izhong Hu

Upregulation of ER signaling as an adaptive mechanism of cell survival in HER2-positive breast tumors treated with anti-HER2 therapy

Purpose: To investigate the direct effect and therapeutic consequences of epidermal growth factor receptor 2 (HER2)-targeting therapy on expression of estrogen receptor (ER) and Bcl2 in preclinical models and clinical tumor samples. Experimental design: Archived xenograft tumors from two preclinical models (UACC812 and MCF7/HER2-18) treated with ER and HER2-targeting therapies and also HER2+ clinical breast cancer specimens collected in a lapatinib neoadjuvant trial (baseline and week 2 posttreatment) were used. Expression levels of ER and Bcl2 were evaluated by immunohistochemistry and Western blot analysis. The effects of Bcl2 and ER inhibition, by ABT-737 and fulvestrant, respectively, were tested in parental versus lapatinib-resistant UACC812 cells in vitro. Results: Expression of ER and Bcl2 was significantly increased in xenograft tumors with acquired resistance to anti-HER2 therapy compared with untreated tumors in both preclinical models (UACC812: ER P = 0.0014; Bcl2 P < 0.001 and MCF7/HER2-18: ER P = 0.0007; Bcl2 P = 0.0306). In the neoadjuvant clinical study, lapatinib treatment for 2 weeks was associated with parallel upregulation of ER and Bcl2 (Spearman coefficient: 0.70; P = 0.0002). Importantly, 18% of tumors originally ER-negative (ER−) converted to ER+ upon anti-HER2 therapy. In ER−/HER2+ MCF7/HER2-18 xenografts, ER reexpression was primarily observed in tumors responding to potent combination of anti-HER2 drugs. Estrogen deprivation added to this anti-HER2 regimen significantly delayed tumor progression (P = 0.018). In the UACC812 cells, fulvestrant, but not ABT-737, was able to completely inhibit anti–HER2-resistant growth (P < 0.0001). Conclusions: HER2 inhibition can enhance or restore ER expression with parallel Bcl2 upregulation, representing an ER-dependent survival mechanism potentially leading to anti-HER2 resistance. Clin Cancer Res; 21(17); 3995–4003. ©2015 AACR.

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies. Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition. Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo. Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

Erratum to Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells (Cell Cycle, 14, 4 (648-655), 10.4161/15384101.2014.994966)

Article title: Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells Authors: Kaleigh Canfield, Jiaqi Li, Owen M. Wilkins, Meghan MMorrison, Matthew Ung, Wendy Wells, Charlotte R Williams, Karen T Liby, Detlef Vullhorst, Andres Buonanno, Huizhong Hu, Rachel Schiff, Rebecca S Cook, and Manabu Kurokawa Journal: Cell Cycle Bibliometrics: Volume 14, Issue 4, Pages 648-55 DOI: 10.4161/15384101.2014.994966

Abstract P3-05-13: Overexpression of insulin receptor substrate 4 can mediate acquired resistance to lapatinib-containing regimens in HER2+ breast cancer cells

