Chemotherapy of breast cancer cells alters susceptibility to complement-mediated opsonization and killing.

HER2-positive breast cancers represent approximately 20-25% of all breast cancers and are characterized by an overexpression of the growth factor receptor HER2. Trastuzumab, a monoclonal antibody, is a molecularly targeted therapeutic used in the treatment of this subtype of breast cancer. However, 30% of eligible patients have intrinsic resistance to trastuzumab and approximately 60% of patients who initially responded to this therapeutic, develop resistance within one year. Calcium transporters and modulators are known to be involved in breast cancer and in chemoresistance. However, their role has not been evaluated in HER2-positive trastuzumab resistant breast cancer cells. The aim of this project was to identify possible calcium related proteins associated with trastuzumab resistance. In the first part of this thesis, the expression of Ca2+ transporters and modulators and their role in trastuzumab activity was assessed in the HER2-positive breast cancer cell line SKBR3. Ca2+ signaling profiling was also assessed using fluorescence imaging plate reader (FLIPR) assays. Inhibition of the expression of the Ca2+ channels TPC2, TRPV1 and the Ca2+ channel modulator STIM1 using siRNA decreased SKBR3 cellular proliferation. Silencing of STIM1, the Ca2+ pump SPCA1 and the Ca2+ permeable ion channel TRPM7 increased the anti-proliferative effects of trastuzumab in SKBR3 cells. In the second part of this thesis, trastuzumab resistant and age-matched control cell lines were established from parental SKBR3 cells through seven months of continuous culturing in the presence of trastuzumab. Two trastuzumab treated colonies were selected for their resistance to trastuzumab (RT1 and RT2). Two other colonies were selected from age-matched controls because of their development of de novo resistance to trastuzumab (RV1 and RV2). Two age-matched cell lines that retained their sensitivity to trastuzumab were selected as controls (SV1 and SV2). Levels of mRNA expression of 45 Ca2+ channels, pumps and channel modulators were evaluated using quantitative RT-PCR. An siRNA screen of selected targets to identify targets that when silenced could restore trastuzumab sensitivity was also performed. Additionally Ca2+ signaling profiling and the quantitation of HER2, EGFR and IGF1R protein expression were conducted. All trastuzumab resistant cell lines maintained their overexpression of the HER2 receptor. Significantly increased mRNA levels of the voltage-gated calcium Ca2+ channel CaV3.2 was observed in both de novo resistant cell lines RV1 and RV2 compared to control cell lines SV1 and SV2. Acquired resistant cell lines RT1 and RT2 showed altered sensitivity to the purinergic receptor activator ATP, indicating a possible remodeling of Ca2+ signaling in these trastuzumab resistant cell lines. In the third part of this thesis, specific experiments were conducted to further evaluate two selected targets, the Ca2+ permeable ion channels CaV3.2 and TRPM7 channel. Pharmacological inhibition and silencing of CaV3.2 channel did not reverse trastuzumab resistance. However, CaV3.2 mRNA levels were higher in the basal HER2-positive trastuzumab resistant HCC1569 breast cancer cell line compared to the luminal HER2-positive trastuzumab sensitive SKBR3 cell line. Partial siRNA-mediated silencing of TRPM7 or pharmacological inhibition of TRPM7 channel activity did not reverse trastuzumab resistance in the trastuzumab resistant cell line RV1. However, the TRPM7 kinase inhibitor NH125 was able to promote trastuzumab activity in the trastuzumab resistant cell line RV1. Further studies are required to definitively associate TRPM7 kinase with trastuzumab resistance, given the reported sensitivity of other atypical α-kinases to NH125. In the last part of this thesis publically available data was mined to identify other potential calcium related proteins associated with trastuzumab resistance. These data sets included cDNA microarray analysis of trastuzumab resistant and sensitive SKBR3 cell lines and trastuzumab resistant breast cancer clinical samples and proteomic analysis of trastuzumab resistant and sensitive SKBR3 cell lines. These analyses indicated that the Ca2+ ATPase pump SERCA3 and galectin-3 may be associated with trastuzumab resistance. Results presented in this thesis suggest that the acquisition of trastuzumab resistance may be associated with the expression and/or activity of specific Ca2+ channels and pumps, including SERCA3, CaV3.2 channel and TRPM7 and the Ca2+-related protein galectin-3. Further studies of these proteins may help identify new approaches to reverse trastuzumab resistance and/or identify new biomarkers for predicting trastuzumab sensitivity in HER2-positive breast cancers.

