Lendahl, Urban

Abstract Background: Despite recent advances in the treatment of triple negative breast cancer (TNBC) many patients are still suffering from early recurrence, therapy resistance, and organ metastasis 1 to 3 years post-chemotherapy. TNBC cells with stem cell-like properties are resistant to standard of care chemotherapy and are responsible for early tumor relapse and poor outcome. Up-regulation of PD-L1 expression in Cancer Stem Cells (CSCs) has been associated with immune evasion capacity and promotion of their stem-like properties. In this study we identify two druggable stemness target, Aurora-A Kinase (AURKA) and NOTCH3, with positive feedback on intra-tumoral PD-L1 expression and cancer cell plasticity. Methods: MDA-MB 231, MDA-MB 231 LM and SUM149-PT TNBC cells were used both in 2D and 3D environment. Unique TNBC cells were established from metastatic PDX models. TNBC cells were treated with Alisertib (AURKA inhibitor, 50nM) or AV353 (NOTCH3 inhibitor, 200ng). Immunofluorescence and immunoblot were used to compare different biomarker expression. ALDH was measured using Aldefluor kit. Apoptotic cells were quantified in real-time using IncuCyte S3. In vivo studies; 1x106 MDA MB 231/LM cells were injected into the mammary fat pad of NSG-female mice and treated with Alisertib or AV353. Control groups were treated with pacebo. Results: Our study shows that AURKA and NOTCH3 pharmacological blockade reduced nuclear/undruggable PD-L1, that is not achieved clinically by using FDA-approved immune checkpoint inhibitors (ICIs). Significantly, following stemness-targeted therapy with alisertib or AV353, reduction of intra-tumoral PD-L1 resulted in inhibition of tumor growth, decreased levels of CD44 expression, increased CD8+ T-cell tumor infiltration, impairment in the enrichment of ALDHhigh CSCs and enhanced chemosensitivity. In vivo pharmacological blockade of AURKA and NOTCH3 signaling pathways induced inhibition of tumor growth, apoptosis, and impairment of organ metastases. Conclusions: This study demonstrates that selective AURKA and NOTCH3 pharmacological blockade results in the inhibition of cancer cell plasticity that is linked to reduction of undruggable nuclear PD-L1 in TNBC cells. Our findings provide the strong preclinical rationale to develop novel clinical trials for patients with advanced TNBC that are refractory to chemotherapy and show limited response to FDA-approved ICIs. Citation Format: Antonino Bonaventura D'Assoro, Matthew Goetz, Carol Lange, Roberto Leon-Ferre, Tufia Haddad, James Ingle, Annapaola Montagner, Andrew Takchi, Ellie Babadi, Lucio Miele, Urban Lendahl, Urban Lendahl. Pharmacological blockade of AURKA and NOTCH3 oncogenic pathways to inhibit cancer cell plasticity in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5978.

Abstract Background: Dysregulated Notch signalling contributes to breast cancer development and progression, but validated tools to measure the level of Notch signalling in breast cancer subtypes and in response to systemic therapy are largely lacking. A transcriptomic signature of Notch signalling would thus be warranted, and in this report, we have established such a classifier. Methods: To generate the signature, we first identified Notch-regulated genes from six basal-like breast cancer cell lines subjected to elevated and reduced Notch signalling by culturing on immobilized Notch ligand Jagged1 or blockade of Notch by g-secretase inhibitors, respectively. From this cadre of Notch-regulated genes, we developed candidate transcriptomic signatures that were trained on a breast cancer patient dataset (the TCGA-BRCA cohort) and a broader breast cancer cell line cohort and sought to validate in independent data sets. Results: An optimal 20-gene transcriptomic signature was selected. We validated the signature on two independent patient datasets (METABRIC and Oslo2) and it showed an improved coherence score and tumour specificity compared with previously published signatures. Furthermore, the signature score was particularly high for basal-like breast cancer, indicating an enhanced level of Notch signalling in this subtype. The signature score was increased after neoadjuvant treatment in the PROMIX and BEAUTY patient cohorts, and a lower signature score generally correlated with better clinical outcome. Conclusions: The 20-gene transcriptional signature has the potential to better stratify patients and to evaluate the response of future Notch-based therapies for breast cancer. Citation Format: Eike-Benjamin Braune, Felix Geist, Xiaojia Tang, Krishna Kalari, Judy Boughey, Liewei Wang, Roberto A. Leon-Ferre, Antonino B. D’Assoro, James N. Ingle, Matthew P. Goetz, Kang Wang, Theodoros Foukakis, Anita Seshire, Dirk Wienke, Urban Lendahl. Identification of a Notch transcriptomic signature for breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B013.

