Introduction: Breast cancer is a heterogeneous disease consisting of uncontrolled growth of epithelial cells originating in the ducts or breast lobules. It is currently the most diagnosed cancer worldwide and the fifth cause of cancer-related death in general population, but the first cause in women. Breast cancer incidence is higher in developed countries due to increased risk factors and to increased and earlier detection by mammography screening, which considerably reduces mortality. This is the reason why transitioning countries have higher mortality despite having lower incidence. Regarding the origin of the disease, it is evidenced that the pathways implicated in normal development of the mammary gland are deregulated in breast cancer disease. Nevertheless, despite several models have been proposed, the specific mechanism by which breast cancer is originated and how its formation and progression occur is still unknown. Between the risk factors, genetic risk factors are responsible of around 10% of breast cancers, being BRCA1 and BRCA2 the most common inherited mutations. Non-genetic risk factors are related to ethnicity, breast conditions or previous breast pathologies and lifestyle factors, such as hormonal therapy, alcohol consumption, overweight, lack of physical activity and late age at giving birth or not breastfeeding. Diagnosis in the very initial steps of the disease is one of the major issues in the clinic and enables efficacy of the therapies and reduces the risk of breast cancer death. For this issue, mammography screening has been demonstrated to be the best strategy and is implemented in most of the developed countries. Mammography together with clinical examination, imaging and needle biopsy are performed for diagnosis. After diagnosis, breast tumors should be classified according to TNM stage, mainly based of tumor size, lymph node involvement and metastasis; histological grading based on differentiation; histological subtypes that include ductal invasive carcinoma, lobular invasive carcinoma and other special subtypes, and pathological or intrinsic subtypes. Currently pathological subtype, which is based on expression of estrogen and progesterone receptors, HER2 and Ki67 by immunohistochemistry, determines treatment decision. Molecular intrinsic subtyping is based on gene expression in tumor tissue and classifies breast cancer into six subtypes. This classification has demonstrated potential value to predict prognosis and treatment response. However, it is not fully applicable to the clinic nowadays. Regarding treatment, locoregional therapy of early breast cancer consists of a surgery and radiation therapy if necessary. For systemic treatment, neoadjuvant or adjuvant strategies could be performed with endocrine therapy in luminal tumors, HER2 blockade in HER2+ breast cancer and chemotherapy, that could be used in all subtypes but is the standard of care for triple negative breast cancer. HER2+ breast cancer presents overexpression of HER2 receptor which, upon dimerization, leads to activation of downstream pathways involved in proliferation, angiogenesis, survival, differentiation, apoptosis, invasion and metastasis. Between them, RAS/RAF, MAPK, JAK/STAT and PI3K/AKT/mTOR are the most studied. HER2+ breast cancer accounts for 15-20% of the cases and is characterized by an aggressive biological and clinical behaviour which correlates with poor prognosis. However, development of targeted therapies against HER2 has considerably improved HER2+ breast cancer patients’ outcome and changed the paradigm of clinical management. Based on this fact, a variety of anti-HER2 agents have been approved for the treatment of HER2+ breast cancer and others are under evaluation. The antibody trastuzumab was the first anti-HER2 agent to be developed and approved and, in combination with pertuzumab, remains the standard of care for HER2+ breast cancer. Despite the evident improvement of HER2+ breast cancer disease management and the effectiveness of anti-HER2 therapies, still around 20% of HER2+ breast cancer patients experience relapse leading to metastatic disease, that nowadays is considered incurable. Disease relapse may occur due to resistance to anti-HER2 agents. Thus, the main challenge in the last years has been to decipher the mechanisms underlying anti-HER2 resistance, to identify biomarkers to predict treatment response and to develop new therapeutic strategies to overcome resistance. Four major mechanisms have been described to be involved in anti-HER2 therapy resistance: impediments of drug binding to HER2 due to upregulation of mucines or HER2 isoforms; failure to trigger immune response because of Fc receptor dysfunctions; activation PI3K/AKT/mTOR downstream signaling pathway due to PIK3CA mutations, loss of PTEN or AKT deregulation; and upregulation of alternative pathways such as cell cycle, SRC or FASN. Crosstalk with other receptors has also been described to occur and to activate alternative pathways. Between them, crosstalk with hormone receptors, HER family members and other receptors tyrosine kinase have been involved in anti-HER2 resistance. Epithelial to mesenchymal process is implicated in metastasis, invasion, migration and colonization and has been related to drug