632 Background: HER-2, a member of the transmembrane receptor tyrosine kinase ErbB family, is over-expressed in approximately 25% of BC. HER-2 targeted therapies, in particular, T, a monoclonal antibody targeting HER-2, and lapatinib (L), a reversible HER-2 tyrosine kinase inhibitor, have been shown to significantly improve the prognosis for HER-2 positive BC patients. However, resistance to T and/or L is a significant clinical problem. The aim of this study is to assess the activity of N (HKI-272), an irreversible HER-2 tyrosine kinase inhibitor, in HER-2 overexpressing BC cell lines, including T and/or L resistant cells. Methods: Using proliferation assays, the effect of N was assessed alone and in combination with T in HER-2 positive BC cell lines, including T and/or L resistant cell lines. The effect of N on HER-2 and downstream signalling molecules, Erk and Akt, was determined by immunoblotting. Results: HER-2 positive BC cell lines, including T and/or L resistant cells, are sensitive to N alone with IC50 values (concentration which inhibits 50% of growth) ranging from 1 to 280 nM. The combination of N and T has additive effects in SkBR3 and BT474 which are sensitive to T and also in SKBR3-Lwhich are resistant to L. In the cell lines HCC1954, HCC1954-L, MDA-MB-453, JIMT1 and SKBR3-HL which are resistant to T, combined treatment with T and N showed no enhancement compared to N alone. Finally, N decreased phosphorylation of HER-2, Erk and Akt in all cell lines tested. Conclusions: Our results suggest that N should be studied in patients with HER-2 positive BC, including patients with T and/or L resistant BC. We also demonstrate that N in combination with T may be more effective than either agent alone in T sensitive cells.

e11572 Background: Human epidermal growth factor receptor 2 (HER2) is a transmembrane kinase receptor, which promotes the RAS/RAF/MEK/ERK and the PI3K/AKT/mTOR pathways. The standard treatment for HER2-positive breast cancer is a combination of chemotherapy plus an anti-HER2 agent, such as trastuzumab and lapatinib. HER2-positive breast cancers are predominantly positive for CD24, which is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein. Overexpression of CD24 in breast cancer is associated with a poor prognosis. We hypothesize that the co-expression of HER2 with CD24 is associated with the therapeutic resistance of HER2-positive breast cancer cells. Methods: We established HER2-expressing cell lines by introducing a wild type HER2 gene into MDA-MB-231 triple-negative (ER-/PgR-/HER2-) breast cancer cells. We used the HER2-positive breast cancer cell lines BT-474, HCC202, SKBR3, and HCC1569. To investigate the relevance of CD24 expression in HER2-positive breast cancer, we analyzed HER2 and...

Breast cancer cell lines are widely used as an in vitro system with which to study the mechanisms underlying biological and chemotherapeutic resistance. In the present study, two novel breast cancer cell lines designated as PC-B-142CA and PC-B-148CA were successfully established from HER2-positive and triple-negative (TN) breast cancer tissues. The cell lines were characterized by cytokeratin (CK), α-smooth muscle actin (α-SMA), fibroblast-activation protein (FAP) and programmed death-ligand 1 (PD-L1). Cell proliferation was assessed using a colony formation assay, an MTS assay, 3-dimensional (3-D) spheroid and 3-D organoid models. Wound healing and Transwell migration assays were used to explore the cell migration capability. The responses to doxorubicin (DOX) and paclitaxel (PTX) were evaluated by 3-D spheroids. The results showed that the PC-B-142CA and PC-B-148CA cell lines were α-SMA-negative, FAP-negative, CK-positive and PD-L1-positive. Both cell lines were adherent with the ability of 3-D-multicellular spheroid and organoid formations; invadopodia were found in the spheroids/organoids of only PC-B-148CA. PC-B-142CA had a faster proliferative but lower metastatic rate compared to PC-B-148CA. Compared to MDA-MB-231, a commercial TN breast cancer cell line, PC-B-148CA had a similar CD44+/CD24− stemness property (96.90%), whereas only 8.75% were found in PC-B-142CA. The mutations of BRCA1/2, KIT, PIK3CA, SMAD4, and TP53 were found in PC-B-142CA cells related to the resistance of several drugs, whereas PC-B-148CA had mutated BRCA2, NRAS and TP53. In conclusion, PC-B-142CA can serve as a novel HER2-positive breast cancer cell line for drug resistance studies; while PC-B-148CA is a novel TN breast cancer cell line suitable for metastatic and stemness-related properties.

