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5-Aminolevulinic acid (ALA) is an intraoperative imaging agent approved for protoporphyrin IX (PpIX) fluorescence-guided resection of glioblastoma (GBM). It is currently under clinical evaluation for photodynamic therapy (PDT) after the completion of GBM surgery. We previously showed that lapatinib, a clinical kinase inhibitor of epidermal growth factor receptor 1 & 2 (EGFR and HER2), enhanced PpIX fluorescence in a panel of GBM cell lines by blocking ABCG2 (ATP-binding cassette super-family G member 2)-mediated PpIX efflux, which suggests its potential for improving ALA for GBM surgery and PDT. Here we show that lapatinib enhanced PDT-induced cytotoxicity by promoting GBM cell death with the induction of apoptosis followed by necrosis. While the induction of tumor cell apoptosis was massive and rapid in the H4 cell line with no detectable Bcl-2 and a low level of Bcl-xL, it was delayed and much less in extent in A172, U-87 and U-118 cell lines with higher levels of pro-survival Bcl-2 family proteins. Lapatinib treatment alone neither reduced GBM cell viability nor had any significant effect on EGFR downstream signaling. Its enhancement of ALA-PDT was largely due to the increase of intracellular PpIX particularly in the mitochondria, resulting in the activation of mitochondria-mediated apoptosis in H4 cells. Our present study demonstrates that lapatinib inhibits ABCG2-mediated PpIX efflux and sensitizes GBM cells to ALA-PDT by inducing tumor cell death., (© 2024. The Author(s).)

Endocrine therapy plays a critical role in patients with hormone receptor-positive breast cancer. Endocrine-resistant breast cancer cells exhibit more HER2 signaling proteins (pAKT and pERK) and mesenchymal biomarkers than wild-type cell lines. In head and neck squamous cell carcinoma, the combination of lapatinib and palbociclib demonstrated synergistic inhibitory effects on cell proliferation and suppressed ERK1/2 phosphorylation. The combination of lapatinib and palbociclib at half-maximal inhibitory concentrations resulted in an increasing cytotoxic effect on cell proliferation. Furthermore, invasion activity was significantly decreased when combining two drugs at nontoxic concentrations more than either single drug alone did. The combination also remarkably suppressed epithelial-mesenchymal transition transcription factors, such as Snail and pAKT, more than monotherapy. Combining drugs, particularly lapatinib and palbociclib for targeting endocrine-resistant breast cancer cells whose tumors overexpressed HER2 after resistance to hormonal therapy, demonstrated better antiproliferative, anti-invasive effects, and suppression of EMT protein and pAKT than a single drug. These results could be from the interruption of the EMT process via the AKT pathway. Thus, this study provides preliminary data for applying this combination to patients with endocrine-resistant breast cancer in further clinical trials., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Deep sequencing of human tumours has uncovered a previously unappreciated role for epigenetic regulators in tumorigenesis. H3K4 methyltransferase KMT2C/MLL3 is mutated in several solid malignancies, including more than 10% of breast tumours. To study the tumour suppressor role of KMT2C in breast cancer, we generated mouse models of Erbb2/Neu, Myc or PIK3CA-driven tumorigenesis, in which the Kmt2c locus is knocked out specifically in the luminal lineage of mouse mammary glands using the Cre recombinase. Kmt2c knock out mice develop tumours earlier, irrespective of the oncogene, assigning a bona fide tumour suppressor role for KMT2C in mammary tumorigenesis. Loss of Kmt2c induces extensive epigenetic and transcriptional changes, which lead to increased ERK1/2 activity, extracellular matrix re-organization, epithelial-to-mesenchymal transition and mitochondrial dysfunction, the latter associated with increased reactive oxygen species production. Loss of Kmt2c renders the Erbb2/Neu-driven tumours more responsive to lapatinib. Publicly available clinical datasets revealed an association of low Kmt2c gene expression and better long-term outcome. Collectively, our findings solidify the role of KMT2C as a tumour suppressor in breast cancer and identify dependencies that could be therapeutically amenable., (© 2023. The Author(s).)

