labeling-breast-cancer-patient-derived-xenografts-with-traceable

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

University of Colorado Anschutz Medical Campus

Helicobacter pylori is recognized as an etiologic agent of several gastric diseases. Bacterial genotypes have been related to clinical outcome in several populations.

[Helicobacter pylori genotypes in non atrophic gastritis are different of the found in peptic ulcer, premalignant lesions and gastric cancer in Colombia].

Spinal cord injury (SCI)-induced functional impairment results from secondary apoptosis regulated in part by SCI-induced decreases in the antiapoptotic protein Bcl-x(L). We assessed the role that Bcl-x(L) subcellular rerouting and posttranslational phosphorylation play in Bcl-x(L) decreases in a contusion model of rat SCI. Immunohistochemical analysis showed the presence of Bcl-x(L) in neurons and oligodendrocytes, but not in astrocytes and microglia, whereas phosphorylated Bcl-x(L) (P-ser(62)-Bcl-x(L)) was present only in neurons. Western blot analyses showed Bcl-x(L) present in mitochondria, endoplasmic reticulum, nuclei, and cytosolic extracts, whereas P-ser(62)-Bcl-x(L) was restricted to organelles. During the first 24 hr after SCI, Bcl-x(L) levels decreased in all fractions but with a different time course, suggesting an independent regulation of Bcl-x(L) shuttling from the cytosol to each compartment after SCI. SCI did not affect P-ser(62)-Bcl-x(L) levels in organelles. However, P-ser(62)-Bcl-x(L), which was not detected in the cytosolic fraction of uninjured spinal cord, appeared in the cytosol as early as 15 min postcontusion, suggesting a role for phosphorylation in SCI-induced Bcl-x(L)-decreases. Using an in vitro model, we observed a correlation between levels of cytosolic phosphorylated Bcl-x(L) and neuronal apoptosis, supporting the hypothesis that Bcl-x(L) phosphorylation is proapoptotic. Activated microglia/macrophages robustly expressed Bcl-x(L) 7 days after SCI, and a subpopulation showing nuclear condensation also expressed P-ser(62)-Bcl-x(L). Therefore, phosphorylation of Bcl-x(L) may have opposite effects in injured spinal cords: 1) it may decrease levels of the antiapoptotic Bcl-x(L) in neurons contributing to neuronal death, and 2) it may promote apoptosis in activated microglia/macrophages, thus curtailing the inflammatory cascades associated with SCI.

Phosphorylation of Bcl-xL after spinal cord injury.

Long-term functional impairments due to spinal cord injury (SCI) in the rat result from secondary apoptotic death regulated, in part, by SCI-induced decreases in protein levels of the antiapoptotic protein Bcl-xL. We have shown that exogenous administration of Bcl-xL spares neurons 24 h after SCI. However, long-term effects of chronic application of Bcl-xL have not been characterized. To counteract SCI-induced decreases in Bcl-xL and resulting apoptosis, we used the TAT protein transduction domain fused to the Bcl-xL protein (Tat-Bcl-xL), or its antiapoptotic domain BH4 (Tat-BH4). We used intrathecal delivery of Tat-Bcl-xL, or Tat-BH4, into injured spinal cords for 24 h or 7 days, and apoptosis, neuronal death and locomotor recovery were assessed up to 2 months after injury. Both, Tat-Bcl-xL and Tat-BH4, significantly decreased SCI-induced apoptosis in thoracic segments containing the site of injury (T10) at 24 h or 7 days after SCI. However, the 7-day delivery of Tat-Bcl-xL, or Tat-BH4, also induced a significant impairment of locomotor recovery that lasted beyond the drug delivery time. We found that the 7-day administration of Tat-Bcl-xL, or Tat-BH4, significantly increased non-apoptotic neuronal loss and robustly augmented microglia/macrophage activation. These results indicate that the antiapoptotic treatment targeting Bcl-xL shifts neuronal apoptosis to necrosis, increases the inflammatory response and impairs locomotor recovery. Our results suggest that a combinatorial treatment consisting of antiapoptotic and anti-inflammatory agents may be necessary to achieve tissue preservation and significant improvement in functional recovery after SCI.