ABSTRACTBreast cancer is a common type of cancer among female cancer patients and the main cause of cancer-related deaths. During the last decades, targeted therapies for breast cancer have been rapidly developing. Among them, MLN4924, a first-in-class NEDD8-activating enzyme (NAE) inhibitor, has performed antitumor activity by inactivating the cullin-RING ligases and causing the accumulation of their substrates to induce apoptosis in a number of studies. In this study, we found that MLN4924 activates the AKT pathway in both HER2-positive and triple-negative breast cancer (TNBC) cell lines. Given that AKT signaling is responsible for tumor progression and drug resistance in some types of cancers, we hypothesized that the AKT inhibitor may synergistically enhance the tumor suppression capability in breast cancer by MLN4924. To demonstrate the sensitizing effect, MK-2206 was chosen as the adjuvant treatment, and cell growth, migration and apoptosis were detected. The results showed that MLN4924 treatment inhibited cell growth and migration and induced apoptosis in both SK-BR3 and MDA-MB231 breast cancer cell lines. More importantly, the combined treatment of MLN4924 and MK-2206 indeed caused stronger cytotoxicity and inhibition of migration and a much higher induction of apoptosis compared with MLN4924 treatment alone. Our study provides the proof-of-concept evidence for strategic drug combination of MLN4924 with an AKT inhibitor for maximal killing of breast cancer cells via the enhancement of apoptosis.

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in HER2-induced signaling and transformation, but its mechanism of action is poorly understood. Given the significance of HER2 in breast cancer, defining a mechanism for SHP2 in the HER2 signaling pathway is of paramount importance. In the current report we show that SHP2 positively modulates the Ras-extracellular signal-regulated kinase 1 and 2 and the phospoinositide-3-kinase-Akt pathways downstream of HER2 by increasing the half-life the activated form of Ras. This is accomplished by dephosphorylating an autophosphorylation site on HER2 that serves as a docking platform for the SH2 domains of the Ras GTPase-activating protein (RasGAP). The net effect is an increase in the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation. In conformity to these findings, the HER2 mutant that lacks the SHP2 target site exhibits an enhanced signaling and cell transformation potential. Therefore, SHP2 promotes HER2-induced signaling and transformation at least in part by dephosphorylating a negative regulatory autophosphorylation site. These results suggest that SHP2 might serve as a therapeutic target against breast cancer and other cancers characterized by HER2 overexpression.

Heregulin (HRG) is a soluble growth factor involved in the control of proliferation, survival, invasion, and differentiation of normal and malignant mammary epithelial cells. HRG binds its receptor, HER3, leading to activation of the HER2-HER3 heterodimer, which acts as an oncogenic factor in breast cancers. Pharmacological inhibitors to HER2 are currently being used in the clinic to treat HER2-positive breast cancer, however, they are associated with resistance after one year of therapy. A more comprehensive understanding of the signaling network downstream of HRG, in HER2-positive breast cancer cells, will be crucial for further development of inhibitors that do not elicit resistance. This study assessed HRG signaling in the HER2-positive breast cancer cell lines BT474 and SKBR3. The RSK (p90 ribosomal S6 Kinase) pathway was activated in an ERK-dependent manner. HRG stimulation increased phosphorylation of RSK at S380, T359, and S363. HRG stimulated the phosphorylation of CREB (cAMP Response-Element Binding Protein), an established substrate of RSK, at S133 and this was blocked by pharmacological inhibition of RSK. HRG induced the expression of c-FOS in an RSK-dependent manner. These data demonstrate that RSK has an important role in transducing HRG signaling to the gene expression program and suggest that inhibitors of RSK may be useful in the treatment of HER2-positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2163. doi:1538-7445.AM2012-2163