resistance, including anti-HER2 resistance. Some receptors tyrosine kinase, such as AXL and MET, have been described to be involved in this process and subsequently take part in metastasis and mechanisms of therapy resistance. AXL can be activated by ligand-dependent or independent mechanism and by dimerization, leading to activation of downstream signaling pathways, such as PI3K/AKT/mTOR, JAK/STAT, NF-κB, and RAS/RAF/MEK/ERK that play major roles in tumor cell survival, migration, invasion, anoikis, angiogenesis and drug resistance. AXL is overexpressed in several types of cancer including breast cancer and correlates with worse prognosis. In breast cancer, it was shown to be a driver for metastasis and to be essential for epithelial to mesenchymal process, besides, AXL has been proposed as a therapeutic target in triple negative breast cancer. Regarding drug resistance, associations between AXL and chemotherapy or targeted drugs resistance have been reported in several cancers. In the particular case of breast cancer, AXL is involved in chemo- and radio-resistance and EGFR-targeted therapy resistance in triple negative breast cancer. However, the role of AXL in anti-HER2 therapy resistance has been poorly investigated. Several AXL-targeted therapies have been shown to supress tumor growth and metastasis and to potentiate the effect of chemotherapy and targeted therapies and some of them are currently under clinical evaluation in solid tumors. Given the knowledge of AXL's role in resistance to therapy, future studies will help to determine the translational application of AXL as a biomarker of treatment response and as a therapeutic target in HER2+ breast cancer. Objectives: In the frame of the antecedents described above, we hypothesize that AXL could be involved in acquisition of trastuzumab resistance in HER2+ breast cancer maybe due to crosstalk with HER2 receptor. Thus, the present work is focused on the study of the role of AXL in acquisition of trastuzumab resistance, as well as its applicability as a therapeutic target and prognostic biomarker. Methods: In this study AU565, BT474 and SKBR3 HER2+ breast cancer cell lines were selected for in vitro experiments and acquired trastuzumab-resistant cell lines (AU565R, BT474R and SKBR3R) were generated by exposure to increasing concentrations of the drug for prolonged time. Innate trastuzumab-resistant HCC1954 HER2+ and MDA-MB-231 triple negative breast cancer cell lines were used as controls. Tumor tissue samples were obtained from patients with early-stage HER2+ breast cancer treated at the Hospital Clínico Universitario de Valencia by standard guidelines, after signature of informed consent and ethical approval of the study. Data from neoadjuvant phase II PAMELA study were used to analyze gene expression in this cohort of HER2+ breast cancer patients. To evaluate changes in gene expression, mRNA levels were measured by real time quantitative PCR on RNA isolated from breast cancer cells and patients’ samples. For protein expression analysis, western blot, immunohistochemistry and flow cytometry were performed and co-immunoprecipitation and proximity ligation assay were used to evaluate dimerization between AXL and HER2 proteins. Apoptosis and viability were analyzed with Annexin V and propidium iodide staining by flow cytometry. To study the effect of the loss and gain of function of specific proteins, small-interfering RNAs or cDNA overexpression plasmids were transfected into the cells by lipofectamine reagent and several functional assays were carried out. Proliferation was measured by WST-1 assay and migration and invasion were studied by wound healing and transwell assays. Mammospheres were generated from BT474R cells and number of live cells was measured by flow cytometry with propidium iodide. Patient-derived xenograft model was obtained from a HER2+ breast cancer patient. Two in vitro trastuzumab-resistant patient-derived xenograft cell lines were established by subculture in presence of increasing concentrations of trastuzumab and five trastuzumab-resistant in vivo patient-derived xenografts were generated by transplantation of tumor tissue into NOD-SCID mice treated with trastuzumab and grown with serial passages. 3D models were also generated from patient-derived xenograft cell lines and in vivo models in which viability and apoptosis were measured after treatment. For the in vivo experiment, NOD-SCID mice were injected in mammary fat pad with trastuzumab-resistant patient-derived xenograft tumors (N=30), randomly divided into 4 groups and treated with either vehicle, trastuzumab, TP-0903 or a combination of both. Tumor size and body weight were measured during follow-up and animals were sacrificed at the end of the experiment or when institutional euthanasia criteria for tumor size and health condition were reached. In-silico analysis were performed for gene expression studies in public databases and for transcription factor binding site prediction. The prognostic value of AXL and GAS6 mRNA expression was analyzed in 252 HER2+ breast cancer patients with available data from Kaplan-Meier Plotter Software. To identify predicted putative binding sites of JUNB in AXL promoter JASPAR 2022 and PROMO software were used. Statistical analysis was performed