Background: The HER2 pathway can be inhibited by potent targeting agents such as lapatinib (L), trastuzumab (T), or their combination (LT), but acquired and de novo resistance still occur. Resistance to these drugs remains a major hurdle in the management of HER2+ breast cancer. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER2-directed therapies is of critical importance. Methods: To obtain clues to the mechanisms for resistance we developed a panel of HER2+ breast cancer cell lines resistant to L, T, or LT. Parental cells and resistant derivatives of the HER2+ BT474 cell line were characterized by RNA-seq. Genes that were overexpressed in resistant compared to parental cells were confirmed by RT-PCR, Western blotting, and immunohistochemistry (IHC). Cell growth and cell signaling were assessed in parental and resistant cell lines after down-regulation (by siRNA) or overexpression (via an inducible cDNA) of IRS4 in the presence or absence of treatment. The effect of IRS4 overexpression on L resistance was assessed in a BT474 xenograft model. The proteins that interact with IRS4 were identified by co-immunoprecipitation with IRS4 followed by separation of the associated proteins by SDS-PAGE and microsequencing by mass spectrometry. Results: RNA-seq analysis revealed that IRS4 was the most up-regulated gene in BT474 L or LT resistant derivatives in which HER2 signaling is effectively inhibited, but not T alone, where HER2 signaling is reactivated. Western blotting and IHC validated this result and identified membrane localization of IRS4. Knockdown of IRS4 in L- or LT-resistant cells reversed resistance and restored growth inhibition. IRS4 knockdown also inhibited downstream signaling, with a reduction in pAKT but not in pMAPK. Induction of the cell cycle regulator p27 and down-regulation of survivin were observed after IRS4 knockdown. Overexpression of IRS4 cDNA in parental BT474 and SKBR3 cells led to resistance to L/LT, increased pAkt, and decreased the apoptotic marker cleaved PARP in the presence of L or the LT combination. The BT474 xenograft model showed that IRS4 overexpression in the absence of treatment had no effect on tumor growth but it significantly reduced the inhibitory effect of lapatinib (p=0.002). A group of proteins that interact with IRS4 in BT474 L-resistant cells were identified by mass spectrometry. The roles of these proteins in IRS4-mediated resistance to lapatinb-containing regimens are under investigation. Conclusion: IRS4 overexpression is a critical factor in causing resistance to lapatinib-containing regimens in BT474 cells. Investigation of IRS4 and its signaling partners in HER2+ human tumors resistant to lapatinib will be important to determine if this mechanism is also operative in patients. Citation Format: Lanfang Qin, Maria B Hahn, Xiaoyong Fu, Martin J Shea, Mario Giuliano, Sarmistha Nanda, Xiaowei Xu, Huizhong Hu, Sung Yun Jung, Laura M Heiser, Nicholas Wang, Joe W Gray, Susan G Hilsenbeck, Chad Creighton, Chad A Shaw, Gary C Chamness, Dean P Edwards, Sabrina Herrera, Carolina Gutierrez, C Kent Osborne, Rachel Schiff. Overexpression of insulin receptor substrate 4 can mediate acquired resistance to lapatinib-containing regimens in HER2+ breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-13.

Abstract P5-05-03: Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib

Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre-treatment baseline specimens from TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in same-model P lines were identified. cDNAs were assessed by targeted Sanger sequencing. Single cells of the BT474AZ-LR line were cloned and their cDNAs were sequenced. Mutant-specific Q-PCR was designed to sensitively quantify mutations. Whole exome sequencing (minimum depth 100X) of 17 baseline tumor/normal pairs from TBCRC006 were performed on Illumina HiSeq. Results: We found and validated the HER2 L755S mutation in the BT474ATCC-LTR line and the BT474AZ-LR line (∼30% of DNA/RNA/cDNA in BT474AZ-LR), in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation was previously shown to induce L resistance in HER2-negative BC cell lines, suggesting a role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ-LR single cell clones found the HER2 L755S mutation in every clone but only in ∼30% of the HER2 copies. Using sensitive mutant-specific Q-PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC-P and BT474AZ-P compared to parentals of other HER2+ BC cell lines (UACC812/AU565/SKBR3/SUM190). These data suggest that this mutation exists subclonally within BT474 parental lines and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 baseline samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient’s baseline tumor. Conclusion: Acquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M Heiser, Nicholas Wang, Kyle R Covington, Edward S Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne AW Fuqua, Gary C Chamness, Chad Shaw, David A Wheeler, Joe W Gray, Susan G Hilsenbeck, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-03.

Abstract LB-166: PARP inhibition effects on endocrine therapy and resistance in estrogen receptor positive (ER+) breast cancer models