15 - 30 % of all breast cancer cases are classified as HER2/neu-positive, a very aggressive form of breast cancer that correlates with a poor prognosis, reduced survival time and increased incidence of metastasis. Kourtidis et al. (2010) have recently shown that the HER2-positive breast cancer cell line BT474 relies on a unique Warburg-like metabolism for survival and aggressive behavior. These cells are dependent on fatty acid synthesis, presumably as a means to regenerate NAD+ for aerobic glycolysis. The nuclear receptors NR1D1 and PPARγ enable these cells to upregulate de novo fatty acid synthesis while avoiding palmitate-induced lipotoxicity by storing neutral fats (TAGs). The cells show markedly increased levels of stored fats compared to other breast cancer cell lines and disruption of the synthetic process results in apoptosis of BT474 cells. Metabolomic analysis of NR1D1 overexpressing cells compared to normal cells reveals profound differences in their metabolite profiles. This metabolic phenotype is operating at its limits in HER2-positive cells, as addition of exogenous palmitate results in cell death, whereas HER2-normal cells are not affected by this treatment. Microarray analysis suggests that palmitate induces an ER-stress response in the HER2-positive cells, ultimately resulting in cell death. Preliminary results suggest that this response is CHOP-dependent. Combination treatments with palmitate and HER2-targeted therapeutic compounds show synergistic effects and markedly increase the cytotoxic effects of either compound alone. The uptake of exogenous fatty acids as well as the de novo fatty acid synthesis are dependent on the essential co-factor coenzyme A (CoA), which is generated from the precursor pantothenate (vitamin B5). Based on our genomic analysis we predicted that HER2-positive breast cancer cells are more sensitive to changes in intracellular CoA levels. Our results show that HER2-positive breast cancer cells are sensitive to shRNA-mediated knockdown of coenzyme A pathway enzymes whereas MCF-7 cells are not. We observe the same pattern of sensitivity using the competitive CoA pathway inhibitor homopantothenate. Interestingly, limiting the supply of CoA in HER2-positive cells reduces the toxicity of exogenous palmitate. Our data suggest that HER2-positive breast cancer cells are metabolically different from other breast cancer cell lines and that these differences offer new possibilities for therapeutic applications. Citation Format: Jan M. Baumann, Antonis Kourtidis, Douglas S. Conklin. The lipogenic phenotype of HER2/neu-positive breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1894. doi:10.1158/1538-7445.AM2013-1894

IntroductionTrastuzumab is widely used for the treatment of HER2-positive breast cancer. Despite encouraging clinical results, a significant fraction of patients are, or become, refractory to the drug. To overcome this, trastuzumab-DM1 (T-DM1), a newer, more potent drug has been introduced. We tested the efficacy and mechanisms of action of T-DM1 in nine HER2-positive breast cancer cell lines in vitro and in vivo. The nine cell lines studied included UACC-893, MDA-453 and JIMT-1, which are resistant to both trastuzumab and lapatinib.MethodsAlamarBlue cell-proliferation assay was used to determine the growth response of breast cancer cell lines to trastuzumab and T-DM1 in vitro. Trastuzumab- and T-DM1-mediated antibody-dependent cellular cytotoxicity (ADCC) was analysed by measuring the lactate dehydrogenase released from the cancer cells as a result of ADCC activity of peripheral blood mononuclear cells. Severe Combined Immunodeficient (SCID) mice were inoculated with trastuzumab-resistant JIMT-1 cells to investigate the tumour inhibitory effect of T-DM1 in vivo. The xenograft samples were investigated using histology and immunohistochemistry.ResultsT-DM1 was strongly growth inhibitory on all investigated HER2-positive breast cancer cell lines in vitro. T-DM1 also evoked antibody-dependent cellular cytotoxicity (ADCC) similar to that of trastuzumab. Outgrowth of JIMT-1 xenograft tumours in SCID mice was significantly inhibited by T-DM1. Histologically, the cellular response to T-DM1 consisted of apoptosis and mitotic catastrophe, the latter evidenced by an increased number of cells with aberrant mitotic figures and giant multinucleated cells.ConclusionsOur results suggest mitotic catastrophe as a previously undescribed mechanism of action of T-DM1. T-DM1 was found effective even on breast cancer cell lines with moderate HER2 expression levels and cross-resistance to trastuzumab and lapatinib (MDA-453 and JIMT-1).

