HER4 Affects Sensitivity to Tamoxifen and Abemaciclib in Luminal Breast Cancer Cells and Restricts Tumor Growth in MCF-7-Based Humanized Tumor Mice.

The impact of the HER4 receptor on the growth and treatment of estrogen receptor-positive breast cancer is widely uncertain. Using CRISPR/Cas9 technology, we generated stable HER4 knockout variants derived from the HER4-positive MCF-7, T-47D, and ZR-75-1 breast cancer cell lines. We investigated tumor cell proliferation as well as the cellular and molecular mechanisms of tamoxifen, abemaciclib, AMG232, and NRG1 treatments as a function of HER4 in vitro. HER4 differentially affects the cellular response to tamoxifen and abemaciclib treatment. Most conspicuous is the increased sensitivity of MCF-7 in vitro upon HER4 knockout and the inhibition of cell proliferation by NRG1. Additionally, we assessed tumor growth and immunological effects as responses to tamoxifen and abemaciclib therapy in humanized tumor mice (HTM) based on MCF-7 HER4-wildtype and the corresponding HER4-knockout cells. Without any treatment, the enhanced MCF-7 tumor growth in HTM upon HER4 knockout suggests a tumor-suppressive effect of HER4 under preclinical but human-like conditions. This phenomenon is associated with an increased HER2 expression in MCF-7 in vivo. Independent of HER4, abemaciclib and tamoxifen treatment considerably inhibited tumor growth in these mice. However, abemaciclib-treated hormone receptor-positive breast cancer patients with tumor-associated mdm2 gene copy gains or pronounced HER4 expression showed a reduced event-free survival. Evidently, the presence of HER4 affects the efficacy of tamoxifen and abemaciclib treatment in different estrogen receptor-positive breast cancer cells, even to different extents, and is associated with unfavorable outcomes in abemaciclib-treated patients.

mdm2 gene amplification is associated with luminal breast cancer progression in humanized PDX mice and a worse outcome of estrogen receptor positive disease.

Estrogen receptor-positive breast cancer is a highly prevalent but heterogeneous disease among women. Advanced molecular stratification is required to enable individually most efficient treatments based on relevant prognostic and predictive biomarkers. First objective of our study was the hypothesis-driven discovery of biomarkers involved in tumor progression upon xenotransplantation of Luminal breast cancer into humanized mice. The second objective was the marker validation and correlation with the clinical outcome of Luminal breast cancer disease within the GeparTrio trial. An elevated mdm2 gene copy number was associated with enhanced tumor growth and lung metastasis in humanized tumor mice. The viability, proliferation and migration capacity of inherently mdm2 positive breast cancer cells in vitro were significantly reduced upon mdm2 knockdown or anti-mdm2 targeting. An mdm2 gain significantly correlated with a worse DFS and OS of Luminal breast cancer patients, albeit it was also associated with an enhanced preoperative pathological response rate. We provide evidence for an enhanced Luminal breast cancer stratification based on mdm2. Moreover, mdm2 can potentially be utilized as a therapeutic target in the Luminal subtype.

Protein kinase C targeting of luminal (T-47D), luminal/HER2-positive (BT474), and triple negative (HCC1806) breast cancer cells in-vitro with AEB071 (Sotrastaurin) is efficient but mediated by subtype specific molecular effects.