Background: Poly [ADP-ribose] polymerase (PARP) is an important mediator of DNA damage repair. Preclinical and clinical studies have indicated PARP inhibitors (i) as promising agents in DNA-repair-defective cancers, especially in the presence of BRCA1/2 alterations. Recent evidence also suggests a role for additional DNA damage-independent functions of PARP, involving transcriptional and epigenetic regulation. Particularly, PARP-1 can interact with the estrogen receptor (ER) and modulate its transcriptional activity in vascular smooth muscle cells, and PARPi can circumvent endocrine resistance in prostate cancer cells. However, the effect of PARPi on the efficacy of endocrine therapy (EndoTx) in ER+ breast cancer is unknown. Here we aimed to establish these effects using the PARPi olaparib (AZD2281, Olap) in our preclinical models. Methods: The effects of Olap (0.01-1μM) alone or in combination with EndoTx [estrogen deprivation (ED) or tamoxifen (Tam)] were tested in vitro in three ER+ parental cell lines (MCF7, ZR75-1, and T47D; BRCAwt) and their endocrine resistant (R) derivatives. Clonogenic assays were used to assess Olap-induced changes in cell growth. In vivo, ovariectomized nude mice bearing 200 mm3 tumors growing in the presence of estrogen supplementation (E2 pellets), were randomized to either continued E2 (control), ED, or ED+Tam; all ± oral Olap (100mg/kg daily). Results: In MCF7 parental cells in vitro, Olap, in a dose dependent manner, inhibited E2-stimulated growth and increased the growth inhibitory effect of EndoTx. In addition, at a clinically relevant dose (1μM), Olap significantly inhibited the growth of MCF7 EndoR cell derivatives. Similar results were also observed in the other 2 ER+ cell models. In vivo, long-term Olap-treatment in the presence of EndoTx (Tam ∼200days, and ED ∼300days) was well tolerated and no apparent drug-related toxicity was observed. Olap did not affect the growth of E2-stimulated MCF7 xenografts. In contrast, Olap enhanced sensitivity to Tam by delaying time to tumor progression (TTP; size doubling) from a median of 95 days to 155 days (p = 0.03), and numerically but not significantly reduced time to tumor regression (TTR; size halving) from 138 to 51 days (p = 0.2). Olap also numerically accelerated median time to tumor regression in the presence of ED (34 ED vs. 25 days ED+Olap; p = 0.23), but had no effect on TTP (P = 0.36). Conclusion: Our results suggest a potential role for Olap in enhancing EndoTx efficacy and circumventing resistance in the absence of BRCA mutations. The absence of any toxicity and negative interaction by adding Olap to EndoTx in the in vivo experiment supports the inclusion of ER+ breast cancer patients in clinical trials using PARPi. Further studies are warranted to better understand the biology of PARP and the efficacy of PARPi in ER+ breast cancer, especially in the presence of EndoTx and resistance. Citation Format: Agostina Nardone, Amit Goldstein, Martin J. Shea, Tamika Mithchell, Xiaoyong Fu, Carmine De Angelis, Huizhong Hu, Xiaowei Xu, Mahitha Rajendran, Mark O9Connor, Gershon Locker, Susan Hilsenbeck, Kent Osborne, Rachel Schiff. PARP inhibition effects on endocrine therapy and resistance in estrogen receptor positive (ER+) breast cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-166. doi:10.1158/1538-7445.AM2015-LB-166

Abstract P5-05-01: Targeting the mevalonate pathway to overcome acquired anti-HER2 treatment resistance

Background: Compelling preclinical and clinical evidence suggests that a more complete blockade of the HER receptor layer and its signaling, by combining anti-HER2 drugs, such as Trastuzumab (T) and Lapatinib (L), is highly effective. However, resistance is still common and remains a challenge. To understand resistance mechanisms and further to identify novel therapeutic strategies, we established a broad panel of L, T, and L+T resistant cell line models. Initial mRNA expression profiling identified upregulation or restoration of the mevalonate (MVA) pathway in some models where HER signaling is completely and sustainably blocked. The MVA pathway is commonly considered as a biosynthetic process primarily for cholesterol and isoprenoid intermediates, particularly farnesyl and geranylgeranyl pyrophosphates (FPP and GGPP, respectively). Statins, widely-used cholesterol-lowering drugs, block this pathway via inhibition of the rate-limiting enzyme, HMG-CoA reductase. While accumulating evidence also suggests a role of the MVA pathway in tumor initiation and progression, its role in anti-HER2 resistance remains elusive. Methods: SKBR3, AU565, and UACC812 parental HER2+ cells and their T, L, and L+T resistant (TR, LR, and LTR respectively) derivatives were used in this study. Cell growth after treatment with statins in the presence or absence of MVA, cholesterol, squalene, FPP, or GGPP was measured by methylene blue staining. Apoptosis was determined by Annexin V staining and the protein level of cleaved PARP. Parallel analysis of molecular signaling was done by western blotting. Results: Blocking the MVA pathway with lipophilic statins, simvastatin or atorvastatin, led to a marked growth inhibition or apoptosis in LR/LTR models, in which the HER signaling remains sustainably inhibited, while cognate parental cells and TR cells, in which HER is (re)activated, were only slightly inhibited. Interestingly, only lipophilic statins (which can be taken up by cancer cells), but not hydrophilic statins such as pravastatin (whose primary target is liver cells), conveyed the inhibitory effect. Prevention of statin-induced apoptosis by adding exogenous MVA indicated that the cell death caused by statin treatment was via its specific blockade of the MVA pathway. Cholesterol or its precursor squalene could not rescue growth inhibition. In contrast, both FPP and GGPP reversed the growth inhibition or apoptosis in SKBR3 and AU565 LR/LTR models, while in the UACC812LTR model only GGPP rescued. Interestingly, mTOR was identified as the downstream signaling target of the MVA pathway in SKBR3 and AU565LTR models, while in the UACC812LTR model, the growth inhibition by statin was due to substantial estrogen receptor (ER) protein reduction. Conclusion: The MVA pathway plays a key role as an escape pathway by activating alternative signaling, including mTOR and ER pathways, in acquired resistance to potent HER2 inhibition in a cholesterol-independent but FPP/GGPP-dependent manner. Targeting the MVA pathway or its downstream effectors could provide a novel therapeutic strategy to overcome anti-HER2 resistance. Citation Format: Huizhong Hu, Lukas M Simon, Agostina Nardone, Chad A Shaw, Gary C Chamness, Laura M Heiser, Nicholas Wang, C Kent Osborne, Rachel Schiff. Targeting the mevalonate pathway to overcome acquired anti-HER2 treatment resistance [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-01.