The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130(Cas) (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain- and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.

Background: Breast tumour kinase (Brk/PTK6) is over-expressed in up to 86% of all breast cancers and has shown to be involved in the processes regulating tumour development and progression. A few Brk inhibitors are in development and our studies indicate potential for a Brk inhibitor to be used in combination with current breast cancer therapies. Furthermore the effect of common breast cancer therapies and Brk inhibition on the expression of ptk6 as well as it's alternatively spliced isoform (ALT-PTK6) have not been previously been shown. Using qPCR we have determined gene expression of both genes before and after treatment. Methods: Breast cancer cell lines T47D, GI101, BT474, SKBR3, MDA-MB 231, and MDA-MB 436 were treated with Brk inhibitor, Compound 4f (Mahmoud et al, 2012) and common breast cancer therapies (Taxol, Doxorubicin, Lapatinib and Tamoxifen) at concentrations ranging from 0µM to 10µM. Western blotting was carried out to determine levels of Brk and activation of Brk substrate; STAT3 after treatment with Brk inhibitor. RNA was extracted using Qiagen Mini RNeasy prep kit, cDNA synthesized using Invitrogen Superscript II Reverse Transcriptase and qPCR carried out using primers specific for PTK6 or primers that recognized both transcripts (PTK6 and ALT-PTK6) for total expression. Results: In all cell lines tested there was a moderate reduction in cell proliferation following treatment with the Brk inhibitor (4f). However a greater effect was observed in combination therapy. Triple negative breast cancer cell lines MDA-MB 231 and MDA-MB 436 were treated with Compound 4f and Taxol or Doxorubicin (n=3) resulting in modest but statistically significant reduction in cell numbers. Cell responses to Taxol in both cell lines were significantly greater in the presence of 4f over a range of doses (P between 0.05 and 0.007). Responses to Doxorubicin were also significantly improved in the presence of 4f (P<0.03 for MDA-MB-231 and P=0.03 for MDA-MB-436). Co-treatment of HER2 positive breast cancer cell lines BT474 and Sk-Br-3 with Lapatinib and 4f showed significant increase in responses over a range of doses between 0.31 and 5µM (n=3, P<0.05 for BT474 and P between 0.03 and 0.0004 for Sk-Br-3). In comparison to untreated cells, there was statistically significant reduction in ptk6 and Total gene expression observed at various time points within multiple breast cancer cell lines in response to Compound 4f treatment. Significant differences between untreated and treated cells for T47D cell line were at 8 hours post treatment (p=0.02), 2 and 4 hours post treatment in GI101 cell line (p=0.04 and p=0.02 respectively), 8 and 24 hours post treatment in Sk-Br-3 (p=0.001 and p=0.017 respectively) and 8 hours post treatment in MDA-MB 231 cell lines (p=0.03). Conclusion: Inhibition of Brk led to a decrease in ptk6 gene expression. Expression ratios of ptk6 and the short isoform ALT-PTK6 were determined and a reduction of ptk6 gene correlated with elevation of ALT-PTK6 and vice versa. Inhibition of Brk also indicated an increase in breast cancer cell sensitivity to current breast cancer therapies. Our studies thus indicate potential for inhibition of Brk kinase activity in combination with current breast cancer treatments. Citation Format: Hussain H, Harvey A. Potential of breast tumour kinase (Brk) as a therapeutic target: Brk modulates drug responses in breast cancer cell lines [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-05.

