Beatriz Morancho

Effect of p95HER2/611CTF on the Response to Trastuzumab and Chemotherapy

Human epidermal growth factor receptor 2 (HER2)-positive breast cancers are currently treated with trastuzumab, an anti-HER2 antibody. About 30% of these tumors express a group of HER2 fragments collectively known as p95HER2. Our previous work indicated that p95HER2-positive tumors are resistant to trastuzumab monotherapy. However, recent results showed that tumors expressing the most active of these fragments, p95HER2/611CTF, respond to trastuzumab plus chemotherapy. To clarify this discrepancy, we analyzed the response to chemotherapy of cell lines transfected with p95HER2/611CTF and patient-derived xenografts (n = 7 mice per group) with different levels of the fragment. All statistical tests were two-sided. p95HER2/611CTF-negative and positive tumors showed different responses to various chemotherapeutic agents, which are particularly effective on p95HER2/611CTF-positive cells. Furthermore, chemotherapy sensitizes p95HER2/611CTF-positive patient-derived xenograft tumors to trastuzumab (mean tumor volume, trastuzumab alone: 906 mm(3), 95% confidence interval = 1274 to 538 mm(3); trastuzumab+doxorubicin: 259 mm(3), 95% confidence interval = 387 to 131 mm(3); P < .001). This sensitization may be related to HER2 stabilization induced by chemotherapy in p95HER2/611CTF-positive cells.

Effect of Cellular Senescence on the Growth of HER2-Positive Breast Cancers

BACKGROUND Oncogene-induced senescence (OIS) is a tumor suppressor mechanism. However, senescent cells remain viable and display a distinct secretome (also known as senescence-associated secretory phenotype [SASP] or senescence messaging secretome, [SMS]) that, paradoxically, includes protumorigenic factors. OIS can be triggered by ectopic overexpression of HER2, a receptor tyrosine kinase and the driving oncogene in a subtype of human breast cancer. However, cellular senescence has not been characterized in HER2-positive tumors. METHODS Using an approach based on their inability to proliferate, we isolated naturally occurring senescent cells from a variety of tumor models including HER2-positive cells, transgenic mice (n = 3), and patient-derived xenografts (PDXs) (n = 6 mice per group from one PDX derived from one patient). Using different biochemical and cell biological techniques, we characterized the secretome of these senescent cells. All statistical tests were two-sided. RESULTS We found that senescent cells arise constantly in different models of advanced breast cancers overexpressing HER2 and constitute approximately 5% of tumor cells. In these models, IL-6 and other cytokines were expressed mainly, if not exclusively, by the naturally occurring senescent cells (95.1% and 45.0% of HCC1954 cells and cells from a HER2-positive PDX expressing a senescent marker expressed IL-6, respectively). Furthermore, inhibition of IL-6 impaired the growth of the HER2-positive PDX (mean tumor volume at day 101, control vs anti-huIL-6 treated, 332.2mm(3) [95% confidence interval {CI} = 216.6 to 449.8] vs 114.4mm(3) [95% CI = 12.79 to 216.0], P = .005). CONCLUSIONS Senescent cells can contribute to the growth of tumors by providing cytokines not expressed by proliferating cells, but required by these to thrive.

Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity in HER2 overexpressing breast cancer

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.

A dominant-negative N-terminal fragment of HER2 frequently expressed in breast cancers

The transmembrane tyrosine kinase HER2 (ErbB2, neu) is a prototypical biomarker for breast cancers and a therapeutic target. Although anti-HER2 therapies are remarkably effective, HER2-positive tumors are heterogeneous and some subtypes do not respond or develop resistance to these therapies. Here we show that H2NTF, a novel N-terminal fragment of HER2, is expressed at variable levels in 60% of the breast cancer samples analyzed. Characterization of H2NTF shows that it is devoid of the tyrosine kinase domain but it readily interacts with full-length HER2 and other HER receptors. As a consequence, H2NTF acts as a dominant-negative, attenuating the signaling triggered by full-length HER receptors. Expression of H2NTF results in resistance to the treatment with low concentrations of trastuzumab in vitro. However, cells expressing H2NTF and non-expressing cells have similar sensitivity to trastuzumab in vivo, indicating that H2NTF/trastuzumab complexes trigger antibody-dependent cell-mediated cytotoxicity.

Patterns of HER2 Gene Amplification and Response to Anti-HER2 Therapies

A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK), is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs), which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.

RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer

Abstract Background BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and methods We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. Conclusion Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence

IntroductionCellular senescence is a terminal cell proliferation arrest that can be triggered by oncogenes. One of the traits of oncogene-induced senescence (OIS) is the so-called senescence-associated secretory phenotype or senescence secretome. Depending on the context, the non-cell autonomous effects of OIS may vary from tumor suppression to promotion of metastasis. Despite being such a physiological and pathologically relevant effector, the mechanisms of generation of the senescence secretome are largely unknown.MethodsWe analyzed by label-free proteomics the secretome of p95HER2-induced senescent cells and compared the levels of the membrane-anchored proteins with their transcript levels. Then, protein and RNA levels of ADAM17 were evaluated by using Western blot and reverse transcription-polymerase chain reaction, its localization by using biotin labeling and immunofluorescence, and its activity by using alkaline phosphatase-tagged substrates. The p95HER2-expressing cell lines, senescent MCF7 and proliferating MCF10A, were analyzed to study ADAM17 regulation. Finally, we knocked down ADAM17 to determine its contribution to the senescence-associated secretome. The effect of this secretome was evaluated in migration assays in vitro and in nude mice by assessing the metastatic ability of orthotopically co-injected non-senescent cells.ResultsUsing breast cancer cells expressing p95HER2, a constitutively active fragment of the proto-oncogene HER2 that induces OIS, we show that the extracellular domains of a variety of membrane-bound proteins form part of the senescence secretome. We determine that these proteins are regulated transcriptionally and, in addition, that their shedding is limited by the protease ADAM17. The activity of the sheddase is constrained, at least in part, by the accumulation of cellular cholesterol. The blockade of ADAM17 abrogates several prometastatic effects of the p95HER2-induced senescence secretome, both in vitro and in vivo.ConclusionsConsidering these findings, we conclude that ectodomain shedding is tightly regulated in oncogene-induced senescent cells by integrating transcription of the shedding substrates with limiting ADAM17 activity. The remaining activity of ADAM17 contributes to the non-cell autonomous protumorigenic effects of p95HER2-induced senescent cells. Because ADAM17 is druggable, these results represent an approximation to the pharmacological regulation of the senescence secretome.

Modeling anti-IL-6 therapy using breast cancer patient-derived xenografts

The pleiotropic cytokine IL-6 accelerates the progression of breast cancer in a variety of preclinical models through the activation of the STAT3 (signal transducer and activator of transcription 3) signaling pathway. However, the proportion of breast cancers sensitive to anti-IL-6 therapies is not known. This study evaluates the efficacy of anti-IL-6 therapies using breast cancer patient derived xenografts (PDXs). During the generation of our collection of PDXs, we showed that the successful engraftment of tumor tissue in immunodeficient mice correlates with bad prognosis. Four PDXs out of six were resistant to anti-IL-6 therapies and the expression of IL-6, its receptor or the levels of phospho-STAT3 (the active form of the signal transducer) did not correlate with sensitivity. Using cell cultures established from the PDXs as well as samples from in vivo treatments, we showed that only tumors in which the activation of STAT3 depends on IL-6 respond to the blocking antibodies. Our results indicate that only a fraction of breast tumors are responsive to anti-IL-6 therapies. In order to identify responsive tumors, a functional assay to determine the dependence of STAT3 activation on IL-6 should be performed.

PTBP1-Mediated Alternative Splicing Regulates the Inflammatory Secretome and the Pro-tumorigenic Effects of Senescent Cells

Summary Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer.

Abstract P4-07-05: PARP inhibition in breast and ovarian patient-derived tumor xenografts (PDX) harboring germline BRCA1/2 mutations unveils mechanisms of primary and acquired resistance that restore homologous recombination (HR)

Background: PARP1/2 inhibitors (PARPi) are active anti-cancer agents in BRCA1 or BRCA2 mutation carriers (BRCA) with advanced breast or ovarian cancer. However, not all BRCA-tumors respond to PARP blockade, and eventually all develop acquired resistance. Little is known about clinically relevant mechanisms of PARPi resistance in BRCA-breast cancer. Here, we sought to identify biomarkers correlating with primary and acquired resistance to PARPi using PDX derived from both the early disease and the metastatic setting. Methods: We have developed a panel of PDX from patients harboring germline BRCA1 or BRCA2 mutations, namely from 12 primary and advanced breast cancer and 1 high-grade serous metastatic ovarian cancer (HGSOC). The antitumor activity of the PARP1/2 inhibitor olaparib as single agent (50 mg/kg) was assessed in all models. To study the mechanisms of acquired resistance, the olaparib-sensitive PDXs were exposed to olaparib for >100 days, until individual tumors regrew. The tumor9s capacity to repair DNA double strand breaks was estimated by quantification of the BRCA1 and RAD51 nuclear foci in the S-phase of the cell cycle. We investigated the correlation between the tumor9s BRCA1/RAD51 foci formation and sensitivity to olaparib, and also identified potential genetic modifiers of PARPi sensitivity by targeted sequencing. Results: Four out of 13 PDX (31%) treated with single agent olaparib exhibited tumor regression or disease stabilization. Nuclear BRCA1/RAD51 foci formation correlated with PARPi resistance in six BRCA1 PDX models investigated, either with primary or acquired resistance. No reversions in BRCA1/2 mutations were identified as the mechanism of olaparib resistance. We identified four potential genetic modifiers of PARPi sensitivity and the corresponding validating studies will be presented. Conclusions: Among our BRCA PDX, reactivation of HR functionality is a frequent event that is associated with PARPi resistance and seems to occur through mechanisms other than secondary mutations in BRCA1/2 in contrast to what it has been reported for HGSOC. Citation Format: Cruz C, Bustos Ld, Gris A, Palafox M, Castroviejo M, Llop A, Morancho B, Diez O, Gutierrez S, Caratu G, Prudkin L, Bruna A, Caldas C, O9Connor MJ, Rubio IT, Arribas J, Baselga J, Cortes J, Serra V, Balmana J. PARP inhibition in breast and ovarian patient-derived tumor xenografts (PDX) harboring germline BRCA1/2 mutations unveils mechanisms of primary and acquired resistance that restore homologous recombination (HR). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-07-05.