Abstract 721: Mevalonate pathway mediates acquired anti-HER2 treatment resistance in HER2+ breast cancer

Background: Compelling evidence suggests that a more complete blockade of the HER receptor layer and its signaling, by combining anti-HER2 drugs such as Trastuzumab (T) and Lapatinib (L), is highly effective. However, resistance remains a challenge. To understand resistance mechanisms, we established a broad panel of L, T, and L+T resistant cell line models. Initial mRNA expression profiling identified upregulation or restoration of the mevalonate (MVA) pathway in some models where HER signaling is completely and sustainably blocked, suggesting a possible role of the MVA pathway in resistance. This pathway is a biosynthetic process for cholesterol and isoprenoid intermediates, particularly farnesyl and geranylgeranyl pyrophosphates (FPP and GGPP). Coordinating gene expression of this pathway is regulated by the master transcription factor, SREBP. Statins, cholesterol-lowering drugs, block this pathway via inhibiting the rate-limiting enzyme, HMG-CoA reductase. While a role of the MVA pathway in tumor initiation and progression was reported, its role in anti-HER2 resistance has remained elusive. Methods: SKBR3, AU565, and UACC812 parental HER2+ cells and their T, L, and L+T resistant derivatives (TR, LR, and LTR) were used. SREBP activity was determined by a reporter luciferase assay. Cell growth was assayed by methylene blue staining. Apoptosis was determined by Annexin V staining and the protein level of cleaved PARP. Molecular signaling analysis was performed by western blotting (WB) and reverse phase protein array (RPPA). Results: Reporter assays showed the inhibition of the SREBP activity upon LT treatment and its restoration at resistance. Blocking the MVA pathway with statins led to a marked growth inhibition or apoptosis in LR/LTR models, in which the HER signaling remains sustainably inhibited, while cognate parental cells and TR cells, in which HER is (re)activated, were slightly inhibited. Prevention of statin-induced growth inhibition or apoptosis by exogenous MVA indicated the specific role of this pathway in resistance. Cholesterol or its precursor squalene could not rescue growth inhibition. In contrast, both FPP and GGPP reversed the growth inhibition or apoptosis in SKBR3 and AU565 LR/LTR models, while in the UACC812LTR model only GGPP rescued. WB showed that mTOR and estrogen receptor (ER) are downstream effectors mediating the MVA pathway to anti-HER2 resistance. Proteomic analysis revealed additional novel MVA effectors which are currently under biochemical and functional validation. Conclusion: The MVA pathway plays a key role as an escape pathway by activating alternative signaling, including mTOR and ER pathways, in acquired resistance to potent HER2 inhibition in a cholesterol-independent but FPP/GGPP-dependent manner. Targeting the MVA pathway or its downstream effectors might provide a novel therapeutic strategy to overcome anti-HER2 resistance. Citation Format: Huizhong Hu, Lukas Simon, Lanfang Qin, Agostina Nardone, Chad Shaw, Gary Chamness, Laura Heiser, Nicholas Wang, Joe W. Gray, C. Kent Osborne, Rachel Schiff. Mevalonate pathway mediates acquired anti-HER2 treatment resistance in HER2+ breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 721. doi:10.1158/1538-7445.AM2015-721