Breast cancer (BC) is the second most common cancer associated with brain metastasis (BM) and among patients with breast cancer brain metastasis (BCBM), human epidermal growth factor receptor 2 (HER2)-positive breast cancer is most likely to develop BM. The overall prognosis of HER2-positive BCBM remains dismal with median survival time after BM of less than a year. Drugs like T-DM1, T-DXd and Tucatinib are reported to deliver a progression-free survival benefit in HER2-positive BCBM patients, but there are still unmet needs for a drug that crosses the blood brain barrier more effectively. We develop VRN101396, a brain penetrant, orally available, and irreversible small molecule inhibitor targeting HER2 to offer a new therapeutic option for the treatment of HER2-positive BCBM. VRN101396 potently inhibits catalytic activity of HER2 but spares wild-type EGFR. In HER2-positive breast cancer cell lines BT474 and SK-BR3 and gastric cancer cell line NCI-N87, VRN101396 inhibited the cell proliferation and phosphorylation of HER2 with single- or double-digit nanomolar IC50 values while inhibiting wild type EGFR with an IC50 value of >100 nM. In both subcutaneous and intracranial efficacy mouse models, once-daily oral dose of VRN101396 significantly inhibited tumor growth and achieved tumor regression. In N87 subcutaneous xenograft model, VRN101396 alone showed superior efficacy to the combination of Tucatinib and Trastuzumab. Additionally, in intracranial xenograft model, VRN101396 showed superior efficacy to Tucatinib. Pharmacokinetics of VRN101396 displayed higher brain exposure than tucatinib and lapatinib, indicating better target engagement in the brain. Pharmacodynamic analysis revealed that VRN101396 reduced phosphorylation of HER2, HER3, AKT, and ERK compared to vehicle group after administration up to 24 hours. In conclusion, VRN101396 is a therapeutic candidate for the treatment of HER2-positive BC, BCBM and gastric cancer. Citation Format: Hong-ryul Jung, Sunghwan Kim, Jihye Yoo, Chan mi Park, Somi Lee, Hyoju Lee, Youngyi Lee, Jinhee Park, Kyungah Seo, Dong-Hyuk Seo, Eunhwa Ko, Jung Beom Son, Deakwon Kim, Hwan Geun Choi, Nam Doo Kim. VRN101396, a brain-permeable HER2 inhibitor, shows the anti-tumor activity in preclinical HER2-positive cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-15.

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK.S100A14,one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens,andthere is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding.Moreover,we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.

14689 Background: There is a potentially therapeutically relevant interaction between the HER2 and VEGF systems. In addition to targeting VEGFR, the multi-target kinase inhibitor (TKI) sunitinib malate targets c-Kit, PDGFR, RET, FLT-3 and CSF-1R. The aim of this study was to assess the direct anti-tumour effects of sunitinib, alone and in combination with trastuzumab in HER2 positive breast cancer. Methods: A panel of HER-2 positive and HER-2 negative BC cell lines were tested with sunitinib malate and compared with imatinib mesylate, which targets Bcr-Abl, PDGFR and c-Kit. IC50s were determined using the acid phosphatase assay. HER-2 positive cell lines were treated with a combination of sunitinib and trastuzumab. Results: IC50 values were similar in the HER-2 positive and negative cell lines for both imatinib and sunitinib. All of the cell lines showed greater sensitivity to sunitinib than to imatinib. Sunitinib was tested in combination with trastuzumab in two trastuzumab sensitive cell lines (SKBR3 and BT474) and two trastuzumab-resistant cell lines (JIMT-1 and HCC-1419). Combined treatment with sunitinib and trastuzumab showed improved response compared to either drug alone, in all of the HER-2 positive cell lines tested (Table). Conclusions: Our findings suggest that sunitinib alone shows modest anti-tumour activity against breast cancer cells but shows a positive interaction with trastuzumab in HER-2 positive BC cell lines in vitro. Together with the anit-angiogenic effects of sunitinib, this combination of targeted therapies may improve response to trastuzumab in HER2 positive breast cancer. Cell Line IC50s (+ std dev) % Growth (+ std dev) Imatinib (μM) Sunitinib (μM) Sunitinib (7.5 μΜ) Trastuzumab (7.5 nΜ) Sunitinib (7.5 μΜ) + trastuzumab (7.5 nΜ) SKBR-3 14.9+2.3 5.4+0.9 45.4+5.1 65.2+5.2 33.3+4.3 JIMT-1 23.4+4.1 7.3+1.9 60.9+11.3 99.5+1.9 50.4+10.9 BT-474 17.7+3.6 5.0+1.3 57.6+6.1 39.6+6.2 20.1+10.1 HCC-1419 32.3+3.7 8.4+2.9 90.9+16.3 86.1+5.6 74.5+7.3 Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Pfizer Oncology, Roche

