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The combination of trastuzumab plus pertuzumab plus docetaxel as a first-line therapy in patients with HER2-positive metastatic breast cancer has provided significant clinical benefits compared to trastuzumab plus docetaxel alone. However, despite the therapeutic success of existing therapies targeting HER2, tumours invariably relapse. Therefore, there is an urgent need to improve our understanding of the mechanisms governing resistance, so that specific therapeutic strategies can be developed to provide improved efficacy. It is well known that the tumour microenvironment (TME) has a significant impact on cancer behaviour. Cancer-associated fibroblasts (CAFs) are essential components of the tumour stroma that have been linked to acquired therapeutic resistance and poor prognosis in breast cancer. For this reason, it would be of interest to identify novel biomarkers in the tumour stroma that could emerge as therapeutic targets for the modulation of resistant phenotypes. Conditioned medium experiments carried out in our laboratory with CAFs derived from HER2-positive patients showed a significant capacity to promote resistance to trastuzumab plus pertuzumab therapies in two HER2-positive breast cancer cell lines (BCCLs), even in the presence of docetaxel. In order to elucidate the components of the CAF-conditioned medium that may be relevant in the promotion of BCCL resistance, we implemented a multiomics strategy to identify cytokines, transcription factors, kinases and miRNAs in the secretome that have specific targets in cancer cells. The combination of cytokine arrays, label-free LC-MS/MS quantification and miRNA analysis to explore the secretome of CAFs under treatment conditions revealed several up- and downregulated candidates. We discuss the potential role of some of the most interesting candidates in generating resistance in HER2-positive breast cancer.

HER2-positive breast cancer is currently managed with chemotherapy in combination with specific anti-HER2 therapies, including trastuzumab. However, a high percentage of patients with HER2-positive tumors do not respond to trastuzumab (primary resistance) or either recur (acquired resistance), mostly due to molecular alterations in the tumor that are either unknown or undetermined in clinical practice. Those alterations may cause the tumor to be refractory to treatment with trastuzumab, promoting tumor proliferation and metastasis. Using continued exposure of a HER2-positive cell line to trastuzumab, we generated a model of acquired resistance characterized by increased expression of several cytokines. Differential gene expression analysis indicated an overexpression of 15 genes, including five different chemokines, and highlighting CCL5/RANTES as the most overexpressed one. Functional studies, either by in vitro gene silencing or by in vitro and in vivo pharmacologic inhibition of the CCL5/CCR5 interaction with maraviroc, confirmed that CCL5 overexpression was implicated in acquired resistance to trastuzumab, which was mediated by ERK activation. In patient samples, increased CCL5 expression significantly correlated with lower rates of complete response after neoadjuvant therapy, confirmed by detection of high serum CCL5 levels by ELISA. Overexpression of CCL5 correlated with ERK phosphorylation in tumor cells and was statistically associated with worse disease-free survival and overall cancer survival in patients with early HER2-positive breast cancer., (©2020 American Association for Cancer Research.)

Purpose: Despite the therapeutic success of existing HER2-targeted therapies, tumors invariably relapse. This study aimed at identifying new mechanisms responsible for HER2-targeted therapy resistance., Experimental Design: We have used a platform of HER2-targeted therapy-resistant cell lines and primary cultures of healthy and tumor-associated fibroblasts (TAF) to identify new potential targets related to tumor escape from anti-HER2 therapies., Results: We have shown that TAFs promote resistance to HER2-targeted therapies. TAFs produce and secrete high levels of FGF5, which induces FGFR2 activation in the surrounding breast cancer cells. FGFR2 transactivates HER2 via c-Src, leading to resistance to HER2-targeted therapies. In vivo , coinoculating nonresistant cell lines with TAFs results in more aggressive and resistant tumors. Resistant cells activate fibroblasts and secrete FGFR ligands, creating a positive feedback loop that fuels resistance. FGFR2 inhibition not only inhibits HER2 activation, but also induces apoptosis in cells resistant to HER2-targeted therapies. In vivo , inhibitors of FGFR2 reverse resistance and resensitize resistant cells to HER2-targeted therapies. In HER2 patients' samples, α-SMA, FGF5, and FGFR2 contribute to poor outcome and correlate with c-Src activation. Importantly, expression of FGF5 and phospho-HER2 correlated with a reduced pathologic complete response rate in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab, which highlights the significant role of TAFs/FGF5 in HER2 breast cancer progression and resistance., Conclusions: We have identified the TAF/FGF5/FGFR2/c-Src/HER2 axis as an escape pathway responsible for HER2-targeted therapy resistance in breast cancer, which can be reversed by FGFR inhibitors., (©2019 American Association for Cancer Research.)

Purpose: We investigated the value of tumor-infiltrating NK (TI-NK) cells and HLA class I tumor expression as biomarkers of response to neoadjuvant anti-HER2 antibody-based treatment in breast cancer., Experimental Design: TI-NK cells and HLA-I were determined by IHC in pretreatment tumor biopsies from two cohorts of patients with HER2-positive breast cancer [discovery cohort ( n = 42) and validation cohort ( n = 71)]. Tumor-infiltrating lymphocytes (TIL) were scored according to international guidelines. Biomarker association with pathologic complete response (pCR) and disease-free survival (DFS) was adjusted for prognostic factors. Gene set variation analysis was used for determining immune cell populations concomitant to NK-cell enrichment in HER2-positive tumors from the Cancer Genome Atlas ( n = 190)., Results: TI-NK cells were significantly associated with pCR in the discovery cohort as well as in the validation cohort ( P < 0.0001), independently of clinicopathologic factors. A ≥3 TI-NK cells/50x high-power field (HPF) cutoff predicted pCR in the discovery and validation cohort [OR, 188 (11-3154); OR, 19.5 (5.3-71.8)]. Presence of TI-NK cells associated with prolonged DFS in both patient cohorts [HR, 0.07 (0.01-0.6); P = 0.01; HR, 0.3 (0.08-1.3); P = 0.1]. NK-, activated dendritic- and CD8 T-cell gene expression signatures positively correlated in HER2-positive tumors, supporting the value of NK cells as surrogates of effective antitumor immunity. Stratification of patients by tumor HLA-I expression identified patients with low and high relapse risk independently of pCR., Conclusions: This study identifies baseline TI-NK cells as an independent biomarker with great predictive value for pCR to anti-HER2 antibody-based treatment and points to the complementary value of tumor HLA-I status for defining patient prognosis independently of pCR., (©2018 American Association for Cancer Research.)