PURPOSE: Protein kinase C (PKC) plays a pivotal role in malignant cell proliferation, apoptosis, invasiveness and migration. However, its exploitation as therapeutic target in breast cancer has been merely explored. Here were evaluated the AEB071 (Sotrastaurin™) treatment efficiency of breast cancer cell lines derived from estrogen receptor positive (T-47D), estrogen/HER2 receptor positive (BT474), and triple negative (HCC1806) breast cancer cells under 2D (monolayer) and 3D (multicellular tumor spheroids) culture conditions. Additionally, spheroid cocultures of BC and N1 fibroblasts were analyzed. METHODS: We quantitatively assessed the proliferation capacity of breast cancer cells and fibroblasts as a function of AEB071 treatment using flow cytometry. The activities of PKC isoforms, substrates, and key molecules of the PKC signaling known to be involved in the regulation of tumor cell proliferation and cellular survival were additionally evaluated. Moreover, a multigene expression analysis (PanCancer Pathways assay) using the nanoString™ technology was applied. RESULTS: All breast cancer cell lines subjected to this study were sensitive to AEB071 treatment, whereby cell proliferation in 2D culture was considerably (BT474) or moderately (HCC1806) retarded in G0/G1 or in G2/M phase (T-47D) of the cell cycle. Regardless of the breast cancer subtype the efficiency of AEB071 treatment was significantly lower in the presence of N1 fibroblast cells. Subtype specific driver molecules, namely IL19, c-myb, and NGFR were mostly affected by the AEB071 treatment. CONCLUSION: A combined targeting of PKC and a subtype specific driver molecule might complement specified breast cancer treatment.

Buparlisib modulates PD-L1 expression in head and neck squamous cell carcinoma cell lines.

High expression of the immune checkpoint receptor PD-L1 is associated with worse patient outcome in a variety of human cancers, including head and neck squamous cell carcinoma (HNSCC). Binding of PD-L1 with its partner PD-1 generates an inhibitory signal that dampens the immune system. Immunotherapy, that is blocking the PD-1/PD-L1 checkpoint, has proven to be an effective tool in cancer therapy. However, not all patients are able to benefit from this immune checkpoint inhibition. Therefore, evidence is growing of intrinsic PD-L1 signaling in cancer cells. For example, intrinsic PD-L1 expression was associated with PI3K/Akt/mTOR signaling, which is part of diverse oncogenic processes including cell proliferation, growth and survival. In this study we demonstrate the effects of PI3K/Akt/mTOR pathway inhibition by buparlisib on PD-L1 expression in HNSCC cell lines. After buparlisib treatment for 72 h, PD-L1 was downregulated in total cell lysates of HNSCC cells. Moreover, flow cytometry revealed a downregulation of PD-L1 membrane expression. Interestingly, the buparlisib mediated effects on PD-L1 expression were reduced by additional irradiation. In PD-L1 overexpressing cells, the buparlisib induced inhibition of proliferation was neutralized. In summary, our findings imply that blocking the PI3K/Akt/mTOR pathway could be a good additional therapy for patients who show poor response to immune checkpoint therapy.

Differential Expression of PD-L1 during Cell Cycle Progression of Head and Neck Squamous Cell Carcinoma.

The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable effects in advanced cancer patients including HNSCC. In this context, irradiation is currently being investigated as a synergistic treatment modality to immunotherapy. However, the majority of HNSCC patients still show little improvement or even hyperprogression. Interestingly, there is increasing evidence for additional cell-intrinsic functions of PD-L1 in tumor cells. In previous studies, we showed that PD-L1 has a strong influence on proliferation, migration, invasion, and survival after irradiation. We demonstrated that cellular expression and localization of PD-L1 differed depending on sensitivity to irradiation. Here, we show that PD-L1 is also differentially expressed during cell cycle progression of HNSCC. Furthermore, cellular localization of PD-L1 also changes depending on a particular cell cycle phase. Moreover, distinct observations occurred depending on the general differentiation status. Overall, the function of PD-L1 cannot be generalized. Rather, it depends on the differentiation status and microenvironment. PD-L1 expression and localization are variable, depending on different factors. These findings may provide insight into why differential response to PD-1/PD-L1 antibody therapy can occur. Detailed understanding of cell-intrinsic PD-L1 functions will further allow antibody-based immunotherapy to be optimized.

A novel rabbit derived anti-HER2 antibody with pronounced therapeutic effectiveness on HER2-positive breast cancer cells in vitro and in humanized tumor mice (HTM).