Background: Targeting the human epidermal growth factor receptor (HER) family of tyrosine kinase receptors with small molecule TKIs lapatinib, neratinib and tucatinib, monoclonal antibodies like trastuzumab and more recently with ADCs like T-DM1 and T-DXd has resulted in improved survival rates for patients with HER2+ BC. TKIs are primarily utilised in trastuzumab-refractory disease. Neratinib, and the pan-HER TKI afatinib, bind irreversibly to all members of the HER family, while lapatinib binds reversibly to HER2 and EGFR, and tucatinib binds reversibly to HER2 alone. Previous work in our lab has shown that TKIs like lapatinib are capable of modulating tumour surface HER2 levels and increasing trastuzumab load on tumours. Thus, there is a rationale for the use of TKI/ADC combinations in HER2+ BC. Using innately trastuzumab-resistant HER2+ BC cell lines, this in vitro study aims to assess the anti-proliferative potential of HER2-targeting ADCs T-DM1 or T-DXd in combination with the TKIs afatinib, lapatinib, neratinib or tucatinib. Methods: HCC1569 and HCC1954 cells (HER2+, estrogen receptor (ER)-negative; innately trastuzumab-resistant) were grown in RPMI1640/10% FBS at 37°C and 5% CO2. The anti-proliferative effects of afatinib, lapatinib, neratinib and tucatinib; of T-DM1 and T-DXd; and of each TKI/ADC combination thereof were assessed in these cells via 5-day acid phosphatase. ADCs were obtained from Saint Vincent’s University Hospital, Dublin, and TKIs were purchased from commercial sources. Calcusyn software was used to generate IC50 values and combination index (CI) values at ED50. CI values > 1 represent an antagonistic combination, CI values = 1 are additive, and CI values < 1 are synergistic. All assays were carried out in triplicate. Results: The HCC1569 cell line was more sensitive to all four TKIs (IC50 values were 12.8 ± 0.3, 453.8 ± 47.1 nM, 4.7 ± 1.4 and 381.5 ± 37.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively) compared to the HCC1954 cell line (IC50 22.3 ± 2.8, 652.4 ± 36.0, 57.0 ± 6.3 and 2365.1 ± 185.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively). Despite their innate trastuzumab resistance, both cell lines displayed high sensitivity to T-DM1 (IC50 25.2 ± 8.7 ng/mL for HCC1569 and IC50 30.4 ± 3.2 ng/mL for HCC1954) and to T-DXd (IC50 16.0 ± 3.2 ng/mL for HCC1569 and IC50 36.8 ± 8.7 ng/mL for HCC1954). In both cell lines, co-treatment with ADCs and the irreversible TKIs afatinib and neratinib resulted in additive or synergistic responses, while the combination of the ADCs with the reversible TKIs lapatinib and tucatinib resulted in mild to moderate antagonism (Table 1). Conclusions: In this pre-clinical study, T-DM1 and T-DXd consistently exhibited antagonistic interactions with reversible HER2-targeting TKIs, and synergy/additivity in combination with the irreversible TKIs tested. Future work will explore the underlying mechanisms of this observed synergy and antagonism, including impacts of TKIs on HER2 expression and real-time ADC internalisation rates. Table 1. CI values +/- Std Dev for each TKI/ADC treatment combination in HCC1569 and HCC1954 cells Citation Format: Niall Ashfield, Amira F. Mahdi, Neil T. Conlon, John Crown, Denis M. Collins. Reversible versus irreversible tyrosine kinase inhibitors (TKIs) combined with antibody-drug conjugates (ADCs) in HER2-positive (HER2+) breast cancer (BC) cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-10.