Justin M. Balko

Phosphatidylinositol 3-Kinase and Antiestrogen Resistance in Breast Cancer

Although antiestrogen therapies targeting estrogen receptor (ER) α signaling prevent disease recurrence in the majority of patients with hormone-dependent breast cancer, a significant fraction of patients exhibit de novo or acquired resistance. Currently, the only accepted mechanism linked with endocrine resistance is amplification or overexpression of the ERBB2 (human epidermal growth factor receptor 2 [HER2]) proto-oncogene. Experimental and clinical evidence suggests that hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway, the most frequently mutated pathway in breast cancer, promotes antiestrogen resistance. PI3K is a major signaling hub downstream of HER2 and other receptor tyrosine kinases. PI3K activates several molecules involved in cell-cycle progression and survival, and in ER-positive breast cancer cells, it promotes estrogen-dependent and -independent ER transcriptional activity. Preclinical tumor models of antiestrogen-resistant breast cancer often remain sensitive to estrogens and PI3K inhibition, suggesting that simultaneous targeting of the PI3K and ER pathways may be most effective. Herein, we review alterations in the PI3K pathway associated with resistance to endocrine therapy, the state of clinical development of PI3K inhibitors, and strategies for the clinical investigation of such drugs in hormone receptor-positive breast cancer.

TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer

After an initial response to chemotherapy, many patients with triple-negative breast cancer (TNBC) have recurrence of drug-resistant metastatic disease. Studies with TNBC cells suggest that chemotherapy-resistant populations of cancer stem-like cells (CSCs) with self-renewing and tumor-initiating capacities are responsible for these relapses. TGF-β has been shown to increase stem-like properties in human breast cancer cells. We analyzed RNA expression in matched pairs of primary breast cancer biopsies before and after chemotherapy. Biopsies after chemotherapy displayed increased RNA transcripts of genes associated with CSCs and TGF-β signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-β signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-β type I receptor kinase inhibitor LY2157299, a neutralizing TGF-β type II receptor antibody, and SMAD4 siRNA all blocked paclitaxel-induced IL8 transcription and CSC expansion. Moreover, treatment of TNBC xenografts with LY2157299 prevented reestablishment of tumors after paclitaxel treatment. These data suggest that chemotherapy-induced TGF-β signaling enhances tumor recurrence through IL-8-dependent expansion of CSCs and that TGF-β pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-β inhibitors and anticancer chemotherapy in patients with TNBC.

Emergence of Constitutively Active Estrogen Receptor-α Mutations in Pretreated Advanced Estrogen Receptor–Positive Breast Cancer

Purpose: We undertook this study to determine the prevalence of estrogen receptor (ER) α (ESR1) mutations throughout the natural history of hormone-dependent breast cancer and to delineate the functional roles of the most commonly detected alterations. Experimental Design: We studied a total of 249 tumor specimens from 208 patients. The specimens include 134 ER-positive (ER+/HER2−) and, as controls, 115 ER-negative (ER−) tumors. The ER+ samples consist of 58 primary breast cancers and 76 metastatic samples. All tumors were sequenced to high unique coverage using next-generation sequencing targeting the coding sequence of the estrogen receptor and an additional 182 cancer-related genes. Results: Recurring somatic mutations in codons 537 and 538 within the ligand-binding domain of ER were detected in ER+ metastatic disease. Overall, the frequency of these mutations was 12% [9/76; 95% confidence interval (CI), 6%–21%] in metastatic tumors and in a subgroup of patients who received an average of 7 lines of treatment the frequency was 20% (5/25; 95% CI, 7%–41%). These mutations were not detected in primary or treatment-naïve ER+ cancer or in any stage of ER− disease. Functional studies in cell line models demonstrate that these mutations render estrogen receptor constitutive activity and confer partial resistance to currently available endocrine treatments. Conclusions: In this study, we show evidence for the temporal selection of functional ESR1 mutations as potential drivers of endocrine resistance during the progression of ER+ breast cancer. Clin Cancer Res; 20(7); 1757–67. ©2014 AACR.

Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers

In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers, and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2(+) breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated nontransformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. Trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, the HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab.