Detrimental effects of antiapoptotic treatments in spinal cord injury.

The vital role of ovarian hormones in the development of the normal breast foreshadowed their importance in mammary stem cell regulation. Two recent papers reveal that 17β-estradiol and progesterone control the size and repopulating ability of the mammary stem cell compartment. This likely occurs via paracrine signaling from steroid receptor-positive luminal cells to steroid receptor-negative stem cells. These findings illuminate roles for the female sex steroids in mobilizing the stem cell pool in the normal breast, and also provide a crucial link between the known hormonal risks of breast cancer and the potential stem cell origin of this disease.

Ovarian steroid hormones: what's hot in the stem cell pool?

Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis.

Oncogenic HER2{Delta}16 suppresses miR-15a/16 and deregulates BCL-2 to promote endocrine resistance of breast tumors.

MicroRNAs (miRNAs) are small non-coding RNAs that serve as post-transcriptional regulators of gene expression. They work predominantly by binding to complementary sequences in target messenger RNA (mRNA) 3' untranslated regions (UTRs) where they prevent translation or cause degradation of the message. Steroid hormone receptors (SHRs) are ligand-activated transcription factors that regulate genes in steroid responsive tissues. Recent studies demonstrate that SHRs regulate miRNAs, and in turn, miRNAs can regulate SHR expression and function. Mounting evidence indicates that miRNAs are intimately involved with SHRs, as they are with other transcription factors, often in double negative feedback loops. Investigators are just beginning to expose the details of these complex relationships and reveal the extent to which miRNAs are involved with SHRs in normal physiology and the pathobiology of steroid hormone responsive tissues.

Steroid receptors and microRNAs: relationships revealed.

Tumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. We have recently demonstrated that the clinically important isoform of HER2, HERΔ16, promotes therapeutically refractory breast cancer including resistance to endocrine therapy. Likewise additional breast tumor cell models of tamoxifen resistance have been developed that do not involve HER2 overexpression. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive.

Downregulation of miR-342 is associated with tamoxifen resistant breast tumors.

To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1- cells (∼100- and 16-fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3' UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1- cells increases Dicer protein. We demonstrate via luciferase reporter assays that these miRNAs directly target the Dicer1 3' UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58-fold higher in the more well-differentiated ERα+ cells, and restoration of miR-200c to ERα- cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1- cells. Together, our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα+ cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R, and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα- breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ERα itself, Dicer, and growth factor receptor levels.

MicroRNAs link estrogen receptor alpha status and Dicer levels in breast cancer.

A therapeutic intervention that could decrease tumor burden and increase sensitivity to chemotherapy would have a significant impact on the high morbidity rate associated with ovarian cancer. miRNAs have emerged as potential therapeutic candidates due to their ability to downregulate multiple targets involved in tumor progression and chemoresistance. miRNA-200c (miR-200c) is downregulated in ovarian cancer cell lines and stage III ovarian tumors, and low miR-200c correlates with poor prognosis. miR-200c increases sensitivity to taxanes in vitro by targeting class III β-tubulin gene (TUBB3), a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors having low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) as compared with those having high TUBB3 (average survival 42.56 ± 3.19 months). miR-200c also targets TrkB, a mediator of resistance to anoikis. We show that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biologic substrates in vitro. Because both chemo- and anoikis-resistance are critical steps in the progression of ovarian cancer, we sought to determine how restoration of miR-200c affects tumor burden and chemosensitivity in an in vivo preclinical model of ovarian cancer. Restoration of miR-200c in an intraperitoneal xenograft model of human ovarian cancer results in decreased tumor formation and tumor burden. Furthermore, even in established tumors, restoration of miR-200c, alone or in combination with paclitaxel, results in significantly decreased tumor burden. Our study suggests that restoration of miR-200c immediately before cytotoxic chemotherapy may allow for a better response or lower effective dose.

