that Sp1, Sp3, and Ets1 are important factors in cathepsin B transcription. The regulation of cathepsin B transcription by Sp1- and Sp1-related factors is mediated through multiple GC boxes.

Differentiating agents regulate cathepsin B gene expression in HL-60 cells.

We utilized HL-60 cells as a model system to examine the regulation of ctsb gene expression by differentiating agents. Inducers of monocytic differentiation [phorbol ester (PMA), calcitriol (D3), and sodium butyrate (NaB)] and inducers of granulocytic differentiation [all-trans retinoic acid (RA) and 9-cis retinoic acid (9-cis RA)] increase ctsb mRNA levels in a dose-dependent manner as determined by Northern blot hybridization. D3 and retinoids exert additive effects, suggesting that these agents act in part through distinct pathways. Actinomycin D decay experiments indicate that D3, NaB, RA, and 9-cis RA do not alter mRNA stability. In contrast, PMA markedly increases the half-life of ctsb mRNA. In transient transfection assays, PMA and NaB both stimulate transcription of the luciferase reporter gene placed under the control of ctsb promoter fragments. Thus, inducers of HL-60 cell differentiation can regulate the expression of the ctsb gene at both transcriptional and posttranscriptional levels.

Exon 2 of human cathepsin B derives from an Alu element.

Transcripts for the cysteine protease cathepsin B are alternatively spliced in the untranslated regions (UTRs). We show that a cathepsin B probe containing 5'-UTR sequences hybridized to an RNA of approximately 300 nt in addition to the typical 2.2 and 4.0 kbp mRNAs. Within this 5'-UTR, exon 2 was found to be homologous to Alu repetitive elements. Specifically, exon 2 was part of an Alu element interspersed with the cathepsin B gene. The approximately 300 nt band that hybridized to our cathepsin B probe likely corresponds to Alu transcripts, which are known to accumulate in human cells. Indeed, a similarly migrating band was detected with an authentic Alu probe. Thus, we suggest that primary transcripts for cathepsin B contain Alu sequences which are preserved as exon 2 in some fully spliced mRNAs.

Cathepsin B expression in human tumors.

Cathepsin B has been linked to tumor progression through observations that its activity, secretion or membrane association are increased. The most malignant tumors, and specifically the cells at the invasive edge of those tumors, express the highest activity. Cathepsin B may facilitate invasion directly by dissolving extracellular matrix barriers like the basement membrane, or indirectly by activating other proteases capable of digesting the extracellular matrix. Cathepsin B also might play a role in tumor growth and angiogenesis. Cathepsin B activity is the result of several levels of regulation: transcription, post-transcription processing, translation and glycosylation, maturation and trafficking, and inhibition. The majority of reports on cathepsin B expression in tumors have focused on measurements of activity or protein staining. In some tumors, e.g. gliomas, a correlation between the amounts of cathepsin B mRNA, protein and activity and tumor progression has been established. Regulation of cathepsin B at the transcriptional and post-transcriptional levels is still poorly understood. Although the putative promoter regions have characteristics of housekeeping-type promoters, cathepsin B mRNA expression varies depending on the cell type and state of differentiation. We have evidence that more than one promoter could direct expression of human cathepsin B. Multiple transcript species have been detected, resulting from alternative splicing in the 5'- or 3'-untranslated regions, and possibly the use of alternative promoter regions. The existence of transcript variants indicates a potential for post-transcriptional control of expression. In support of this, ras-transformation of MCF-10A human breast epithelial cells results in an increase in protein levels without a concomitant increase in mRNA levels. Cathepsin B mRNA species with distinct 5'- or 3'-untranslated regions may differ in their stability and translatability. Variations in the coding region may also alter cathepsin B properties. We and Frankfater's group have observed transcript species that would encode a truncated protein, lacking the prepeptide and about half of the propeptide. This truncated protein, if synthesized in cells, would be expected to be cytosolic; therefore its function is unclear. Once the several mechanisms of regulation of cathepsin B expression and activity are better understood, they could provide us with new strategies to specifically reduce cathepsin B activity in tumors.

Identification of two new exons and multiple transcription start points in the 5'-untranslated region of the human cathepsin-B-encoding gene.

Transcripts for cysteine protease cathepsin B (CTSB) were found to be highly variable in the 5'-UTR (untranslated region). In cDNA clones from a human gastric adenocarcinoma cDNA library, we have identified two new exons (designated 2a and 2b) between exons 2 and 3 in the 5'-UTR of the gene. All of the exons of the 5'-UTR could be alternatively spliced to produce several transcript species. In addition, transcription was initiated from more than one promoter region. Using RT-PCR (reverse transcription-polymerase chain reaction) and primer extension assays, CTSB mRNA species were found to differ among tissues and between a glioblastoma sample and a cell line derived from it. Exons 2a and 2b were detected more frequently in tumor samples than in matched normal samples. Thus, factors related to the cell differentiation and environment seem likely to determine the types of transcripts that are expressed which in turn could influence the overall steady-state level of CTSB mRNAs and their rate of translation. Interestingly, at least three upstream translation initiation codons were observed and could constitute a means of controlling translation initiation.

Human gastric adenocarcinoma cathepsin B: isolation and sequencing of full-length cDNAs and polymorphisms of the gene.

Four full-length cDNA clones coding for preprocathepsin B were isolated from a human gastric adenocarcinoma cDNA library (AGS 1-6-30-1) and analyzed for possible sequence modifications that might be linked to altered intracellular trafficking and secretion of cathepsin B (CTSB) in malignant tumors. Comparison of AGS 1-6-30-1 cDNAs with human kidney/hepatoma cDNAs revealed: (1) three potential N-glycosylation sites instead of two, (2) a nucleotide (nt) substitution in the coding region for the propeptide from GTG to CTG which would result in a Val26-->Leu change, (3) three silent nt replacements in the coding region for the mature protein, (4) five single-nt differences in the 5'- and 3'-UTR (untranslated regions), (5) heterogeneity in the 5'-UTR, and (6) a 10-bp insertion in the 3'-UTR. The 10-bp insertion in the 3'-UTR may alter the stability of CTSB mRNA transcripts and thereby the expression of CTSB. These clones should be useful for expressing human tumor CTSB and analyzing the function of this enzyme in malignant progression. Two restriction-fragment length polymorphisms (RFLPs), EcoRI and TaqI, were detected by Southern blot analysis of genomic DNA from 36 unrelated Caucasians. Inheritance and distribution of the EcoRI alleles (13.0 and 11.0 kb) and the TaqI alleles (5.7 and 4.6 kb) indicated they were independent polymorphisms. In contrast to the EcoRI alleles of 13.0 and 11.0 kb observed in the population survey, genomic DNA from two AGS gastric adenocarcinoma subclones revealed two EcoRI alleles of 13.0 and 7.8 kb.(ABSTRACT TRUNCATED AT 250 WORDS)