Oncolytic Adenovirus for the Targeting of Paclitaxel-Resistant Breast Cancer Stem Cells.

Adjuvant systemic therapies effectively reduce the risk of breast cancer recurrence and metastasis, but therapy resistance can develop in some patients due to breast cancer stem cells (BCSCs). Oncolytic adenovirus (OAd) represents a promising therapeutic approach as it can specifically target cancer cells. However, its potential to target BCSCs remains unclear. Here, we evaluated a Cox-2 promoter-controlled, Ad5/3 fiber-modified OAd designed to encode the human sodium iodide symporter (hNIS) in breast cancer models. To confirm the potential of OAds to target BCSCs, we employed BCSC-enriched estrogen receptor-positive (ER+) paclitaxel-resistant (TaxR) cells and tumorsphere assays. OAd-hNIS demonstrated significantly enhanced binding and superior oncolysis in breast cancer cells, including ER+ cells, while exhibiting no activity in normal mammary epithelial cells. We observed improved NIS expression as the result of adenovirus death protein deletion. OAd-hNIS demonstrated efficacy in targeting TaxR BCSCs, exhibiting superior killing and hNIS expression compared to the parental cells. Our vector was capable of inhibiting tumorsphere formation upon early infection and reversing paclitaxel resistance in TaxR cells. Importantly, OAd-hNIS also destroyed already formed tumorspheres seven days after their initiation. Overall, our findings highlight the promise of OAd-hNIS as a potential tool for studying and targeting ER+ breast cancer recurrence and metastasis.

Stress sensing within the breast tumor microenvironment: how glucocorticoid receptors live in the moment.

The classification and treatment of breast cancer is largely defined by the expression of steroid hormone receptors (HRs), namely estrogen receptor (ER) and progesterone receptor (PR), and gene amplification/overexpression of human epidermal growth factor receptor 2 (HER2). More recently, studies of androgen receptor (AR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) have revealed that targeting these related HRs may be a promising strategy for a more personalized approach to the treatment of specific subtypes of HR+ breast cancer. For example, GR expression is associated with a good prognosis in ER+ breast cancer, but predicts poor prognosis in triple-negative breast cancer (TNBC). GR, like ER, PRs, and AR, is a ligand-activated transcription factor, but also has significant ligand-independent signaling activities. GR transcriptional activity is classically regulated by circulating glucocorticoids (GCs; ligand-dependent). Recent studies demonstrate that GR transcriptional activity is also regulated by a variety of cellular stress stimuli that input to GR Ser134 phosphorylation via rapid activation of the p38 mitogen activated protein kinase (MAPK) signaling pathway (ligand-independent). Furthermore, ligand-independent GR activation promotes feedforward signaling loops that mediate sustained activation of stress signaling pathways to drive advanced cancer biology (i.e. migration, invasion, chemoresistance, survival, and cellular growth). In this review, we will focus on the role of GR as a key sensor and mediator of physiologic and tumor microenvironment (TME)-derived cellular stress signaling in TNBC and discuss how targeting GR and/or associated signaling pathways may provide a strategy to inhibit deadly TNBC progression.

Optical redox ratio differentiates breast cancer cell lines based on estrogen receptor status.

Autofluorescence spectroscopy is a powerful imaging technique that exploits endogenous fluorophores. The endogenous fluorophores NADH and flavin adenine dinucleotide (FAD) are two of the principal electron donors and acceptors in cellular metabolism, respectively. The optical oxidation-reduction (redox) ratio is a measure of cellular metabolism and can be determined by the ratio of NADH/FAD. We hypothesized that there would be a significant difference in the optical redox ratio of normal mammary epithelial cells compared with breast tumor cell lines and that estrogen receptor (ER)-positive cells would have a higher redox ratio than ER-negative cells. To test our hypothesis, the optical redox ratio was determined by collecting the fluorescence emission for NADH and FAD via confocal microscopy. We observed a statistically significant increase in the optical redox ratio of cancer compared with normal cell lines (P < 0.05). Additionally, we observed a statistically significant increase in the optical redox ratio of ER(+) breast cancer cell lines. The level of ESR1 expression, determined by real-time PCR, directly correlated with the optical redox ratio (Pearson's correlation coefficient = 0.8122, P = 0.0024). Furthermore, treatment with tamoxifen and ICI 182,870 statistically decreased the optical redox ratio of only ER(+) breast cancer cell lines. The results of this study raise the important possibility that fluorescence spectroscopy can be used to identify subtypes of breast cancer based on receptor status, monitor response to therapy, or potentially predict response to therapy. This source of optical contrast could be a potentially useful tool for drug screening in preclinical models.

