Co-targeting of the PI3K pathway improves the response of BRCA1 deficient breast cancer cells to PARP1 inhibition
Introduction
Major improvements in breast cancer therapy have been achieved with the development of treatments targeting estrogen receptor signaling (anti-estrogens and aromatase inhibitors) and epidermal growth factor receptors (e.g. HER2 signaling using monoclonal antibodies and tyrosine kinase inhibitors), respectively. However, “triple-negative” (i.e. ER, PR and HER2 negative) tumors remain a treatment challenge and the survival of these patients is poor compared to patients with other subtypes of breast cancer. Systemic treatment for patients with triple-negative disease is currently limited to chemotherapy. Gene expression profiling and immunohistochemical studies have repeatedly classified most BRCA1-associated tumors together with the triple-negative and basal-like tumors [1], [2], [3]. This suggests a common pathogenesis for BRCA1-associated cancers and a subset of triple negative cancers, considering the marked heterogeneity within the triple negative class. Furthermore, this indicates the possibility of a common therapeutic approach for these patients. The inherently aggressive behavior, poor prognosis and limited therapeutic options urgently warrant new, targeted therapies for this group of patients.
Poly(ADP-ribose) polymerase 1 (PARP1), is the most abundant and well-studied member of the PARP family of proteins. It plays a crucial role in the repair of both single- and double-stranded DNA breaks (SSB and DSB) by binding to the DNA lesions and activating downstream repair proteins [4]. PARP inhibitors gained ground as potential anti-cancer agents due to their ability to sensitize tumor cells to DNA damaging radio- and chemotherapy [5]. More interestingly, the demonstration that PARP inhibitors could selectively kill cells with defective DSB repair (such as BRCA1/2 mutated cells) [6], [7] when used as a single agent inspired their rapid development and prompt entry into a series of clinical trials [8], [9], [10]. The proposed rationale for the selective sensitivity of BRCA1/2 deficient cells to PARP1 inhibition is their increased dependency on PARP1 for efficient repair of spontaneous SSB to maintain genomic integrity and stability. In the past 5 years several studies have been published reporting promising effects of combining PARP inhibitors with radiotherapy and conventional DNA damaging chemotherapy (reviewed in [11]) as well as when used as single agents for targeting BRCA-associated tumors [9]. However, as for most single agent cancer therapies, the obvious risk that some patients may acquire resistance cannot be neglected and needs to be addressed. Also, because the consequence of long-term treatment and the overall effect of PARP inhibitors on other cellular pathways are widely unknown it is important to investigate options for limiting toxicity and establishing a more rational design of the treatment course.
BRCA1-dependent breast tumors, like all triple-negative breast tumors, harbor a variety of de-regulated pathways and it has been suggested that management of these tumors with multiple targeted therapies may be a superior therapeutic approach [12]. Specifically, informed combination treatments targeting different key pathways have the potential of both increasing the efficacy and reducing the risk of resistance. Aberrant signaling of the phospho-inositol-3 kinase (PI3K) pathway is frequently observed in many cancer types. Over-activation of this pathway in breast cancer may result from genetic abnormalities including gain of function of oncogenes (e.g. PIK3CA) or the loss of function of tumor suppressor genes (e.g. PTEN). Tumorigenic advantages driven by inappropriate activation of the PI3K/AKT pathway include cell transformation, proliferation, increased migration, angiogenesis, evasion of apoptosis and genome instability [13]. It has been suggested that among other mechanisms, increased signaling through the PI3K/AKT pathway may constitute a mechanism of resistance to cancer therapeutic agents (reviewed in [13]).
PTEN is a tumor suppressor gene and its function is crucial for regulating and maintaining accurate PI3K/AKT signaling. More recently, a nuclear role of PTEN has been elucidated, in which dysfunctional PTEN, e.g. by mutations, contributes to defective homologous recombination [14] resulting in increased sensitivity to PARP inhibition [15]. Intriguingly, gross PTEN mutations have been shown to be a specific and recurrent oncogenic consequence of a malfunctioning DNA repair pathway and are strongly associated with BRCA1 mutations [16]. It has further been shown that BRCA1 is able to bind to phosphorylated AKT, thereby functioning as a negative regulator of AKT activity [17]. In the same study, loss of BRCA1 expression was found to increase AKT activation. These separate links between BRCA1 and the PI3K pathway would then provide a mechanistic explanation to the negative correlation between BRCA1 expression and AKT phosphorylation found in human breast cancers [18]. Taken together, this suggests an addiction of BRCA1 deficient tumors to aberrant PI3K/AKT signaling. This implies that a combination of PI3K inhibitors with PARP inhibitors for targeting BRCA1 mutant cells should be more effective than targeting a single molecular abnormality. It is also possible that this combination may circumvent the development of resistance without compromising the specificity of the treatment. Furthermore, rational design of the combination regimen may also avoid the exacerbation of toxicity.