using GraphPad Prism and R software. Results were expressed as means ± SD, means ± SEM for in vivo tumor size and median and interquartile for mRNA expression in patients’ samples. Normality was checked using Shapiro-Wilks’s test. Mean comparison was carried out using two-tailed t-Student test. All experiments were reproduced at least three times. mRNA expression in patients’ cohort was dichotomized by the median and ROC analysis was carried out. Disease-free survival and overall survival were described graphically using Kaplan-Meier curves and differences were evaluated using log-rank test. Selection of the best model was done according to Akaike’s Information Criterion. To identify AXL gene expression changes in PAMELA trial we used two-tailed paired t-tests. Two-way ANOVA with Bonferroni correction post-hoc was used for in vivo statistical analysis. The cut-off for statistical significance in all tests was p-value <0.05. Results: To study the mechanisms of acquired resistance to trastuzumab in our models, we first characterized the three generated trastuzumab-resistant cell lines. We checked response to trastuzumab and, as expected, treatment reduced proliferation in resistant cells by less than 20% in comparison with approximately 50% of reduction observed in parental trastuzumab-sensitive cell lines, thus confirming the acquisition of resistance in these in vitro models. Given that the standard of care for HER2+ breast cancer consists of trastuzumab plus pertuzumab, we next evaluated the effect of pertuzumab in our models and observed that those cell lines with resistance to trastuzumab, were also resistant to pertuzumab and combination of both drugs, thus giving higher clinical value to our study. In addition, the acquired trastuzumab-resistant cell lines were significantly more proliferative at basal level than parental cell lines. No substantial changes were found between resistant and parental cell lines in ER, PR and HER2 by immunohistochemistry. However, despite still HER2-amplified, ERBB2 mRNA and protein expression were decreased in resistant versus parental cells. Given that AXL has been previously associated with resistance to therapies, we compared AXL expression between trastuzumab-sensitive and resistant cell lines, finding that both AXL mRNA and protein expression, together with AXL phosphorylation, were upregulated in resistant cell lines compared to their respective parental cell lines, suggesting its role in trastuzumab resistance. To address this question, AXL knockdown and pharmacological inhibition by TP-0903 were carried out and cells were treated with trastuzumab. Our data demonstrated that both strategies were able to resensitize trastuzumab-resistant cells, reaching IC50 values similar to those observed in parental cell lines. AXL downregulation or inhibition had no effect on trastuzumab response in sensitive or innate resistant HER2+ breast cancer cell lines. Conversely, these results were confirmed by AXL gain of function in parental cell lines, which significantly reduced growth rate inhibition by trastuzumab, thus leading to trastuzumab resistance. Basal proliferation rates were not affected by AXL gain and loss of function. These results suggest that AXL promotes acquired resistance to trastuzumab, and that AXL inhibition restores trastuzumab sensitivity in acquired resistant in vitro models. Given that the mesenchymal phenotype is related to worse response to trastuzumab and that AXL has been described as a component of the acquired mesenchymal phenotype, we next analyzed the association between AXL expression and epithelial to mesenchymal transition. First, we observed that trastuzumab-resistant cell lines showed a mesenchymal-like phenotype by upregulation mesenchymal markers and downregulation of epithelial marker, besides, they presented increased migration and invasion ability compared to parental cells. We next demonstrate that AXL gain and loss of function was sufficient to modulate a mesenchymal-like phenotype. To determine the biological mechanism underlying the resistance to trastuzumab mediated by AXL overexpression, we checked expression of its ligand GAS6 in our models, are no overexpression was observed in acquired trastuzumab-resistant cells at mRNA or protein level, nor in percentage of GAS6+ cells, indicating a ligand-independent activation of AXL. On the basis of these data, we hypothesized heterodimerization between AXL and HER2 as the trigger in our trastuzumab-resistant models. Co-immunoprecipitation and proximity ligation assays confirmed the presence of HER2-AXL dimer in our resistant cell lines. We next studied signaling on HER2-AXL downstream pathways and observed hyperphosphorylation in our resistant models compared to sensitive parental cell lines. Furthermore, genetic knockdown or pharmacological inhibition of AXL plus trastuzumab achieved the greatest inactivation of HER2 downstream pathways by reducing phosphorylation in both PI3K/AKT and MAPK/ERK pathways, even in acquired trastuzumab-resistant cells. Conversely, pathway inhibition by trastuzumab was considerably less effective in AXL overexpressed cell lines than in control cell lines. These data indicate that AXL-HER2 dimerization activates PI3K/AKT and MAPK/ERK signaling pathways, thus