Light scattering measurements of subcellular structure provide noninvasive early detection of chemotherapy-induced apoptosis.

We present a light scattering study using angle-resolved low coherence interferometry (a/LCI) to assess nuclear morphology and subcellular structure within MCF-7 cells at several time points after treatment with chemotherapeutic agents. Although the nuclear diameter and eccentricity are not observed to change, the light scattering signal reveals a change in the organization of subcellular structures that we interpret using fractal dimension (FD). The FD of subcellular structures in cells treated with paclitaxel and doxorubicin is observed to increase significantly compared with that of control cells as early as 1.5 and 3 hours after application, respectively. The FD is then found to decrease slightly at 6 hours postapplication for both agents only to increase again from 12 to 24 hours posttreatment when the observations ceased. The changes in structure appear over two time scales, suggesting that multiple mechanisms are evident in these early apoptotic stages. Indeed, quantitative image analysis of fluorescence micrographs of cells undergoing apoptosis verifies that the FD of 4',6-diamidino-2-phenylindole-stained nuclear structures does not change significantly in cells until 12 hours after treatment, whereas that of MitoTracker stained mitochondria is seen to modulate as early as 3 hours after treatment. In contrast, cells receiving an increased dose of paclitaxel that induced G(2)-M arrest, but not apoptosis, only exhibited the early change in subcellular structure but did not show the later change associated with changes in nuclear substructure. These results suggest that a/LCI may have utility in detecting early apoptotic events for both clinical and basic science applications.

Breast tumor kinase (protein tyrosine kinase 6) regulates heregulin-induced activation of ERK5 and p38 MAP kinases in breast cancer cells.

Total tyrosine kinase activity is often elevated in both cytosolic and membrane fractions of malignant breast tissue and correlates with a decrease in disease-free survival. Breast tumor kinase (Brk; protein tyrosine kinase 6) is a soluble tyrosine kinase that was cloned from a metastatic breast tumor and found to be overexpressed in a majority of breast tumors. Herein, we show that Brk is overexpressed in 86% of invasive ductal breast tumors and coexpressed with ErbB family members in breast cancer cell lines. Additionally, the ErbB ligand, heregulin, activates Brk kinase activity. Knockdown of Brk by stable expression of short hairpin RNA (shRNA) in T47D breast cancer cells decreases proliferation and blocks epidermal growth factor (EGF)- and heregulin-induced activation of Rac GTPase, extracellular signal-regulated kinase (ERK) 5, and p38 mitogen-activated protein kinase (MAPK) but not Akt, ERK1/2, or c-Jun NH(2)-terminal kinase. Furthermore, EGF- and heregulin-induced cyclin D1 expression is dependent on p38 signaling and inhibited by Brk shRNA knockdown. The myocyte enhancer factor 2 transcription factor target of p38 MAPK and ERK5 signaling is also sensitive to altered Brk expression. Finally, heregulin-induced migration of T47D cells requires p38 MAPK activity and is blocked by Brk knockdown. These results place Brk in a novel signaling pathway downstream of ErbB receptors and upstream of Rac, p38 MAPK, and ERK5 and establish the ErbB-Brk-Rac-p38 MAPK pathway as a critical mediator of breast cancer cell migration.

