Resistance to CDK4/6 inhibition: Mechanisms and strategies to overcome a therapeutic problem in the treatment of hormone receptor-positive metastatic breast cancer

Selective CDK4/6 inhibitors, such as palbociclib, ribociclib, and abemaciclib, have been approved in combination with hormone therapy for the treatment of patients with HR+, HER2-negative advanced or metastatic breast cancer (mBC). Despite their promising activity, approximately 10 % of patients have de novo resistance, while the rest of them will develop acquired resistance after 24–28 months when used as first-line therapy and after a shorter period when used as second-line therapy. Various mechanisms of resistance to CDK4/6 inhibitors have been described, including cell cycle-related mechanisms, such as RB loss, p16 amplification, CDK6 or CDK4 amplification, and cyclin E-CDK2 amplification. Other bypass mechanisms involve the activation of FGFR or PI3K/AKT/mTOR pathways. Identifying the different mechanisms by which resistance to CDK4/6 inhibitors occurs may help to design new treatment strategies to improve patient outcomes. This review presents the currently available knowledge on the mechanisms of resistance to CDK4/6 inhibitors, explores possible treatment strategies that could overcome this therapeutic problem, and summarizes relevant recent clinical trials.     

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2? breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Ų), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpK_a = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse K_p,uu = 0.20–0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.     

Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor‐positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib‐induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib‐induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence‐like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse‐free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.     

Enhanced dermatologic toxicity following concurrent treatment with palbociclib and radiation therapy: A case report

BackgroundCyclin-dependent kinase (CDK) 4/6 inhibitors represent a new class of targeted therapy options for the treatment of estrogen receptor-positive (ER+) human epidermal growth factor 2-negative (HER2-) metastatic breast cancer. There are currently no published prospective data on the safety of use of radiation treatment with palbociclib.CaseWe describe the case of a patient with metastatic breast cancer who received radiation treatment to a metastatic supraclavicular lymph node to planned 60 Gy in 30 fractions while on palbociclib, a selective inhibitor of CDK4/6. The patient developed early radiation toxicities including esophagitis and dermatitis that progressed to a severe left neck skin breakdown in the radiation field, resulting in the need for hospitalization. She had a break in treatment but was able to finish the radiation without palbociclib. Her tumor responded well to the treatment and her side effects healed.DiscussionTo our knowledge this is the first case to report on concurrent palbociclib and radiation use, with resultant enhanced radiation effects that required hospitalization for symptom management. Several preclinical studies have shown synergistic effects of radiation and both in vivo and in vitro experiments resulting in improved survival and decreased cell proliferation, respectively, through enhanced G1 cell cycle arrest.ConclusionThis case highlights the importance of using caution when combining radiation with the new targeted therapies. Until more data becomes available, physicians are recommended to exercise clinical judgment when deciding on whether to continue or discontinue a CDK4/6 inhibitor in a patient who may need radiation.     

Efficacy of a combination therapy targeting CDK4/6 and autophagy in a mouse xenograft model of t(8;21) acute myeloid leukemia

One of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML) is t(8;21). Although patients with t(8;21) AML have a more favorable prognosis than other cytogenetic subgroups, relapse is still common and novel therapeutic approaches are needed. A recent study showed that t(8;21) AML is characterized by CCND2 deregulation and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells. In this study, we examined the in vivo effects of co-inhibiting CDK4/6 and autophagy. We used a mouse model in which t(8;21)-positive Kasumi-1 cells were subcutaneously inoculated into NOD/Shi-scid IL2Rg^null mice. The mice were treated with the autophagy inhibitor chloroquine (CQ), a CDK4/6 inhibitor (either abemaciclib or palbociclib), or a CDK4/6 inhibitor plus CQ. After 20 days of treatment, tumor volume was measured, and immunostaining and transmission electron microscopy observations were performed. There was no change in tumor growth in CQ-treated mice. However, mice treated with a CDK4/6 inhibitor plus CQ had significantly less tumor growth than mice treated with a CDK4/6 inhibitor alone. CDK4/6 inhibitor treatment increased the formation of autophagosomes. The number of single-strand DNA-positive (apoptotic) cells was significantly higher in the tumors of mice treated with a CDK4/6 inhibitor plus CQ than in mice treated with either CQ or a CDK4/6 inhibitor. These results show that CDK4/6 inhibition induces autophagy, and that co-inhibition of CDK4/6 and autophagy induces apoptosis in t(8;21) AML cells in vivo. The results suggest that inhibiting CDK4/6 and autophagy could be a novel and promising therapeutic strategy in t(8;21) AML.     