leading to reduced trastuzumab treatment efficacy. In order to obtain more translational results, we tested the combination of trastuzumab and AXL inhibition in different in vitro and in vivo acquired trastuzumab-resistant patient-derived xenograft models from a HER2+ breast cancer patient. Assays performed in the two acquired trastuzumab-resistant patient-derived xenograft cell lines (PDX118-TR1 and TR2) confirmed results from previous cell lines. PDX118-TR1 and TR2 cell lines were resistant to trastuzumab, pertuzumab and combination of pertuzumab plus trastuzumab and overexpressed AXL together with VIM. The in vitro combination of TP-0903 plus trastuzumab resensitized PDX118-TR1 and TR2 cells and significantly decreased trastuzumab IC50 in 2D and 3D models, while this strategy did not increase trastuzumab response in parental cell line. In accordance with the results from HER2+ breast cancer cell lines, AXL inhibition plus trastuzumab significantly inhibited phosphorylation of PI3K/AKT and MAPK/ERK pathways in patient-derived xenograft trastuzumab-resistant cell lines. Next, we analyzed AXL expression in five different in vivo acquired trastuzumab-resistant patient-derived xenograft models and observed AXL overexpression in all of them compared to parental tumor. In addition, AXL correlated with VIM expression in the selected resistant model (PDX118-TR4). First, AXL inhibition plus trastuzumab treatment strategy was tested in organoids from PDX118-TR4: trastuzumab or TP-0903 alone did not significantly reduce cell numbers, whereas combination decreased cell numbers to 38.2% compared to control. In a PDX118-TR4 in vivo experiment, mice were treated with vehicle, trastuzumab, TP-0903 or a combination of these two drugs. Results showed that trastuzumab and the AXL inhibitor alone had no significant effect on tumor growth; however, the two drugs in combination abrogated tumor growth and achieved a complete regression after 21 days of treatment with no evidence of tumor growth after therapy during a total of 321 days follow-up; likewise, treatment did not affect animal weight, showing that AXL inhibition did not lead to a significant toxic effect in preclinical models. Histopathological analysis in tumor tissue at endpoint confirmed on-target effect of AXL inhibitor by decreased phosphorylation of AXL in the tumor of TP-0903 treated animals. In accordance with tumor size, no differences in Ki67 nor c-CASP3 were found between control and trastuzumab. However, despite tumor size did not achieve statistical difference between control and TP-0903 treated groups, percentage of Ki67+ cells was significantly decreased while percentage of c-CASP3+ cells was increased in AXL inhibitor group. With these results, we conclude that the combination of TP-0903 with trastuzumab is a promising therapeutic strategy to treat HER2+ breast cancer resistant to standard care. On the basis of these preclinical data, we hypothesized that upregulation of AXL might promote acquired resistance to trastuzumab treatment in HER2+ breast cancer patients. To test this hypothesis, we analyzed AXL expression in a retrospective cohort of 50 HER2+ breast cancer patients. AXL expression was significantly upregulated in primary tumor tissue from patients who later experienced relapse and ROC analysis revealed that AXL expression may robustly discriminate between HER2+ breast cancer patients who will relapse from those who will be free of disease. Moreover, AXL significantly correlated to shorter disease-free survival and overall survival. The prognostic value of AXL was also confirmed in-silico in an independent public early HER2+ breast cancer patients’ cohort. Interestingly, we found a strong positive correlation between VIM and AXL expression and, indeed, VIM expression was associated with worse prognosis. Furthermore, there was no significant correlation between GAS6 levels and disease-free survival or overall survival. In the multivariable model, AXL was selected the best model for both disease-free survival and overall survival. Collectively, these findings reveal a ligand-independent association between high AXL expression and worse prognosis in patients with HER2+ breast cancer treated with trastuzumab and taxanes plus anthracycline-based chemotherapy. To identify early molecular changes induced by dual HER2 blockade in HER2+ breast cancer patients, gene expression profiling was performed in tumor samples from phase II PAMELA clinical trial. Of particular interest was that AXL significantly increased at day 14 of treatment compared to baseline. Interestingly, in agreement with previous gene expression analysis in the PAMELA trial, we found a rebound effect in AXL expression between day 14 and surgery samples, potentially due to discontinuation of dual HER2 inhibition which reverses its biological effects. According to our previous preclinical data, VIM expression presented similar biological changes to AXL, whereas ERBB2 expression decreased after dual HER2 blockade. These observations were particularly significant as the biological changes observed in our preclinical models recapitulated the biological changes observed in patients from a phase II clinical trial. The increase in AXL mRNA expression upon trastuzumab resistance indicates