Linkage of progestin and epidermal growth factor signaling: phosphorylation of progesterone receptors mediates transcriptional hypersensitivity and increased ligand-independent breast cancer cell growth.

Progesterone receptor (PR) action is linked to epidermal growth factor (EGF) initiated signaling pathways at multiple levels; mitogen-activated protein kinases (MAPKs) are key mediators of this important cross-talk. Herein, we probed the effects of EGF on PR function and regulation of breast cancer cell growth. EGF stimulated rapid and transient phosphorylation of PR-B Ser294 relative to persistent phosphorylation of this site induced by the synthetic progestin, R5020. EGF induced nuclear translocation and DNA binding of unliganded wild-type, but not mutant PRs containing an Ala at position 294 (S294A). However, EGF alone induced little to no PR-B transcriptional activity; S294A PR-B was transcriptionally impaired. In contrast, pretreatment of cells with EGF (30min) significantly increased the potency and efficacy of wild-type, but not S294A PR transcriptional activity in response to progestin, and enhanced ligand-dependent downregulation of wild-type but not S294A PR. Replacement of Ser294 with aspartic acid (S294D) to mimic phosphorylation at this site decreased receptor stability and, as predicted, heightened progestin-induced transcription relative to wild-type PR-B. RT-PCR demonstrated the Ser294 phosphorylation-dependence of selected PR target genes (TGFalpha and HB-EGF). Surprisingly, PR-B expressing cells growing in soft agar were highly responsive to EGF or progestin, and this was further stimulated by the combination of both hormones. Cells expressing S294A PR exhibited reduced soft agar growth, and were also sensitive to R5020 alone, but failed to respond to EGF. These results suggest that PR Ser294 is an important "sensor" for growth factor inputs that affects PR function and breast cancer cell growth in the absence of progestin or in the presence of low or "sub-threshold" progestin concentrations. PR function likely contributes to breast cancer progression when EGFR family members or their ligands are overexpressed, a condition that predicts low abundance, but highly active and nuclear PR.

Regulated association of protein kinase B/Akt with breast tumor kinase.

Increased protein-tyrosine kinase activity is a prognostic indicator of decreased disease-free survival in patients with advanced breast tumors. Breast tumor kinase (Brk) is a soluble protein-tyrosine kinase overexpressed in the majority of breast cancers and also in normal skin and gut epithelium, but not in normal breast epithelial cells. Herein, we show that Brk interacts with protein kinase B/Akt, a serine/threonine kinase involved in cell growth and survival. Epidermal growth factor (EGF) treatment of human keratinocytes or Brk-transfected COS-1 cells leads to the dissociation of the Brk.Akt complex, whereas a constitutively active Brk mutant containing a point mutation at Tyr-447 (YF-Brk) failed to dissociate from Akt upon EGF treatment. In addition, Brk.Akt dissociation was blocked by the inhibition of phosphatidylinositol 3-kinase. Similar to ectopic Brk, endogenous Brk in T47D breast cancer cells was less phosphorylated upon EGF treatment, but it remained constitutively associated with Akt in the presence of EGF. Overexpression of wild-type (wt)-Brk, kinase-inactive (KM)-Brk, or YF-Brk increased the Tyr phosphorylation of multiple signaling molecules including EGF receptor. However, only wt- and YF-Brk, but not KM-Brk, induced phosphorylation of Akt and inhibited the kinase activity of Akt in unstimulated cells. Similarly, overexpression of wt- or YF-, but not KM-Brk, blocked the phosphorylation of the forkhead transcription factor, a downstream Akt target. These results suggest that Brk may function as a signaling molecule whose kinase activity normally limits the activity of Akt in unstimulated cells. Additionally, these results suggest that in breast cancer cells Brk behaves similarly to a constitutively active Brk mutant (YF-Brk) and associates with tyrosine-phosphorylated proteins in deregulated signaling complexes. Together these data provide clues to the possible proto-oncogenic and oncogenic functions of Brk.