Downregulation of stromal BRCA1 drives breast cancer tumor growth via upregulation of HIF-1α, autophagy and ketone body production

Generating BRCA1 knockdown stromal fibroblasts

Several previous studies have suggested that DNA damage and chromosomal instability in the tumor stroma are potently tumorigenic.^1,25,26 To investigate the possible tumor-promoting effects of a defective DNA-damage response in the tumor stroma, we stably transduced hTERT-immortalized human fibroblasts (hTERT-BJ1 cells) with four different constructs of short hairpin RNA’s targeting BRCA1 (shBRCA1).

Figure 1 shows that only one shBRCA1 construct (designated as #2) effectively knocked-down BRCA1 expression. Thus, all our subsequent studies utilized this fibroblast cell line transduced with shBRCA1 construct #2.

BRCA1 knockdown accelerates fibroblasts proliferation

Previous studies have shown BRCA1 knockdown increases the epithelial cancer cell proliferation rate.²⁷ To validate this result in fibroblasts, we plated shBRCA1 fibroblasts in a 12-well dish. For 10 d, cells were trypsinized and counted using a hemocytometer chamber. At day 10, shBRCA1 fibroblasts demonstrated a 5-fold increase in cell number, as compared with control fibroblasts (Fig. 2).

shBRCA1 fibroblasts display higher levels of autophagy and mitophagy markers

Several studies indicate that knocking-down BRCA1 induces autophagy.^22-24 Using immunoblot analysis, shBRCA1 fibroblasts displayed an increase in the autophagy marker (LC3) and the mitophagy marker (BNIP3) (Fig. 3A). Moreover, immunofluorescence shows that shBRCA1 fibroblasts overexpressed BNIP3 and LAMP1 (autophagy marker) when co-cultured with a triple-negative breast cancer cell line (namely, MDA-MB-231 cells) (Fig. 3B).

shBRCA1 fibroblasts express HIF-1α, under chemical pseudo-hypoxia

Stable overexpression of HIF-1α mediates mitophagy by triggering overexpression of BNIP3.²⁸ As shBRCA1 fibroblasts displayed an elevation of BNIP3 expression, we wanted to examine HIF-1α expression levels in these cells. Interestingly, shBRCA1 fibroblasts showed an increase of HIF-1α after 5 h of chemically induced pseudo-hypoxia (Fig. 4A). Concurrently, these pseudo-hypoxic fibroblasts demonstrated an elevation in an autophagy marker (LC3) (Fig. 4A).

Knocking-down BRCA1 downregulates SDHB

While BRCA1 functions mainly as a DNA guardian, it is also important in maintaining the integrity of mitochondria.^22,29 Although BRCA1 is mainly present in the cell nucleus, phosphorylated BRCA1 has also been found to be localized in the mitochondrial matrix.³⁰ Interestingly, in BRCA1-mutated cancers, succinate dehydrogenase B (SDHB, mitochondrial complex II subunit) was found to be a hot spot gene that frequently showed loss of heterozygosity and allelic instability in cancer stroma.³¹ Notably, shBRCA1 fibroblast lysates showed a downregulation of SDHB expression (Fig. 4B). Complex II is critical in mitochondrial metabolism; upon downregulation, it disturbs both the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Consequently, it is likely that disrupted mitochondrial metabolism allowed succinate accumulation and elevated ROS levels to stabilize HIF-1α expression.³² Moreover, Figure 5 shows that a loss of BRCA1 induces the activation of the Akt-mTOR signaling pathway, likely as a compensatory mechanism to survive the induction of autophagy.

shBRCA1 fibroblasts display increased ketone body generation

To evaluate if BRCA1 knockdown induces mitochondrial dysfunction, ketone body generation was measured in the conditioned media of control and shBRCA1 fibroblasts. Figure 6A shows that ketone body generation is increased up to ~5.5-fold in shBRCA1 fibroblasts relative to control cells.

Elevated mitochondrial activity in cancer cells co-cultured with shBRCA1 fibroblasts

In previous studies, we have shown that autophagic fibroblasts, when co-cultured with cancer cells, induce cancer cell mitochondrial activity.³³ This is due to the production of L-lactate and ketone bodies by autophagic fibroblasts, which are then metabolized by cancer cell mitochondria.³³ Moreover, it has been shown that BRCA1 knockdown decreases mitochondrial activity, which shifts metabolism toward glycolysis.²² As shBRCA1 fibroblasts show an elevation of mitophagy, we expected shBRCA1 fibroblasts to induce mitochondrial activity when co-cultured with breast cancer cells. As expected, the mitochondrial activity of MDA-MB-231 cells was significantly elevated by ~1.4-fold when they were co-cultured with shBRCA1 fibroblasts (Fig. 6B).

shBRCA1 fibroblasts promote xenograft tumor growth

We hypothesized that a BRCA1-deficient cancer stroma can promote tumor growth. To test our hypothesis, we used a pre-clinical model in which we subcutaneously co-injected shBRCA1 fibroblasts with MDA-MB-231 cancer cells into the flanks of nude mice. After 31 d, the tumors were excised and weighed. Importantly, shBRCA1 fibroblasts markedly increased tumor growth by ~2.2-fold, as compared with control fibroblasts (Fig. 7). This study highlights the potential lethality of a BRCA1-deficient tumor stroma.