We hypothesized that the effect of PARP inhibition on BRCA1 mutant cells would be potentiated by co-targeting PI3K signaling. As an experimental model, we have used the human breast cancer cell lines MDA-MD-436, SUM149, HCC1937 and L56Br-C1, all of which harbor BRCA1 mutations [19], [20] and, with the exception of L56Br-C1, gross PTEN mutations [16]. MCF7 cells, with wild type BRCA1, served as control. The drugs tested in combination were the PARP inhibitors AG14361 or AG014699 and the PI3K inhibitor LY294002. Our in vitro results suggest that the cytotoxic effect of combining a PARP inhibitor and a PI3K inhibitor in a sequential regimen is superior over PARP inhibition alone and may represent an improved selective targeted treatment strategy for breast cancers with concomitant DNA damage repair defects and de-regulated PI3K signaling, and potentially also for sporadic tumors with a “BRCAness” [21] phenotype.
Section snippets
Drugs
AG14361 was synthesized by Istvan Jablonski, Institute of Biomolecular Chemistry, Hungarian Academy of Science and a 78 mM stock solution was prepared. AG014699 and AZD2881 (olaparib) were purchased from Selleck Chemicals (Houston, TX) and 20 mM stocks were prepared. LY294002 was purchased from Invitrogen (Carlsbad, CA) and diluted to a 10 mM stock solution. All stock solutions were prepared using dimethyl sulfoxide (DMSO) as solvent and stored at −20 °C.
Cell lines
The human breast cancer cell lines
Sensitivity to PARP inhibition
Initially, we were interested to validate the observation that BRCA1 mutated cells are sensitive to PARP inhibition. Following continuous treatment with the PARP inhibitor AG014699 alone for seven days, three out of four BRCA1 deficient breast cancer cell lines (MDA-MB-436, L56Br-C1 and SUM149) were more sensitive to the treatment compared to the BRCA1 expressing MCF7 cell line (Fig. 1A). Growth inhibition of approximately 50% was reached at 0.07 μM, 2 μM and 2.8 μM for MDA-MB-436, L56Br-C1 and
Discussion
PARP inhibitors are showing great promise as a selective targeted therapy for breast and ovarian cancers with mutations in the BRCA genes [8], [9], [10]. At the same time, these clinical trials clearly show that there is significant variation in the response to these agents even in selected (i.e. BRCA1/2 mutation positive) patient cohorts. It is possible that prior exposure to chemotherapy may affect the biological behavior and responsiveness to PARP inhibition. In fact, patients with previous
Acknowledgements
This study was supported through grants from the Swedish Cancer Society, the G Nilsson Cancer Foundation, the B Kamprad Foundation and the Lund University Hospital Research Foundation. Study sponsors did not participate in any part of the work reported herein.
References (40)
- et al.
Poly(ADP-ribose) polymerase inhibitors in cancer treatment: a clinical perspective
Eur. J. Cancer
(2010) - et al.
Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: a proof-of-concept trial
Lancet
(2010) - et al.
Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial
Lancet
(2010) - et al.
Essential role for nuclear PTEN in maintaining chromosomal integrity
Cell
(2007) - et al.
Characterization of a novel breast carcinoma xenograft and cell line derived from a BRCA1 germ-line mutation carrier
Lab. Invest.
(2003) - et al.
DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139
J. Biol. Chem.
(1998) - et al.
PARP-1 inhibition-induced activation of PI-3-kinase-Akt pathway promotes resistance to taxol
Biochem. Pharmacol.
(2009) - et al.
Repeated observation of breast tumor subtypes in independent gene expression data sets
Proc. Natl Acad. Sci. USA
(2003) - et al.
Gene-expression profiles in hereditary breast cancer
N. Engl. J. Med.
(2001) - et al.
An immune response gene expression module identifies a good prognosis subtype in estrogen receptor negative breast cancer
Genome Biol.
(2007)