Purpose: Standard therapy for HER2+ breast cancers includes HER2 inhibition. While HER2 inhibitors have significantly improved therapeutic outcomes, many patients remain resistant to therapy. An important intrinsic resistance mechanism to HER2 inhibition in some breast cancers is dynamic upregulation of HER3. Increase in HER3 expression that occurs in response to HER2 inhibition allows for continued growth signaling through HER2/HER3 heterodimers, promoting tumor escape. We hypothesized that a non-invasive method to image changes in HER3 expression would be valuable to identify those breast cancers that dynamically upregulate HER3 in response to HER2 inhibition. We further hypothesized that this imaging method could identify those tumors that would benefit by additional HER3 knockdown., Procedures: In a panel of HER2+ breast cancer cell lines treated with the HER2 inhibitor lapatinib, we evaluate changes in HER3 expression and viability. Mouse HER2+ breast cancer models treated with lapatinib were imaged with a peptide-based HER3-specific PET imaging agent [ 68 Ga]HER3P1 to assess for dynamic changes in tumoral HER3 expression and uptake confirmed by biodistribution. Subsequently, HER2+ cell lines were treated with the HER2 inhibitor lapatinib as well HER3-specific siRNA to assess for changes in viability and correlate with HER3 expression upregulation. For all statistical comparisons, P<0.05 was considered statistically significant., Results: Lapatinib treatment of a panel of HER2+ breast cancer cell lines increased HER3 expression in the lapatinib-resistant cell line MDA-MB 453 but not the lapatinib-resistant cell-line HCC-1569. Evaluation of [ 68 Ga]HER3P1 uptake in mice implanted with the HER2+ breast cancer cell lines MDA-MB453 or HCC-1569 prior to and after treatment with lapatinib demonstrated a significant increase in MDA-MB453 tumors only, consistent with in vitro findings. The additional knockdown of HER3 increased therapeutic efficacy of lapatinib only in MDA-MB453 cells, but not in HCC-1569 cells., Conclusion: HER3 PET imaging can be used to visualize dynamic changes in HER3 expression that occur in HER2+ breast cancers with HER2 inhibitor treatment and identify those likely to benefit by the addition of combination HER3 and HER2 inhibition., (© 2021. World Molecular Imaging Society.)

HER2-positive breast cancer (BC) is a rapidly growing and aggressive BC subtype that predominantly affects younger women. Despite improvements in patient outcomes with anti-HER2 therapy, primary and/or acquired resistance remain a major clinical challenge. Here, we sought to use a quantitative systems pharmacological (QSP) approach to evaluate the efficacy of lapatinib (LAP), abemaciclib (ABE) and 5-fluorouracil (5-FU) mono- and combination therapies in JIMT-1 cells, a HER2+ BC cell line exhibiting intrinsic resistance to trastuzumab. Concentration-response relationships and temporal profiles of cellular viability were assessed upon exposure to single agents and their combinations. To quantify the nature and intensity of drug-drug interactions, pharmacodynamic cellular response models were generated, to characterize single agent and combination time course data. Temporal changes in cell-cycle phase distributions, intracellular protein signaling, and JIMT-1 cellular viability were quantified, and a systems-based protein signaling network model was developed, integrating  protein dynamics to drive the observed changes in cell viability. Global sensitivity analyses for each treatment arm were performed, to identify the most influential parameters governing cellular responses. Our QSP model was able to adequately characterize protein dynamic and cellular viability trends following single and combination drug exposure. Moreover, the model and subsequent sensitivity analyses suggest that the  activation of the stress pathway, through pJNK, has the greatest impact over the observed declines of JIMT-1 cell viability in vitro. These findings suggest that dual HER2 and CDK 4/6 inhibition may be a promising novel treatment strategy for refractory HER2+ BC, however, proof-of-concept in vivo studies are needed to further evaluate the combined use of these therapies.