BRCA1 and the tumor microenvironment

Here, we successfully knocked-down BRCA1 expression using a lentiviral shRNA approach in hTERT-immortalized fibroblasts. shBRCA1 fibroblasts displayed increased proliferation, as expected. Moreover, our immunoblot and immunofluorescence studies demonstrate that shBRCA1 fibroblasts display an increase in markers of autophagy (LAMP1 and LC3) and mitophagy (BNIP3). Interestingly, shBRCA1 fibroblasts show an increase in HIF-1α expression when treated with chemical inhibitors to prevent HIF-1 α degradation. Furthermore, we demonstrate a decrease in succinate dehydrogenase subunit B (SDHB, a complex II subunit) in shBRCA1 fibroblasts. Under normoxia, prolyl-4-hydroxylase (PHD) promotes the ability of von Hippel-Lindau (VHL) to target HIF-1α for proteasomal-degradation.³⁴ Guzy et al. showed that knockdown of SDHB allowed excess succinate to migrate to the cytoplasm and inhibit PHD oxidation of HIF-1α, resulting in the stabilization of HIF-1α.³² Alternatively, stabilized HIF-1α mediates the expression of BNIP3, which is responsible for mitochondrial autophagy.³⁵ Therefore, the relationship between BRCA1 and mitochondrial integrity is emerging.^22,30 Also, when shBRCA1 fibroblasts were co-cultured with breast cancer cells, the cancer cells displayed an increase in mitochondrial activity. Similar results were obtained when we co-cultured glycolytic fibroblasts with cancer cells, as the fibroblasts secrete ketone bodies, which are utilized in cancer cell mitochondria.³³ Interestingly, shBRCA1 fibroblasts increased tumor growth by ~2.2-fold when co-injected with MDA-MD-231 human breast cancer cells. For the first time, our current study demonstrates the potential lethal effects of a BRCA1-deficient tumor stroma on breast cancer tumor growth.

In previous studies, we provided evidence for the pro-tumorigenic effects of an autophagic and glycolytic tumor stroma.^5,11 To better explain epithelial-stromal interactions in cancer progression, we suggested a new paradigm to describe this co-evolution termed the “autophagic tumor stroma model of cancer metabolism” (Fig. 8).¹³ In this model, we hypothesize that the first step is the release of hydrogen peroxide (H₂O₂) by cancer cells, which then activates neighboring stromal cells, thereby inducing more oxidative stress.¹³ Accordingly, several authors have indicated that an activated stroma displays higher DNA damage than cancer cells. Following DNA damage, several molecules trigger autophagy and mitophagy pathways.³⁶ As a result, CAFs switch their metabolism toward glycolysis and secrete energy-rich molecules (such as L-lactate and ketone bodies) into the tumor microenvironment. Moreover, elevated ROS or mitochondrial dysfunction stabilizes HIF-1α, which will act as a feed-forward mechanism for more mitophagy and glycolysis.^32,37 In turn, cancer cells overexpress MCT1 to import stromally derived nutrients (L-lactate and ketone bodies) and to employ them in oxidative mitochondrial metabolism.³⁸ This vicious cycle provides a steady stream of high-energy metabolites that directly supports cancer cell growth.

PARP inhibitors in clinical trials

BRCA1 is a homologous recombination (HR) protein that is involved in the repair of DNA double-strand breaks (DSB’s). When BRCA1 is downregulated by mutation or methylation, HR fails to repair the DSB’s, which will be subsequently repaired by non-homologous end joining (NHEJ). As NHEJ fixes DSB’s randomly, it induces chromosomal instability, which can lead to cancer development or cell death.³⁹