The Notch signaling pathway is an architecturally simple signaling mechanism, well known for its role in cell fate regulation during organ development and in tissue homeostasis. In keeping with its importance for normal development, dysregulation of Notch signaling is increasingly associated with different types of tumors, and proteins in the Notch signaling pathway can act as oncogenes or tumor suppressors, depending on the cellular context and tumor type. In addition to a role as a driver of tumor initiation and progression in the tumor cells carrying oncogenic mutations, it is an emerging realization that Notch signaling also plays a role in non-mutated cells in the tumor microenvironment. In this review, we discuss how aberrant Notch signaling can affect three types of cells in the tumor stroma—cancer-associated fibroblasts, immune cells and vascular cells—and how this influences their interactions with the tumor cells. Insights into the roles of Notch in cells of the tumor environment and the impact on tumor-stroma interactions will lead to a deeper understanding of Notch signaling in cancer and inspire new strategies for Notch-based tumor therapy.

Abstract We have investigated the distribution of PDGFR-a and -b in the stroma of normal breast tissue, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) by immunohistochemistry. Both receptors were expressed by fibroblasts of normal breast tissue. While the PDGFRb remained high during cancer progression, the PDGFRa was lost in most of the DCIS samples and not detectable in IDC. The patient group with PDGFRa(low)/PDGFRb(high) fibroblasts had the highest risk for local recurrences or metastasis. The characteristic loss of stromal PDGFRa expression was also observed in the PyMT mouse breast cancer model. In vitro co-culture experiments confirmed a tumor cell-induced differentiation of fibroblasts, resulting in a fibroblast population marked by PDGFRa(low)/PDGFRb(high) expression. We could identify the interaction of Jagged-1 expressed by the tumor cells with Notch-2 expressed on the fibroblasts, as an essential activator of TGFb signaling in fibroblasts. Gene expression analysis and reporter-gene assays of sorted cells from co-cultures revealed, that fibroblasts switch from a more anti-tumorigenic signaling (expression of BMPs and Wnt antagonists) towards a pro-tumorigenic signaling (expression of BMP inhibitors, TGFb and Wnt ligands). Co-cultured tumor cells show an increased migratory activity and the induction of a mesenchymal phenotype. Inhibition of Notch or TGF signaling in fibroblasts could block these phenotypes. To investigate the impact of fibroblasts on the progression of DCIS to IDC, a DCIS xenograft model was used. Co-injection of MCF10.DCIS cells with human breast fibroblasts, expressing both PDGFRs, into the mouse mammary fat pad resulted in an earlier tumor take with faster tumor growth. The corresponding co-injection experiments with fibroblast and tumor cells, that were depleted of Notch2 respectively Jagged-1 expression by CRISPR/CAS technology is currently ongoing. Collectively the study identifies a potentially druggable paracrine cross-talk between epithelial and stromal cells, which promotes DCIS progression and involves a Notch mediated up-regulation of TGFb signaling. The study also suggests that loss of PDGFRa marks a subset of fibroblasts with specific tumor-promoting activities. Citation Format: Carina Strell, Janna Paulsson, Artur Mezheyeuski, Shao-Bo Jin, Pernilla Roswall, Johanna Andrea, Oliver Frings, Fredrick Warnberg, Hemming Johansson, Karin Jirstrom, Kristian Pietras, Per Hall, Urban Lendahl, Christer Betsholtz, Derek C. Radisky, Arne Ostman. Cell-contact dependent epithelial-stromal cross talk, including a modulation of stromal PDGFR expression, drives the progression of early-stage breast cancer lesions. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B41.