抗乳腺癌新药 palbociclib

Palbociclib 是 2015 年 2 月 3 日获美国食品药品监督管理局加速批准的乳腺癌新药,联合来曲唑作为以内分泌治疗为基础的初 始方案,用于治疗绝经期女性雌激素受体阳性（ER+）、人表皮生长因子受体 2 阴性（HER2-）的绝经期女性晚期乳腺癌。Palbociclib 是首个口服、靶向性 CDK4/6 抑制剂,阻止细胞周期从生长期（G1 期）到 DNA 复制期（S1 期）的转变,从而抑制肿瘤增殖,其上市为 晚期乳腺癌患者提供了新的治疗选择。介绍 palbociclib 的化学合成、临床前药理学研究、临床研究及专利保护情况等 , 为抗乳腺癌新药 研发提供参考。     

RB-E2F信号通路与乳腺癌及其治疗

视网膜母细胞瘤基因（retinoblastoma gene, RB1）突变或调节CDK-RB-E2F通路其他成分的突变存在于几乎所有人类恶性肿瘤中。因此,通过抑制细胞周期蛋白激酶（CDK）来实现对细胞周期的调控,在肿瘤治疗中越来越显示出其优势。目前,CDK4/6抑制剂帕博西尼（palbociclib）联合芳香酶抑制剂,治疗ER 阳性乳腺癌是很有效的临床应用。研究显示,CDK-RB-E2F信号通路,对控制乳腺细胞增殖发挥关键作用。近期的研究结果,揭示了该通路在肿瘤发展、血管生成及转移中的作用。并且,E2Fs是不依赖于其他临床参数的乳腺癌预后指标。本综述总结了乳腺癌中RB E2F通路的最新研究进展,并且讨论应用高通量基因组学研究,筛选获得乳腺癌中CDK4/6抑制剂重要的作用靶点,旨在发展更有效的联合治疗手段。     

Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.   In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.     

Risk assessment of disease recurrence in early breast cancer: A serum metabolomic study focused on elderly patients

BackgroundWe previously showed that metabolomics predicts relapse in early breast cancer (eBC) patients, unselected by age. This study aims to identify a “metabolic signature” that differentiates eBC from advanced breast cancer (aBC) patients, and to investigate its potential prognostic role in an elderly population.MethodsSerum samples from elderly breast cancer (BC) patients enrolled in 3 onco-geriatric trials, were retrospectively analyzed via proton nuclear magnetic resonance (1H NMR) spectroscopy. Three nuclear magnetic resonance (NMR) spectra were acquired for each serum sample: NOESY1D, CPMG, Diffusion-edited. Random Forest (RF) models to predict BC relapse were built on NMR spectra, and resulting RF risk scores were evaluated by Kaplan–Meier curves.ResultsSerum samples from 140 eBC patients and 27 aBC were retrieved. In the eBC cohort, median age was 76 years; 77% of patients had luminal, 10% HER2-positive and 13% triple negative (TN) BC. Forty-two percent of patients had tumors >2 cm, 43% had positive axillary nodes. Using NOESY1D spectra, the RF classifier discriminated free-from-recurrence eBC from aBC with sensitivity, specificity and accuracy of 81%, 67% and 70% respectively. We tested the NOESY1D spectra of each eBC patient on the RF models already calculated. We found that patients classified as "high risk" had higher risk of disease recurrence (hazard ratio (HR) 3.42, 95% confidence interval (CI) 1.58–7.37) than patients at low-risk.ConclusionsThis analysis suggests that a “metabolic signature”, identified employing NMR fingerprinting, is able to predict the risk of disease recurrence in elderly patients with eBC independently from standard clinicopathological features.     

