Sonja Thaler

RASSF1A mediates p21Cip1/Waf1-dependent cell cycle arrest and senescence through modulation of the Raf-MEK-ERK pathway and inhibition of Akt.

Promoter hypermethylation preventing expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is among the most abundant epigenetic deregulations in human cancer. Restoration of RASSF1A inhibits tumor cell growth in vitro and in murine xenograft models. Rassf1a-deficient mice feature increased spontaneous and carcinogen-induced tumor formation. Mechanistically, RASSF1A affects several cellular functions, such as microtubule dynamics, migration, proliferation, and apoptosis; however, its tumor-suppressive mechanism is incompletely understood. To study the functional consequences of RASSF1A expression in human cancer cells, we made use of a doxycycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G(1) phase and senescence in vitro and in tumors established in immunodeficient mice. RASSF1A-mediated growth inhibition was accompanied by the up-regulation of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) and proceeded independently of p53, p14(Arf), and p16(Ink4a). Loss of p21(Cip1/Waf1) or coexpression of the human papilloma virus 16 oncoprotein E7 was found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A affected mitogen-activated protein kinase and protein kinase B/Akt signaling to up-regulate p21(Cip1/Waf1) and to facilitate its nuclear localization. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21(Cip1/Waf1).

Targeting AKT Signaling Sensitizes Cancer to Cellular Immunotherapy

The promise of cancer immunotherapy is long-term disease control with high specificity and low toxicity. However, many cancers fail immune interventions, and secretion of immunosuppressive factors, defective antigen presentation, and expression of death ligands or serpins are regarded as main escape mechanisms. Here, we study whether deregulation of growth and survival factor signaling, which is encountered in most human cancers, provides another level of protection against immunologic tumor eradication. We show in two models that activated cell autonomous protein kinase B (PKB)/AKT signaling mediates resistance against tumor suppression by antigen-specific CTLs in vitro and adoptively transferred cellular immune effectors in vivo. PKB/AKT-dependent immunoresistance of established tumors is reversed by genetic suppression of endogenous Mcl-1, an antiapoptotic member of the Bcl-2 family. Mechanistically, deregulated PKB/AKT stabilizes Mcl-1 expression in a mammalian target of rapamycin (mTOR)-dependent pathway. Treatment with the mTOR inhibitor rapamycin effectively sensitizes established cancers to adoptive immunotherapy in vivo. In conclusion, cancer cell-intrinsic PKB/AKT signaling regulates the susceptibility to immune-mediated cytotoxicity. Combined targeting of signal transduction pathways may be critical for improvement of cancer immunotherapies.

The proteasome inhibitor Bortezomib (Velcade) as potential inhibitor of estrogen receptor‐positive breast cancer

Around 70% of breast cancers express the estrogen receptor α (ERα) and depend on estrogen for growth, survival and disease progression. The presence of hormone sensitivity is usually associated with a favorable prognosis. Use of adjuvant anti‐endocrine therapy has significantly decreased breast cancer mortality in patients with early‐stage disease, and anti‐endocrine therapy also plays a central role in the treatment of advanced stages. However a subset of hormone receptor‐positive breast cancers do not benefit from anti‐endocrine therapy, and nearly all hormone receptor‐positive metastatic breast cancers ultimately develop resistance to anti‐hormonal therapies. Despite new insights into mechanisms of anti‐endocrine therapy resistance, e.g., crosstalk between ERα and Her2/neu, the management of advanced hormone‐receptor‐positive breast cancers that are resistant to anti‐endocrine agents remains a significant challenge. In the present study, we demonstrate that the proteasome inhibitor Bortezomib strongly inhibits ERα and HER2/neu expression, increases expression of cyclin‐dependent kinase inhibitors, inhibits expression of multiple genes associated with poor prognosis in ERα+ breast cancer patients and induces cell death in ER+ breast cancer cells in both the presence and absence of functional p53. Although Bortezomib increased the levels of p53 and increased the expression of pro‐apoptotic target genes in ERα+ breast cancer cells harboring wild‐type p53, Bortezomib also exerts anti‐tumoral effects on ERα+ breast cancer cells through suppression of ERα expression and inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) and ERK signaling independently of functional p53. These findings suggest that Bortezomib might have the potential to improve the management of anti‐endocrine therapy resistant ERα+ breast cancers independently of their p53 status.

Delphinidin is a novel inhibitor of lymphangiogenesis but promotes mammary tumor growth and metastasis formation in syngeneic experimental rats

We have recently demonstrated that the anthocyanidin delphinidin (DEL), one of the most abundant dietary flavonoids, inhibits activation of ErbB and vascular endothelial growth factor receptor family members. These receptors play crucial roles in the context of tumor progression and the outgrowth of blood and lymphatic vessels. Here, we have developed an improved chemical synthesis for DEL in order to study the effects of the aglycon and its degradation product gallic acid (GA) on endothelial and tumor cells in vitro and in vivo. We found that DEL blocked the proliferation in vitro of primary human blood and lymphatic endothelial cells as well as human HT29 colon and rat MT-450 mammary carcinoma cells in a dose-dependent manner. In contrast, its degradation product GA had little effect. At higher concentrations, DEL induced apoptosis of endothelial and tumor cells. Furthermore, DEL potently blocked the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In the MT-450 rat syngeneic breast tumor model, it also significantly reduced angiogenesis and tumor-induced lymphangiogenesis when administered in vivo. These data reveal DEL to be a novel antilymphangiogenesis reagent. Surprisingly, however, the application of DEL unexpectedly promoted tumor growth and metastasis in the MT-450 tumor model, suggesting that the antiproliferative effect of DEL on cultured cells does not necessarily reflect the response of tumors to this anthocyanidin in vivo. Furthermore, while DEL may have utility as a cancer chemopreventative agent, its ability to promote tumor growth once a neoplasm develops also needs to be taken into consideration.