BACKGROUND: Antibody based cancer therapies have achieved convincing success rates combining enhanced tumor specificity and reduced side effects in patients. Trastuzumab that targets the human epidermal growth factor related receptor 2 (HER2) is one of the greatest success stories in this field. For decades, trastuzumab based treatment regimens are significantly improving the prognosis of HER2-positive breast cancer patients both in the metastatic and the (neo-) adjuvant setting. Nevertheless, ≥ 50% of trastuzumab treated patients experience de-novo or acquired resistance. Therefore, an enhanced anti-HER2 targeting with improved treatment efficiency is still aspired. METHODS: Here, we determined cellular and molecular mechanisms involved in the treatment of HER2-positive BC cells with a new rabbit derived HER2 specific chimeric monoclonal antibody called "B100″. We evaluated the B100 treatment efficiency of HER2-positive BC cells with different sensitivity to trastuzumab both in vitro and in the presence of a human immune system in humanized tumor mice. RESULTS: B100 not only efficiently blocks cell proliferation but more importantly induces apoptotic tumor cell death. Detailed in vitro analyses of B100 in comparison to trastuzumab (and pertuzumab) revealed equivalent HER2 internalization and recycling capacity, similar Fc receptor signaling, but different HER2 epitope recognition with high binding and treatment efficiency. In trastuzumab resistant SK-BR-3 based humanized tumor mice the B100 treatment eliminated the primary tumor but even more importantly eradicated metastasized tumor cells in lung, liver, brain, and bone marrow. CONCLUSION: Overall, B100 demonstrated an enhanced anti-tumor activity both in vitro and in an enhanced preclinical HTM in vivo model compared to trastuzumab or pertuzumab. Thus, the use of B100 is a promising option to complement and to enhance established treatment regimens for HER2-positive (breast) cancer and to overcome trastuzumab resistance. Extended preclinical analyses using appropriate models and clinical investigations are warranted.

HER4 expression in estrogen receptor-positive breast cancer is associated with decreased sensitivity to tamoxifen treatment and reduced overall survival of postmenopausal women.

BACKGROUND: The sensitivity of estrogen receptor-positive breast cancers to tamoxifen treatment varies considerably, and the molecular mechanisms affecting the response rates are manifold. The human epidermal growth factor receptor-related receptor HER2 is known to trigger intracellular signaling cascades that modulate the activity of coregulators of the estrogen receptor which, in turn, reduces the cell sensitivity to tamoxifen treatment. However, the impact of HER2-related receptor tyrosine kinases HER1, HER3, and, in particular, HER4 on endocrine treatment is largely unknown. METHODS: Here, we retrospectively evaluated the importance of HER4 expression on the outcome of tamoxifen- and aromatase inhibitor-treated estrogen receptor-positive breast cancer patients (n = 258). In addition, we experimentally analyzed the efficiency of tamoxifen treatment as a function of HER4 co-expression in vitro. RESULTS: We found a significantly improved survival in tamoxifen-treated postmenopausal breast cancer patients in the absence of HER4 compared with those with pronounced HER4 expression. In accordance with this finding, the sensitivity to tamoxifen treatment of estrogen and HER4 receptor-positive ZR-75-1 breast cancer cells can be significantly enhanced by HER4 knockdown. CONCLUSION: We suggest an HER4/estrogen receptor interaction that impedes tamoxifen binding to the estrogen receptor and reduces treatment efficiency. Whether the sensitivity to tamoxifen treatment can be enhanced by anti-HER4 targeting needs to be prospectively evaluated.

Diagnostic pathology of early systemic cancer: ERBB2 gene amplification in single disseminated cancer cells determines patient survival in operable esophageal cancer.