Restoration of miR-200c to ovarian cancer reduces tumor burden and increases sensitivity to paclitaxel.

Anoikis is apoptosis initiated upon cell detachment from the native extracellular matrix. Since survival upon detachment from basement membrane is required for metastasis, the ability to resist anoikis contributes to the metastatic potential of breast tumors. miR-200c, a potent repressor of epithelial to mesenchymal transition, is expressed in luminal breast cancers, but is lost in more aggressive basal-like, or triple negative breast cancers (TNBC). We previously demonstrated that miR-200c restores anoikis sensitivity to TNBC cells by directly targeting the neurotrophic receptor tyrosine kinase, TrkB. In this study, we identify a TrkB ligand, neurotrophin 3 (NTF3), as capable of activating TrkB to induce anoikis resistance, and show that NTF3 is also a direct target of miR-200c. We present the first evidence that anoikis resistant TNBC cells up-regulate both TrkB and NTF3 when suspended, and show that this up-regulation is necessary for survival in suspension. We further demonstrate that NF-κB activity increases 6 fold in suspended TNBC cells, and identify RelA and NF-κB1 as the transcription factors responsible for suspension-induced up-regulation of TrkB and NTF3. Consequently, inhibition of NF-κB activity represses anoikis resistance. Taken together, our findings define a critical mechanism for transcriptional and post-transcriptional control of suspension-induced up-regulation of TrkB and NTF3 in anoikis resistant breast cancer cells.

miR-200c targets a NF-κB up-regulated TrkB/NTF3 autocrine signaling loop to enhance anoikis sensitivity in triple negative breast cancer.

The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

Dicer is an RNase III enzyme responsible for cleaving double-stranded RNAs into small interfering RNAs and microRNAs, which either target messenger RNA transcripts for degradation or inhibit translation. Dicer protein levels have been examined in breast cancer with contradictory results. Our goal was to resolve whether Dicer levels differ in breast cancer versus normal breast epithelium and between estrogen receptor-α-positive (ER+) or estrogen receptor-α-negative (ER-) primary breast cancers. We compared 3 different Dicer antibodies: Abcam 4A6, Abcam ab5818, and Sigma HPA000694, using immunohistochemistry and Western blot analyses. All 3 Dicer antibodies detected higher levels of Dicer in ER+ breast cancer cell lines versus ER-, and all 3 recognized exogenous overexpressed Dicer. In clinical specimens, all 3 antibodies detected higher Dicer in ER+ breast cancers versus triple-negative breast cancer (TNBC) but had very different staining patterns by immunohistochemistry on the same tumor samples. Using the optimal antibody, ab5818, selected for its sensitivity and specificity, Dicer protein expression was significantly higher in ER+ versus TNBC clinical specimens of primary tumor (P<.0001, unpaired t test). Dicer was also significantly higher in adjacent normal breast epithelium versus TNBC (P<.0001, paired t test; n=18 pairs). Differences in antibody performance may explain contrasting results observed in the literature regarding Dicer protein in breast cancer. If Dicer becomes more clinically relevant as a prognostic indicator, further antibody optimization and standardization will be critical.

Dicer expression in estrogen receptor-positive versus triple-negative breast cancer: an antibody comparison.

Anti-endocrine therapy remains the most effective treatment for ER+ breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer-associated fibroblasts (CAFs), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and anti-endocrine resistance.

Diana M. Cittelly

Department of Pathology,
School of Medicine,
Department of Pathology, School of Medicine

University of Colorado Anschutz Medical Campus

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

Diana M. Cittelly

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathology,School of Medicine,Department of Pathology, School of Medicine

University of Colorado Anschutz Medical Campus

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

Department of Pathology,
School of Medicine,
Department of Pathology, School of Medicine