Assessment of HIV-1 entry inhibitors by MLV/HIV-1 pseudotyped vectors

BackgroundMurine leukemia virus (MLV) vector particles can be pseudotyped with a truncated variant of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) and selectively target gene transfer to human cells expressing both CD4 and an appropriate co-receptor. Vector transduction mimics the HIV-1 entry process and is therefore a safe tool to study HIV-1 entry.ResultsUsing FLY cells, which express the MLV gag and pol genes, we generated stable producer cell lines that express the HIV-1 envelope gene and a retroviral vector genome encoding the green fluorescent protein (GFP). The BH10 or 89.6 P HIV-1 Env was expressed from a bicistronic vector which allowed the rapid selection of stable cell lines. A codon-usage-optimized synthetic env gene permitted high, Rev-independent Env expression. Vectors generated by these producer cells displayed different sensitivity to entry inhibitors.ConclusionThese data illustrate that MLV/HIV-1 vectors are a valuable screening system for entry inhibitors or neutralizing antisera generated by vaccines.

Abstract P6-12-14: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

Background:Aberrant signaling through HER2 and other members of the HER family has been identified as mediator of endocrine resistance in estrogen receptor alpha (ERα) positive breast cancer. On the other hand, ERα co-expression has been shown to attenuate the efficiency of anti-HER2 targeted therapies. These findings indicate that HER2 and ERα synergize to allow breast cancer cells to escape from both anti-ERα and anti-HER2-targeted therapies. Rationally designed clinical trials that combine endocrine therapy with anti-HER2 agents to interfere with HER2/ERα cross-talk have been conducted. However, the outcome of these trials suggests that novel therapeutic approaches are needed to further improve inhibition of HER2 and other HER family members in conjunction with a more efficient ERα blockade. We examined the ability of proteasome inhibitors (PIs) to disrupt HER2/ERα cross-talk in HER2+/ER+ breast cancer (BC) cells. Furthermore we investigated the potential of PIs to suppress the activity of a constitutively active HER2 variant resistant to trastuzumab and lapatinib. Methode: HER2+/ER+ BC cells and fulvestrant resistant ER+ BC cells that overexpress a constitutively active HER2 variant resistant to trastuzumab and lapatinib have been treated with the PIs carfilzomib and bortezomib. The potential of these PIs to suppress ERα expression, to block HER2 activation and to inhibit the HER2 downstream pathways PI3K/Akt and Ras/MAPK was monitored by western blotting. Induction of cell death upon PI treatment was measured by quantification of SubG1 cells using propidium iodide staining or the use of colony formation assays. Results: Carfilzomib and bortezomib markedly inhibit bi-directional HER2/ERα signaling pathways in HER2/ER+ BC cells. Both PIs suppress ERα expression, inhibit HER2 activity and subsequently suppress the HER2 downstream pathways PI3K/Akt and Ras/MAPK that are major executors for endocrine resistance. Furthermore we observed that both PIs stabilize the HER2 specific tyrosine phosphatase BDP1 (PTPN18), thereby suppressing the activity of even a constitutive active HER2 variant that cause resistance to trastuzumab and lapatinib. Based on these findings we hypothesize that PIs inhibits ERα and HER2 activity through different mechanisms as currently used therapeutic regiments. Conclusion: These findings demonstrate that PIs disrupt the cross-talk between HER2 and ERα signaling pathways and therefore might have the potential to expand treatment opportunities for HER2+/ER+ and possibly also for other groups of BC patients. Citation Format: Thaler S, Schmidt M, Thiede G, Schad A, Sleeman JP. Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-14.

Detection of cellular senescence within human invasive breast carcinomas distinguishes different breast tumor subtypes

Oncogene-induced senescence is thought to act as a barrier to tumorigenesis by arresting cells at risk of malignant transformation. Nevertheless, numerous findings suggest that senescent cells may conversely promote tumor progression through the development of the senescence-associated secretome they produce. It is likely that the composition and the physiological consequences mediated by the senescence secretome are dependent on the oncogenes that trigger the senescence program. Breast cancer represents a heterogenous disease that can be divided into breast cancer subtypes due to different subsets of genetic and epigenetic abnormalities. As tumor initiation and progression of these breast cancer subtypes is triggered by diverse oncogenic stimuli, differences in the senescence secretomes within breast tumors might be responsible for tumor initiation, progression, metastasis and therapeutic response. Many studies have addressed the role of senescence as a barrier to tumor progression using murine xenograft models. However, few investigations have been performed to elucidate the degree to which senescent tumor cells are present within untreated human tumors, and if present, whether these senescent tumor cells may play a role in disease progression. In the present study we analysed the appearance of senescent cells within invasive breast cancers. Detection of cellular senescence by the use of SAβ-galactosidase (SAβ-gal) staining within invasive breast carcinoms from 129 untreated patients revealed differences in the amount of SAβ-gal+ tumor cells between breast cancer subtypes. The highest percentages of SAβ-gal+ tumor cells were found in HER2-positive and luminal A breast carcinomas whereas triple negative tumors showed either little or no positivity.