Profiling of residual breast cancers after neoadjuvant chemotherapy identifies DUSP4 deficiency as a mechanism of drug resistance

Neoadjuvant chemotherapy (NAC) induces a pathological complete response (pCR) in ∼30% of patients with breast cancer. However, many patients have residual cancer after chemotherapy, which correlates with a higher risk of metastatic recurrence and poorer outcome than those who achieve a pCR. We hypothesized that molecular profiling of tumors after NAC would identify genes associated with drug resistance. Digital transcript counting was used to profile surgically resected breast cancers after NAC. Low concentrations of dual specificity protein phosphatase 4 (DUSP4), an ERK phosphatase, correlated with high post-NAC tumor cell proliferation and with basal-like breast cancer (BLBC) status. BLBC had higher DUSP4 promoter methylation and gene expression patterns of Ras-ERK pathway activation relative to other breast cancer subtypes. DUSP4 overexpression increased chemotherapy-induced apoptosis, whereas DUSP4 depletion dampened the response to chemotherapy. Reduced DUSP4 expression in primary tumors after NAC was associated with treatment-refractory high Ki-67 scores and shorter recurrence-free survival. Finally, inhibition of mitogen-activated protein kinase kinase (MEK) synergized with docetaxel treatment in BLBC xenografts. Thus, DUSP4 downregulation activates the Ras-ERK pathway in BLBC, resulting in an attenuated response to anti-cancer chemotherapy.

A Kinome-Wide Screen Identifies the Insulin/IGF-I Receptor Pathway as a Mechanism of Escape from Hormone Dependence in Breast Cancer

Estrogen receptor α (ER)-positive breast cancers adapt to hormone deprivation and become resistant to antiestrogens. In this study, we sought to identify kinases essential for growth of ER(+) breast cancer cells resistant to long-term estrogen deprivation (LTED). A kinome-wide siRNA screen showed that the insulin receptor (InsR) is required for growth of MCF-7/LTED cells. Knockdown of InsR and/or insulin-like growth factor-I receptor (IGF-IR) inhibited growth of 3 of 4 LTED cell lines. Inhibition of InsR and IGF-IR with the dual tyrosine kinase inhibitor OSI-906 prevented the emergence of hormone-independent cells and tumors in vivo, inhibited parental and LTED cell growth and PI3K/AKT signaling, and suppressed growth of established MCF-7 xenografts in ovariectomized mice, whereas treatment with the neutralizing IGF-IR monoclonal antibody MAB391 was ineffective. Combined treatment with OSI-906 and the ER downregulator fulvestrant more effectively suppressed hormone-independent tumor growth than either drug alone. Finally, an insulin/IGF-I gene expression signature predicted recurrence-free survival in patients with ER(+) breast cancer treated with the antiestrogen tamoxifen. We conclude that therapeutic targeting of both InsR and IGF-IR should be more effective than targeting IGF-IR alone in abrogating resistance to endocrine therapy in breast cancer.

Stand Up to Cancer Phase Ib Study of Pan-Phosphoinositide-3-Kinase Inhibitor Buparlisib With Letrozole in Estrogen Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Metastatic Breast Cancer

PURPOSE Buparlisib, an oral reversible inhibitor of all class I phosphoinositide-3-kinases, has shown antitumoral activity against estrogen receptor (ER)-positive breast cancer cell lines and xenografts, alone and with endocrine therapy. This phase Ib study evaluated buparlisib plus letrozoles safety, tolerability, and preliminary activity in patients with metastatic ER-positive breast cancer refractory to endocrine therapy. PATIENTS AND METHODS Patients received letrozole and buparlisib in two different administration schedules. Outcomes were assessed by standard solid-tumor phase I methods. [(18)F]fluorodeoxyglucose-positron emission tomography/computed tomography ([(18)F]FDG-PET/CT) scans were done at baseline and 2 weeks after treatment initiation. Tumor blocks were collected for phosphoinositide-3-kinase pathway mutation analysis. RESULTS Fifty-one patients were allocated sequentially to continuous or intermittent (five on/two off days) buparlisib administration on an every-4-week schedule. Buparlisibs maximum-tolerated dose (MTD) was 100 mg/d. Common drug-related adverse events included ≤ grade 2 hyperglycemia, nausea, fatigue, transaminitis, and mood disorders. The clinical benefit rate (lack of progression ≥ 6 months) among all patients treated at the MTD was 31%, including two objective responses in the continuous dose arm. Of seven patients remaining on treatment ≥ 12 months, three had tumors with PIK3CA hot-spot mutation. Patients exhibiting metabolic disease progression by [(18)F]FDG-PET/CT scan at 2 weeks progressed rapidly on therapy. CONCLUSION The letrozole and buparlisib combination was safe, with reversible toxicities regardless of schedule administration. Clinical activity was observed independent of PIK3CA mutation status. No metabolic response by [(18)F]FDG-PET/CT scan at 2 weeks was associated with rapid disease progression. Phase III trials of buparlisib and endocrine therapy in patients with ER-positive breast cancer are ongoing.

PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors

IntroductionTriple negative breast cancer (TNBC) is a heterogeneous collection of biologically diverse cancers, which contributes to variable clinical outcomes. Previously, we identified a TNBC subtype that has a luminal phenotype and expresses the androgen receptor (AR+). TNBC cells derived from these luminal AR + tumors have high frequency phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations. The purpose of this study was to determine if targeting phosphoinositide 3-kinase (PI3K) alone or in combination with an AR antagonist is effective in AR + TNBC.MethodsWe determined the frequency of activating PIK3CA mutations in AR + and AR- TNBC clinical cases. Using AR + TNBC cell line and xenograft models we evaluated the effectiveness of PI3K inhibitors, used alone or in combination with an AR antagonist, on tumor cell growth and viability.ResultsPIK3CA kinase mutations were highly clonal, more frequent in AR + vs. AR- TNBC (40% vs. 4%), and often associated with concurrent amplification of the PIK3CA locus. PI3K/mTOR inhibitors had an additive growth inhibitory effect when combined with genetic or pharmacological AR targeting in AR + TNBC cells. We also analyzed the combination of bicalutamide +/- the pan-PI3K inhibitor GDC-0941 or the dual PI3K/mTOR inhibitor GDC-0980 in xenograft tumor studies and observed additive effects.ConclusionsWhile approximately one third of TNBC patients respond to neoadjuvant/adjuvant chemotherapy, recent studies have shown that patients with AR + TNBC are far less likely to benefit from the current standard of care chemotherapy regimens and novel targeted approaches need to be investigated. In this study, we show that activating PIK3CA mutations are enriched in AR + TNBC; and, we show that the growth and viability of AR + TNBC cell line models is significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist. These results provide rationale for pre-selection of TNBC patients with a biomarker (AR expression) to investigate the use of AR antagonists in combination with PI3K/mTOR inhibitors.

Mutant PIK3CA accelerates HER2-driven transgenic mammary tumors and induces resistance to combinations of anti-HER2 therapies

Human epidermal growth factor receptor 2 (HER2; ERBB2) amplification and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations often co-occur in breast cancer. Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway has been shown to correlate with a diminished response to HER2-directed therapies. We generated a mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared with mice expressing either oncogene alone. HER2 and mutant PIK3CA also cooperated to promote lung metastases. By microarray analysis, HER2-driven tumors clustered with luminal breast cancers, whereas mutant PIK3CA tumors were associated with claudin-low breast cancers. PIK3CA and HER2+/PIK3CA tumors expressed elevated transcripts encoding markers of epithelial-to-mesenchymal transition and stem cells. Cells from HER2+/PIK3CA tumors more efficiently formed mammospheres and lung metastases. Finally, HER2+/PIK3CA tumors were resistant to trastuzumab alone and in combination with lapatinib or pertuzumab. Both drug resistance and enhanced mammosphere formation were reversed by treatment with a PI3K inhibitor. In sum, PIK3CAH1047R accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers, and generates resistance to approved combinations of anti-HER2 therapies.

RAS/MAPK Activation Is Associated with Reduced Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer: Therapeutic Cooperation Between MEK and PD-1/PD-L1 Immune Checkpoint Inhibitors

Purpose: Tumor-infiltrating lymphocytes (TIL) in the residual disease (RD) of triple-negative breast cancers (TNBC) after neoadjuvant chemotherapy (NAC) are associated with improved survival, but insight into tumor cell-autonomous molecular pathways affecting these features are lacking. Experimental Design: We analyzed TILs in the RD of clinically and molecularly characterized TNBCs after NAC and explored therapeutic strategies targeting combinations of MEK inhibitors with PD-1/PD-L1–targeted immunotherapy in mouse models of breast cancer. Results: Presence of TILs in the RD was significantly associated with improved prognosis. Genetic or transcriptomic alterations in Ras–MAPK signaling were significantly correlated with lower TILs. MEK inhibition upregulated cell surface MHC expression and PD-L1 in TNBC cells both in vivo and in vitro. Moreover, combined MEK and PD-L1/PD-1 inhibition enhanced antitumor immune responses in mouse models of breast cancer. Conclusions: These data suggest the possibility that Ras–MAPK pathway activation promotes immune-evasion in TNBC, and support clinical trials combining MEK- and PD-L1–targeted therapies. Furthermore, Ras/MAPK activation and MHC expression may be predictive biomarkers of response to immune checkpoint inhibitors. Clin Cancer Res; 22(6); 1499–509. ©2015 AACR.