The process of random repair in BRCA1-deficient cancer cells led to the idea to employ poly (ADP-ribose) polymerase (PARP) inhibitors, in treating familial BRCA1 cancers or triple-negative breast cancers (TNBC).³⁹ The PARP enzyme is responsible for base excision repair (BER) in single-strand breaks (SSB’s). BRCA1-mutation carriers have heterozygous BRCA1 mutations in normal tissue, while tumor cells display homozygous BRCA1 mutations after loss of heterzygosity (LOH).³¹ Hence, when BRCA1-deficient cancer cells are treated with PARP inhibitors, SSBs develop DSBs, and then NHEJ haphazardly repairs the damaged DNA. Increased DNA repair errors selectively kills BRCA1-deficient cancer cells.³⁹ This “synthetic lethality” theory was promising for the treatment lethal TNBC. Thus, several pharmaceutical companies conducted phase I/II trials using PARP inhibitors to treat BRCA1 tumors. Unfortunately, a phase III trial of Iniparib (a PARP inhibitor produced by Sanofi-Aventis) failed to offer any significant benefits for the treatment of metastatic TNBC patients, when combined with cytotoxic agents.⁴⁰ This drawback discouraged other companies, such as AstraZeneca, from performing a phase III trial for the PARP inhibitor, Olaparib. We speculate that this failure is due to the fact that PARP inhibitors do not only target epithelial cancer cells, but they will also target the BRCA1-deficient tumor stroma.³¹ Inhibiting PARP in the BRCA1-deficient tumor stroma can elevate or activate the autophagy pathway.⁴¹ According to our current study, inducing autophagy in the tumor stroma will promote the production of recycled nutrients that increase cancer cell growth. Thus, we suggest that PARP inhibitors may benefit breast cancer patients when combined with autophagy inhibitors, such as chloroquine. To test this hypothesis, we will require further clinical trials.

Materials

Antibodies used in this paper were purchased from commercial sources: anti-BRCA1 (Santa Cruz, C-20, SC-642), anti-BNIP3 (ab10433, Abcam), anti-LC3A/B (Ab58610, Abcam), anti-HIF-1 α (NB100–123, Novus), anti-β actin (A5441, Sigma), anti-SDHB (NBP-54154, Novus), and anti-LAMP1 (Santa Cruz, SC-17768). Other reagents were purchased as follows; Hoechst-33258 nuclear stain was from Sigma, Anti-fade reagent (S2828) was from Invitrogen, MG-132 purchased from Calbiochem and DMOG was from Cayman Chemical.

Cell culture and transfection

MDA-MB-231 cells (a human triple-negative breast cancer cell line) and hTERT-immortalized human fibroblasts (hTERT-BJ1) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum in a 37°C humidified atmosphere containing 5% CO₂, unless otherwise noted. Lentiviral particles were produced after 48 h of transfecting GeneCopoeia 293Ta lentiviral packaging cell line with lentiviral plasmids (CSHCTR001-HIVmH1) (empty vector) or shBRCA1–1 [HSH017713–1-HIVmH1(OS224289)] or shBRCA1–2 [HSH017713–2-HIVmH1(OS224290)] or shBRCA1–3 [HSH017713–3-HIVmH1(OS224291)] or shBRCA1–4 [HSH017713–4-HIVmH1(OS224292)] (all obtained from GeneCopoeia.Inc.), using the Lenti-Pac HIV Expression Packing Kit (GeneCopoeia.Inc.) according to manufacturer’s recommendation. hTERT-immortalized fibroblasts were transduced by lentivirus particles in the presence of 5 μg/ml of Polybrene (Santa Cruz Biotech). Transduced cells were then selected with 1.5 μg/ml of puromycin.

Growth curves

Two thousand fibroblasts per well were plated in a 12-well tissue-culture dish. For each time point, the cells were trypsinized, stained with trypan blue and then counted using a hemocytometer. The average of each cell count was then calculated from three independent representative wells.

Immunoblot analysis

For chemically induced pseudo-hypoxia, one million fibroblasts were plated in 10-cm dish. The next day, the media was discarded and replaced with complete DMEM, supplemented with 10 μM MG-132 (a proteaosome inhibitor) plus 1 mM dimethyloxallyl glycine (DMOG, a stabilizer of HIF-1 α) for 5 h. Then, the cells were lysed by scraping into lysis-buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and 60 mM n-octylglucoside) containing protease inhibitors (Boehringer Mannheim). Samples were incubated on a rotating platform at 4°C and were then centrifuged at 12,000x g for 10 min (at 4°C) to remove insoluble debris. Protein concentrations were analyzed using the BCA reagent (Pierce). Samples were then separated by SDS-PAGE (10% acrylamide) and transferred to nitrocellulose. All subsequent wash buffers contained 10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20, which was supplemented with 5% nonfat dry milk (Carnation) for the blocking solution and 1% bovine serum albumin (Sigma) for the antibody diluent. Horseradish peroxidase-conjugated secondary antibodies were used to visualize bound primary antibodies with an ECL detection kit (Pierce).

Immunofluorescence

MDA-MB-231 cells and hTERT-fibroblasts were plated on coverslips in 12-well plates for 48 h in DMEM supplemented with 10% