Anja Rockstroh

Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment

Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.

Cellular stress triggers the human topoisomerase I damage response independently of DNA damage in a p53 controlled manner

The ‘human topoisomerase I (htopoI) damage response’ was reported to be triggered by various kinds of DNA lesions. Also, a high and persistent level of htopoI cleavage complexes correlated with apoptosis. In the present study, we demonstrate that DNA damage-independent induction of cell death using colcemid and tumor necrosis factor α is also accompanied by a strong htopoI response that correlates with the onset of apoptotic hallmarks. Consequently, these results suggest that htopoI cleavage complex formation may be caused by signaling pathways independent of the kind of cellular stress. Thus, protein interactions or signaling cascades induced by DNA damage or cellular stress might lead to the formation of stabilized cleavage complexes rather than the DNA lesion itself. Finally, we show that p53 not only plays a key role in the regulation of the htopoI response to UV-C irradiation but also to treatment with colcemid.

The ascidian natural product eusynstyelamide B is a novel topoisomerase II poison that induces DNA damage and growth arrest in prostate and breast cancer cells

As part of an anti-cancer natural product drug discovery program, we recently identified eusynstyelamide B (EB), which displayed cytotoxicity against MDA-MB-231 breast cancer cells (IC50 = 5 μM) and induced apoptosis. Here, we investigated the mechanism of action of EB in cancer cell lines of the prostate (LNCaP) and breast (MDA-MB-231). EB inhibited cell growth (IC50 = 5 μM) and induced a G2 cell cycle arrest, as shown by a significant increase in the G2/M cell population in the absence of elevated levels of the mitotic marker phospho-histone H3. In contrast to MDA-MB-231 cells, EB did not induce cell death in LNCaP cells when treated for up to 10 days. Transcript profiling and Ingenuity Pathway Analysis suggested that EB activated DNA damage pathways in LNCaP cells. Consistent with this, CHK2 phosphorylation was increased, p21CIP1/WAF1 was up-regulated and CDC2 expression strongly reduced by EB. Importantly, EB caused DNA double-strand breaks, yet did not directly interact with DNA. Analysis of topoisomerase II-mediated decatenation discovered that EB is a novel topoisomerase II poison.

Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1-RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader.

Extracellular Vesicles in the Adaptive Process of Prostate Cancer during Inhibition of Androgen Receptor Signaling by Enzalutamide

Current treatments for advanced prostate cancer focus on inhibition of the androgen receptor (AR) by androgen deprivation therapy (ADT). However, complex interactions mediated by tumor suppressors, oncogenes, aberrations of AR expression, or de novo androgen production have been shown to induce the adaptive response of prostate cancer, leading to the development of castration resistant prostate cancer. In this study, we report the effects of AR antagonist, enzalutamide on the protein contents of extracellular vesicles (EVs). EVs mediate cell‐to‐cell communication and increasing evidence shows the role of EVs in promoting cancer survival and metastasis. We found that treatment with enzalutamide alters the secretion of EVs, one of which is a plasma membrane calcium pump, ATP2B1/PMCA ATPase, as an AR‐regulated EV protein. We highlight the networks of interactions between AR, Ca2+, and ATP2B1, where the extracellular proteins thrombospondin‐1, gelsolin, and integrinß1 were previously reported as regulators for cancer progression and metastasis, indicating the potential role of EV‐derived proteins in mediating calcium homoeostasis under AR inhibition by enzalutamide. Our data further highlight the cross‐talk between AR signaling and EV pathways in mediating resistance toward ADT.

6α-Acetoxyanopterine: A Novel Structure Class of Mitotic Inhibitor Disrupting Microtubule Dynamics in Prostate Cancer Cells

The lack of a cure for metastatic castrate-resistant prostate cancer (mCRPC) highlights the urgent need for more efficient drugs to fight this disease. Here, we report the mechanism of action of the natural product 6α-acetoxyanopterine (6-AA) in prostate cancer cells. At low nanomolar doses, this potent cytotoxic alkaloid from the Australian endemic tree Anopterus macleayanus induced a strong accumulation of LNCaP and PC-3 (prostate cancer) cells as well as HeLa (cervical cancer) cells in mitosis, severe mitotic spindle defects, and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. DNA microarray of 6-AA–treated LNCaP cells combined with pathway analysis identified very similar transcriptional changes when compared with the anticancer drug vinblastine, which included pathways involved in mitosis, microtubule spindle organization, and microtubule binding. Like vinblastine, 6-AA inhibited microtubule polymerization in a cell-free system and reduced cellular microtubule polymer mass. Yet, microtubule alterations that are associated with resistance to microtubule-destabilizing drugs like vinca alkaloids (vinblastine/vincristine) or 2-methoxyestradiol did not confer resistance to 6-AA, suggesting a different mechanism of microtubule interaction. 6-AA is a first-in-class microtubule inhibitor that features the unique anopterine scaffold. This study provides a strong rationale to further develop this novel structure class of microtubule inhibitor for the treatment of malignant disease. Mol Cancer Ther; 16(1); 3–15. ©2016 AACR.

A molecular portrait of epithelial–mesenchymal plasticity in prostate cancer associated with clinical outcome

The propensity of cancer cells to transition between epithelial and mesenchymal phenotypic states via the epithelial–mesenchymal transition (EMT) program can regulate metastatic processes, cancer progression, and treatment resistance. Transcriptional investigations using reversible models of EMT, revealed the mesenchymal-to-epithelial reverting transition (MErT) to be enriched in clinical samples of metastatic castrate resistant prostate cancer (mCRPC). From this enrichment, a metastasis-derived gene signature was identified that predicted more rapid cancer relapse and reduced survival across multiple human carcinoma types. Additionally, the transcriptional profile of MErT is not a simple mirror image of EMT as tumour cells retain a transcriptional “memory” following a reversible EMT. This memory was also enriched in mCRPC samples. Cumulatively, our studies reveal the transcriptional profile of epithelial–mesenchymal plasticity and highlight the unique transcriptional properties of MErT. Furthermore, our findings provide evidence to support the association of epithelial plasticity with poor clinical outcomes in multiple human carcinoma types.

Kallikrein-related peptidases 4, 5, 6 and 7 regulate tumour-associated factors in serous ovarian cancer

BackgroundTissue kallikrein-related peptidases 4, 5, 6 and 7 (KLK4–7) strongly increase the malignancy of ovarian cancer cells. Deciphering their downstream effectors, we aimed at finding new potential prognostic biomarkers and treatment targets for ovarian cancer patients. KLK4–7-transfected (OV-KLK4–7) and vector-control OV-MZ-6 (OV-VC) ovarian cancer cells were established to select differentially regulated factors.MethodsWith three independent approaches, PCR arrays, genome-wide microarray and proteome analyses, we identified 10 candidates (MSN, KRT19, COL5A2, COL1A2, BMP5, F10, KRT7, JUNB, BMP4, MMP1). To determine differential protein expression, we performed western blot analyses, immunofluorescence and immunohistochemistry for four candidates (MSN, KRT19, KRT7, JUNB) in cells, tumour xenograft and patient-derived tissues.ResultsWe demonstrated that KLK4–7 clearly regulates expression of MSN, KRT19, KRT7 and JUNB at the mRNA and protein levels in ovarian cancer cells and tissues. Protein expression of the top-upregulated effectors, MSN and KRT19, was investigated by immunohistochemistry in patients afflicted with serous ovarian cancer and related to KLK4–7 immunoexpression. Significant positive associations were found for KRT19/KLK4, KRT19/KLK5 and MSN/KLK7.ConclusionThese findings imply that KLK4–7 exert key modulatory effects on other cancer-related genes and proteins in ovarian cancer. These downstream effectors of KLK4–7, MSN and KRT19 may represent important therapeutic targets in serous ovarian cancer.

A 3D tumor microenvironment regulates cell proliferation, peritoneal growth and expression patterns

Peritoneal invasion through the mesothelial cell layer is a hallmark of ovarian cancer metastasis. Using tissue engineering technologies, we recreated an ovarian tumor microenvironment replicating this aspect of disease progression. Ovarian cancer cell-laden hydrogels were combined with mesothelial cell-layered melt electrospun written scaffolds and characterized with proliferation and transcriptomic analyses and used as intraperitoneal xenografts. Here we show increased cancer cell proliferation in these 3D co-cultures, which we validated using patient-derived cells and linked to peritoneal tumor growth in vivo. Transcriptome-wide expression analysis identified IGFBP7, PTGS2, VEGFC and FGF2 as bidirectional factors deregulated in 3D co-cultures compared to 3D mono-cultures, which we confirmed by immunohistochemistry of xenograft and patient-derived tumor tissues and correlated with overall and progression-free survival. These factors were further increased upon expression of kallikrein-related proteases. This clinically predictive model allows us to mimic the complexity and processes of the metastatic disease that may lead to therapies that protect from peritoneal invasion or delay the development of metastasis.