Palbociclib can overcome mutations in cyclin dependent kinase 6 that break hydrogen bonds between the drug and the protein

Inhibition of cyclin dependent kinases (CDKs) 4 and 6 prevent cells from entering the synthesis phase of the cell cycle. CDK4 and 6 are therefore important drug targets in various cancers. The selective CDK4/6 inhibitor palbociclib is approved for the treatment of breast cancer and has shown activity in a cellular model of mixed lineage leukaemia (MLL)‐rearranged acute myeloid leukaemia (AML). We studied the interactions of palbociclib and CDK6 using molecular dynamics simulations. Analysis of the simulations suggested several interactions that stabilized the drug in its binding site and that were not observed in the crystal structure of the protein‐drug complex. These included a hydrogen bond to His 100 that was hitherto not reported and several hydrophobic contacts. Evolutionary‐based bioinformatic analysis was used to suggest two mutants, D163G and H100L that would potentially yield drug resistance, as they lead to loss of important protein–drug interactions without hindering the viability of the protein. One of the mutants involved a change in the glycine of the well‐conserved DFG motif of the kinase. Interestingly, CDK6‐dependent human AML cells stably expressing either mutant retained sensitivity to palbociclib, indicating that the protein‐drug interactions are not affected by these. Furthermore, the cells were proliferative in the absence of palbociclib, indicating that the Asp to Gly mutation in the DFG motif did not interfere with the catalytic activity of the protein.     

p63 suppresses the ability of pregnancy-identified mammary epithelial cells (PIMECs) to drive HER2-positive breast cancer

While pregnancy is known to reduce a woman’s life-long risk of breast cancer, clinical data suggest that it can specifically promote HER2 (human EGF receptor 2)-positive breast cancer subtype (HER2+ BC). HER2+ BC, characterized by amplification of HER2, comprises about 20% of all sporadic breast cancers and is more aggressive than hormone receptor-positive breast cancer (the majority of cases). Consistently with human data, pregnancy strongly promotes HER2+ BC in genetic mouse models. One proposed mechanism of this is post-pregnancy accumulation of PIMECs (pregnancy-identified mammary epithelial cells), tumor-initiating cells for HER2+ BC in mice. We previously showed that p63, a homologue of the tumor suppressor p53, is required to maintain the post-pregnancy number of PIMECs and thereby promotes HER2+ BC. Here we set to test whether p63 also affects the intrinsic tumorigenic properties of PIMECs. To this end, we FACS-sorted YFP-labeled PIMECs from p63+/−;ErbB2 and control p63+/+;ErbB2 females and injected their equal amounts into immunodeficient recipients. To our surprise, p63+/− PIMECs showed increased, rather than decreased, tumorigenic capacity in vivo, i.e., significantly accelerated tumor onset and tumor growth, as well as increased self-renewal in mammosphere assays and proliferation in vitro and in vivo. The underlying mechanism of these phenotypes seems to be a specific reduction of the tumor suppressor TAp63 isoform in p63+/− luminal cells, including PIMECs, with concomitant aberrant upregulation of the oncogenic ΔNp63 isoform, as determined by qRT-PCR and scRNA-seq analyses. In addition, scRNA-seq revealed upregulation of several cancer-associated (Il-4/Il-13, Hsf1/HSP), oncogenic (TGFβ, NGF, FGF, MAPK) and self-renewal (Wnt, Notch) pathways in p63+/−;ErbB2 luminal cells and PIMECs per se. Altogether, these data reveal a complex role of p63 in PIMECs and pregnancy-associated HER2+ BC: maintaining the amount of PIMECs while suppressing their intrinsic tumorigenic capacity.Subject terms: Breast cancer, Mechanisms of disease     

Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; K _i<0.5 nM; cellular IC₅₀ 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser₁₀ levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser₁₀). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G₁ cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.     