Ablation of protein acyltransferase DHHC3 selectively enhanced the anti-cancer cell activities of several chemotherapeutic agents, but not kinase inhibitors. To understand why this occurs, we used comparative mass spectrometry-based palmitoyl-proteomic analysis of breast and prostate cancer cell lines, ± DHHC3 ablation, to obtain the first comprehensive lists of candidate protein substrates palmitoylated by DHHC3. Putative substrates included 22-28 antioxidant/redox-regulatory proteins, thus predicting that DHHC3 should have antioxidant functions. Consistent with this, DHHC3 ablation elevated oxidative stress. Furthermore, DHHC3 ablation, together with chemotherapeutic drug treatment, (a) elevated oxidative stress, with a greater than additive effect, and (b) enhanced the anti-growth effects of the chemotherapeutic agents. These results suggest that DHHC3 ablation enhances chemotherapeutic drug potency by disabling the antioxidant protections that contribute to drug resistance. Affirming this concept, DHHC3 ablation synergized with another anti-cancer drug, PARP inhibitor PJ-34, to decrease cell proliferation and increase oxidative stress. Hence, DHHC3 targeting can be a useful strategy for selectively enhancing potency of oxidative stress-inducing anti-cancer drugs. Also, comprehensive identification of DHHC3 substrates provides insight into other DHHC3 functions, relevant to in vivo tumor growth modulation.

MET is a receptor tyrosine kinase known to drive neoplastic transformation and aggressive tumor phenotypes. Crizotinib is an oral multi-targeted tyrosine kinase inhibitor of MET, ALK, RON, and ROS1 kinases. In this study, the anticancer effects of crizotinib on breast cancer cells were investigated in vitro along with the molecular mechanisms associated with these effects. Besides, the antiproliferative effects of crizotinib in combination with chemotherapy, hormonal drugs, and targeted agents were examined. Results showed that crizotinib produced dose-dependent antiproliferative effects in BT-474 and SK-BR-3 breast cancer cells with IC 50 values of 1.7 μM and 5.2 μM, respectively. Crizotinib inhibited colony formation of BT-474 cells at low micromolar concentrations (1-5 μM). Immunofluorescence and Western blotting indicated that crizotinib reduced total levels of MET and estrogen receptor (ERα) in BT-474 cells. Also, crizotinib reduced the levels of phosphorylated (active) MET and HER2 in BT-474 cells. The combined treatment of crizotinib with doxorubicin and paclitaxel resulted in synergistic growth inhibition of BT-474 cells with combination index values of 0.46 and 0.35, respectively. Synergy was also observed with the combination of crizotinib with the hormonal drugs 4-hydroxytamoxifen and fulvestrant in BT-474 cells. Alternatively, the combination of crizotinib with lapatinib produced antagonistic antiproliferative effects in both BT-474 and SK-BR-3 cells. Collectively, these findings demonstrate the anticancer effects of crizotinib in breast cancer cells and reveal ERα as a potential therapeutic target of the drug apart from its classical kinase inhibitory activity. Crizotinib could be an appealing option in combination with chemotherapy or hormonal drugs for the management of breast cancer.

Receptor tyrosine kinases (RTKs) play key roles in various aspects of cell biology, including cell-to-cell communication, proliferation and differentiation, survival, and tissue homeostasis, and have been implicated in various diseases including cancer and neurodevelopmental disorders. Ligand-activated RTKs recruit adapter proteins through a phosphotyrosine (p-Tyr) motif that is present on the RTK and a p-Tyr-binding domain, like the Src homology 2 (SH2) domain found in adapter proteins. Notably, numerous combinations of RTK/adapter combinations exist, making it challenging to compare receptor activities in standardised assays. In cell-based assays, a regulated adapter recruitment can be investigated using genetically encoded protein-protein interaction detection methods, such as the split TEV biosensor assay. Here, we applied the split TEV technique to robustly monitor the dynamic recruitment of both naturally occurring full-length adapters and artificial adapters, which are formed of clustered SH2 domains. The applicability of this approach was tested for RTKs from various subfamilies including the epidermal growth factor (ERBB) family, the insulin receptor (INSR) family, and the hepatocyte growth factor receptor (HGFR) family. Best signal-to-noise ratios of ligand-activated RTK receptor activation was obtained when clustered SH2 domains derived from GRB2 were used as adapters. The sensitivity and robustness of the RTK recruitment assays were validated in dose-dependent inhibition assays using the ERBB family-selective antagonists lapatinib and WZ4002. The RTK split TEV recruitment assays also qualify for high-throughput screening approaches, suggesting that the artificial adapter may be used as universal adapter in cell-based profiling assays within pharmacological intervention studies.