Background: Overexpression and amplification of the human epidermal growth factor receptor 2 (HER2) occur in 20% of total breast carcinomas. HER2-overexpression is implicated in disease initiation and progression and associated with poor prognosis. Trastuzumab, a humanized monoclonal antibody, is the standard HER2-targeted therapy for early and metastatic HER2-amplified breast cancer patients. Trastuzumab has significantly increased clinical benefit in HER2+ metastatic and adjuvant settings; however, it is not effective for many patients due to primary or acquired resistance to the drug. During the last decade, many studies have revealed a number of novel molecular traits of HER2+ breast cancer, allowing us to uncover the molecular mechanisms involved in trastuzumab resistance and develop strategies to overcome resistance to therapy., Objective: In this review, we comprehensively addressed the current achievements in preclinical studies; we discussed molecular mechanisms of acquired trastuzumab resistance in HER2+ breast cancer models and potential therapeutic approaches based on the molecular features for HER2+ breast cancer., Conclusion: Enhanced understanding of the molecular profiles in HER2+ breast cancer may lead to the identification of novel biomarkers for the development of diagnostic approaches and improvement of therapeutic targets for the prevention and treatment of trastuzumab resistant HER2+ breast cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance. Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2 -positive breast cancer explants. Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation. Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006-19. ©2017 AACR ., (©2017 American Association for Cancer Research.)

A subset of HER2+ breast cancer patients manifest clinical resistance to trastuzumab. Recently, miR-26a and miR-30b have been identified as trastuzumab response regulators, and their target gene CCNE2 seems to play an important role in resistance to trastuzumab therapy. Cell viability was evaluated in trastuzumab treated HER2+ BT474 wt (sensitive), BT474r (acquired resistance), HCC1954 (innate resistance), and MDA-MB-231 (HER2-) cell lines, and the expression of miR-26a, miR-30b, and their target genes was measured. BT474 wt cell viability decreased by 60% and miR-26a and miR-30b were significantly overexpressed (~3-fold, p = 0.003 and p = 0.002, respectively) after trastuzumab treatment, but no differences were observed in resistant and control cell lines. Overexpression of miR-30b sensitized BT474r cells to trastuzumab (p = 0.01) and CCNE2, was significantly overexpressed after trastuzumab treatment in BT474r cells (p = 0.032), but no significant changes were observed in sensitive cell line. When CCNE2 was silenced BT474r cell sensitivity to trastuzumab increased (p = 0.03). Thus, the molecular mechanism of trastuzumab action in BT474 cell line may be regulated by miR-26a and miR-30b and CCNE2 overexpression might play an important role in acquired trastuzumab resistance in HER2+ breast cancer given that resistance was diminished when CCNE2 was silenced.

Aim: Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies., Methods: We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells., Results: In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis., Conclusion: Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The HER family of transmembrane tyrosine kinase receptors is composed of four members, BER1 to HER4. HER2 is a ligand-orphan receptor expressed in many human tumors and overexpressed in 25-30% of breast cancers. HER2 amplifies the signal provided by other receptors of the HER family by forming heterodimers. The essential role of HER2 in the HER signaling network led to the development of anti-HER2 monoclonal antibodies (MAbs) for cancer therapy. In particular, the humanized MAb trastuzumab (Herceptin) has antitumor activity against HER2-overexpressing human breast tumor cells and is widely used for the treatment of women with HER2 overexpressing breast cancers. Trastuzumab induces HER2 receptor downmodulation and, as a result, inhibits critical signalling pathways (i.e. ras-Raf-MAPK and PI3K/Akt) and blocks cell cycle progression by inducing the formation of p27/Cdk2 complexes. Trastuzumab also inhibits HER2 cleavage, preceding antibody-induced receptor downmodulation, and this effect might contribute to its antitumor activity in some cancers. In vivo, trastuzumab inhibits angiogenesis and induces antibody-dependent cellular cytotoxicity. A limitation of trastuzumab is that its activity is largely restricted to breast cancers with the highest level of HER2 overexpression or HER2 gene amplification. However, there is a large population of breast cancers and of many other tumors that have low or moderate HER2 expression. In such tumors, HER2 functions as a preferred coreceptor to form heterodimers with HER1 (EGFR), HER3 or HER4. For this reason, a humanized monoclonal antibody, called 2C4, that targets the role of HER2 as a coreceptor is under active development. 2C4 binds to a different epitope of HER2 ectodomain than trastuzumab and sterically hinders HER2 recruitment in heterodimers with other HER receptors. This results in the inhibition of signalling by HER2-based heterodimers both in cells with low and high HER2 expression. In vitro and in vivo antitumor activity has been reported in a range of breast and prostate tumor models. Therefore, 2C4 may have potential against a wide variety of solid tumors. Phase I trials are underway.