Palbociclib (PD 0332991) Interaction with Kinases. Theoretical and Comparative Molecular Docking Study

The optimized geometry of palbociclib, (PD 0332991) (8-cyclopentyl-6-ethanoyl-5-methyl-2-(5-(piperazin-1-yl)pyridin-2-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one), electrostatic potential map, molecular orbitals were calculated using the density functional theory. The geometry was used in a molecular docking study of palbociclib-kinase complexes, results could be explained by the charge of the nitrogen and oxygen atoms within the palbociclib. Energy gap of HOMO-LUMO surfaces, could help to explain the reactivity of the ligand and the hydrogen bonding with three different kinases, two of CDK6 and one of CDK4 type. Docking results are similar and complementary with literature reports using molecular dynamics, were hydrogen bonding was obtained and analyzed. The promiscuity of three kinases with palbociclib was detected by the docking results, thus, palbociclib could be used in other types of cancer besides myeloid leukemia. Some similarities are found with CDK4/CDK6 kinases which allow us to determine that palbociclib could be used to control other resistant inhibitor types of cancer.     

Outcomes following stereotactic radiosurgery or whole brain radiation therapy by molecular subtype of metastatic breast cancer

BackgroundThis study quantified clinical outcomes by molecular subtype of metastatic breast cancer (BC) following whole brain radiation therapy (WBRT) or stereotactic radiosurgery (SRS). Doing so is important for patient counseling and to assess the potential benefit of combining targeted therapy and brain radiotherapy for certain molecular subtypes in ongoing trials.Materials and methodsThe National Cancer Database was queried for BC (invasive ductal carcinoma) cases receiving brain radiotherapy (divided into WBRT and SRS ). Statistics included multivariable logistic regression to determine factors associated with SRS delivery, Kaplan-Meier analysis to evaluate overall survival (OS), and Cox proportional hazards modeling.ResultsOf 1,112 patients, 186 (16.7%) received SRS and 926 (83.3%) underwent WBRT. Altogether, 410 (36.9%), 195 (17.5%), 162 (14.6%), and 345 (31.0%) were ER+/HER2−, ER+/HER2+, ER−/HER2+, and ER−/HER2−, respectively. In the respective molecular subtypes, the proportion of subjects who underwent SRS was 13.4%, 19.4%, 24.1%, and 15.7%. Respective OS for WBRT patients were 12.9, 22.8, 10.6, and 5.8 months; corresponding figures for the SRS cohort were 28.3, 40.7, 15.0, and 12.9 months (p < 0.05 for both). When comparing OS between treatment different histologic subtypes, patients with ER−/HER2+ and ER−/HER2− disease had worse OS than patients with ER+/HER2− disease, for both patients treated with SRS and for patients treated with WBRT.ConclusionsMolecular subtype may be a useful prognostic marker to quantify survival following SRS/WBRT for metastatic BC. Patients with HER 2-enriched and triple-negative disease had the poorest survival following brain irradiation, lending credence to ongoing studies testing the addition of targeted therapies for these subtypes.     

Implementation of a combined CDK inhibition and arginine-deprivation approach to target arginine-auxotrophic glioblastoma multiforme cells

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and β-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.Subject terms: CNS cancer, DNA damage and repair, Cell death

The complex effects of combined CDki/SPyADI application on arginine-auxotrophic glioblastoma multiforme cells. CDki/SPyADI combination therapy impairs cell proliferation, invasiveness, gene expression, induces mitochondrial impairment, and vacuole formation. Abemaciclib-SPyADI-treatment suppresses DNA repair, dinaciclib-SpyADI-treatment enhances γ-H2AX accumulation and activates the stress sensor GADD45 and β-catenin antagonist AXIN2. Both CDKi/SpyADI combinations significantly boost cell death.     