Early metastatic dissemination and evolution of disseminated cancer cells (DCCs) outside the primary tumor is one reason for the failure of adjuvant therapies because it generates molecular genotypes and phenotypes different from primary tumors, which still underlie therapy decisions. Since ERBB2 amplification in esophageal DCCs but not in primary tumor cells predict outcome, we aimed to establish an assay with diagnostic reliability for single DCCs or circulating tumor cells. For this, we evaluated copy number alterations of more than 600 single DCCs from multiple cancer types to define reference regions suitable for quantification of target regions, such as ERBB2. We then compared ERBB2 quantitative PCR (qPCR) measurements with fluorescent in situ hybridization (FISH) data of various breast cancer cell lines and identified the aberration-calling threshold. The method was applied to two independent cohorts of esophageal cancer patients from Hamburg (n = 59) and Düsseldorf (n = 53). We found a high correlation between the single cell qPCR assay and the standard FISH assay (R = 0.98) and significant associations between amplification and survival for both patient cohorts (Hamburg (HH), p = 0.033; Düsseldorf (D), p = 0.052; pooled HH + D, p = 0.002) when applied to DCCs of esophageal cancer patients. Detection of a single ERBB2-amplified DCC was the most important risk factor for death from esophageal cancer (relative risk = 4.22; 95% CI = 1.91-9.32; p < 0.001). In our study, we detected ERBB2-amplified cells in 7% of patients. These patients could benefit from anti-ERBB2 targeting therapies.

Regulation of Programmed Death Ligand 1 (PD-L1) Expression in Breast Cancer Cell Lines In Vitro and in Immunodeficient and Humanized Tumor Mice.

Programmed death ligand 1 (PD-L1) expression is an efficient strategy of tumor cells to escape immunological eradiation. However, only little is known about the factors that affect the cellular expression levels. Here we assessed the PD-L1 expression on different breast cancer cell lines under standard in vitro culture conditions and as a function of Epirubicin or Paclitaxel treatment. Moreover, we evaluated the expression in immunodeficient tumor mice as well as in humanized tumor mice (i.e., in the presence of a human immune system). We found highest PD-L1 levels in JIMT-1 and MDA-MB-231 cells. Epirubicin treatment caused a decrease and Paclitaxel treatment an increased PD-L1 expression in MDA-MB-231 cells. In addition, we identified nuclear PD-L1 in MDA-MB-231 cells. All in vivo transplanted breast cancer cell lines downregulated PD-L1 expression compared to their in vitro counterpart. Neither the gene copy number nor the presence of human immune system in humanized tumor mice had an effect on the PD-L1 content. We demonstrate that the degree of PD-L1 expression amongst breast cancer cell lines varies considerably. In addition, cytotoxic treatments and other extrinsic parameters differentially affect the expression. Hence, further investigations including in vivo evaluations are necessary to understand PD-L1 regulation for advanced breast cancer stratification.

HER2 FISH results in breast cancers with increased CEN17 signals using alternative chromosome 17 probes - reclassifying cases in the equivocal category.

AIMS: HER2 testing of invasive breast cancer by in-situ hybridization guides therapy decisions. Probing HER2 and centromere of chromosome 17 (cen17) simultaneously is supposed to reveal both a potential HER2 gene amplification and polysomy 17. However, a considerable number of breast cancer patients with quasi polysomy 17 are considered 'equivocal', which is diagnostically meaningless. Moreover, patients with equivocal/false polysomic tumours are prevented from a potentially beneficial anti-HER2 treatment. Here we evaluated the RAI1, D17S122 and TP53 hybridization markers to indicate true polysomy reliably and to reclassify equivocal samples accurately as HER2-positive. METHODS AND RESULTS: Samples with (n = 82) and without (n = 52) increased cen17 copy numbers and 78 evidently HER2-amplified specimens were analysed using dual and triple marker hybridization probes. Selected putative polysomic samples were subjected to array-based comparative genomic hybridization (aCGH). We found that 37.8% samples with putative polysomy 17 did not show any gain in RAI1, D17S122 or TP53. Accordingly, aCGH revealed evidence for the presence of HER2/cen17 co-amplification rather than for true polysomy in those cases. Reflex testing using alternate 17p markers reclassified up to 56.3% equivocal cases as HER2-positive and the combined assessment of a 17p and 17q locus allows the differentiation of true versus false polysomy. CONCLUSIONS: An increased cen17 copy number does not necessarily reflect polysomy, and reflex testing facilitates the reclassification of 'equivocals'. Nevertheless, the prognostic and predictive value of a HER2/cen17 co-amplification versus HER2 gene amplification alone must still be evaluated prospectively.