for enhanced transcription activity. Thus, to better understand the mechanism underlying trastuzumab resistance through AXL, we aimed to investigate potential transcription factors regulating AXL. Results from in-silico analysis, JUNB overexpression in acquired trastuzumab-resistant cell lines and patient-derived xenograft models and gain and loss of function experiments indicated that JUNB transcription factor might be regulating AXL as a mechanism of trastuzumab resistance. Discussion: Trastuzumab is an essential and effective anticancer targeted agent for HER2+ breast cancer patients. Unfortunately, despite the efficacy of this anti-HER2 treatment, a significant number of patients develop progressive disease, requiring additional therapeutic strategies. Given the urgent need to develop new therapeutic approaches for this group of patients, the current study was conducted to investigate the potential role of AXL activation in acquired resistance to trastuzumab with the aim of exploring AXL inhibition as a new potential therapeutic strategy for HER2+ breast cancer patients and evaluating the performance of AXL as a prognostic biomarker. Based on the obtained results, we demonstrated that acquired trastuzumab-resistant HER2+ breast cancer cells have significantly higher AXL expression than their parental counterparts and that AXL upregulation occurs in the context of a mesenchymal-associated transcriptional program, which is consistent with previous studies. Moreover, we demonstrated AXL implication on trastuzumab resistance and, interestingly, AXL inhibition by TP-0903 was able to restore response to anti-HER2 therapy in vitro. After exploring the mechanism leading to trastuzumab resistance through AXL, results provided evidence that AXL activation arises through AXL-HER2 heterodimerization followed by the trigger of PI3K and MAPK cascades, which is consistent with previous studies showing that AXL can drive oncogenic signaling of these pathways in other cancer types. Simultaneous AXL inhibition and trastuzumab treatment achieved a significantly great inhibition of these pathways in all cell lines, including resistant. Besides, we demonstrated that AXL activation in our models occurs in a ligand-independent manner. Moreover, we were able to confirm these results in trastuzumab-resistant patient-derived xenograft models. Trastuzumab-resistant patient-derived xenograft cell lines and tumors showed overexpression of AXL and its pharmacological inhibition restored sensitivity to trastuzumab in 2D and 3D in vitro models. Importantly, we reported that simultaneous therapeutical ablation of AXL and HER2 activity in vivo results in complete regression of trastuzumab-resistant tumors with no evidence of tumor regrowth nor symptoms of toxicity after follow-up. These data support the potential of AXL inhibitors in the treatment of cancer previously reported in other studies and suggest that the combination of HER2 and AXL inhibition might avoid treatment escape by tumor cells with acquired trastuzumab resistance, leading to a potential long-term benefit for HER2+ BC patients. Analysis of AXL expression in HER2+ breast cancer patients’ samples evidenced its potential as a prognostic biomarker. Besides, rapid changes in AXL expression during HER2 blockade observed in PAMELA trial suggest the importance of AXL overexpression in development of anti-HER2 resistance and the consequent high risk of metastases. The increased in AXL mRNA upon trastuzumab resistance indicates increased transcription activity. Previous works demonstrated AP-1 transcription factor family as a main regulator of AXL, and specifically, JUNB has been related to drug resistant in other types of cancer. Here, observed overexpression of JUNB in trastuzumab-resistant models and demonstrate that JUNB regulates AXL, thus suggesting the role of JUNB in anti-HER2 resistance though AXL regulation. This work has shed the light on how HER2+ breast cancer cells acquire resistance to trastuzumab through AXL activation. However, future work directed at better understanding AXL activation in HER2+ breast cancer will extend our knowledge of its contribution of acquisition to anti-HER2 resistance. Future studies regarding JUNB involvement in trastuzumab resistance, role of AXL in tumor microenvironment in the context of HER2+ breast cancer and trastuzumab resistance and validation of AXL as a biomarker in a prospective clinical trial would be of special interest. Conclusions: In conclusion, this study has demonstrated the substantial biological significance of AXL activation in acquired trastuzumab resistance in HER2+ breast cancer. Our findings demonstrate AXL-HER2 heterodimerization that induces resistance through PI3K and MAPK pathways activation. Importantly, the combination of trastuzumab with AXL inhibition overcomes resistance to trastuzumab both in vitro and in vivo. This dual inhibition induced complete tumor regression in in vivo trastuzumab-resistant patient-derived xenograft models without tumor regrowth after follow-up. The strong independent correlation of AXL expression with reduced disease-free survival and overall survival in HER2+ breast cancer patients suggests that patients with high AXL expression could benefit from AXL-targeted therapy.