Metaplastic breast cancer: Treatment and prognosis by molecular subtype

BackgroundMetaplastic breast cancer (MBC) is a rare and aggressive subtype of breast. However, the effect of molecular subtype on treatment and prognosis of MBC remains unclear.Patients and methodsThe Surveillance, Epidemiology, and End Results database was used to analyze patients with MBC between 2010 and 2016. Molecular subtype was stratified to TN group (ER and PR-/HER2-), HER2 group (ER and PR-/HER2+, ER/PR+ and HER2+), and HR group (ER/PR+ and HER2-). The breast cancer-specific survival (BCSS) differences were estimated using multivariate Cox regression model and Kaplan-Meier curves.ResultsWe included 1665 patients with median follow-up time of 27 months (range 0–83 months). 1154 (69.3%), 65 (3.9%), and 446 (26.8%) patients presented in TN group, HER2 group, and HR group, respectively. On multivariate Cox analysis, the prognosis was related to age, tumor size, regional node metastasis, and surgery. Molecular subtype remained no impact on BCSS. Radiotherapy (RT) was associated with better prognosis. Patients cannot benefit from chemotherapy. In Kaplan-Meier curve, triple-negative (P = 0.047) and HR-positive (P = 0.006) patients receiving RT had a superior BCSS than that not RT. HER2-positive patients cannot benefit from RT. However, adjusted Kaplan-Meier survival model showed that triple-negative (P = 0.019) but not HER2-positive (P = 0.575) or HR-positive (P = 0.574) patients receiving RT had a superior BCSS than that not RT.ConclusionsMolecular subtype is not associated with the better prognosis of MBC. Patients could benefit from RT. However, triple-negative but not HR-positive or HER2-positive patients have superior survival after receiving RT.     

Loss of lkb1 Expression Reduces the Latency of ErbB2-Mediated Mammary Gland Tumorigenesis,Promoting Changes in Metabolic Pathways

The tumor suppressor kinase LKB1 is mutated in a broad range of cancers however, the role of LKB1 mammary gland tumorigenesis is not fully understood. Evaluation of human breast cancer tissue microarrays, indicate that 31% of HER2 positive samples lacked LKB1 expression. To expand on these observations, we crossed STK11^fl/fl mice with mice genetically engineered to express activated Neu/HER2-MMTV-Cre (NIC) under the endogenous Erbb2 promoter, to generate STK11^−/−/NIC mice. In these mice, the loss of lkb1 expression reduced the latency of ErbB2-mediated tumorigenesis compared to the latency of tumorigenesis in NIC mice alone. Analysis of STK11^−/−/NIC mammary tumors revealed hyperactivation of mammalian target of rapamycin (mTOR) through both mTORC1 and mTORC2 pathways as determined by the phosphorylation status of ribosomal protein S6 and AKT. Furthermore, STK11^−/−/NIC mammary tumors had elevated ATP levels along with changes in metabolic enzymes and metabolites. The treatment of primary mammary tumor cells with specific mTOR inhibitors AZD8055 and Torin1, that target both mTOR complexes, attenuated mTOR activity and decreased expression of glycolytic enzymes. Our findings underscore the existence of a molecular interplay between LKB1-AMPK-mTORC1 and ErbB2-AKT-mTORC2 pathways with mTOR at its epicenter, suggestive that loss of LKB1 expression may serve as a marker for hyperactivated mTOR in HER2 positive breast cancer and warranting further investigation into therapeutics that target LKB1-AMPK-mTOR and glycolytic pathways.     

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼15–20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2−) breast cancer tissues, whereas both HER2+ and HER2− tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser⁶⁷⁴ by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.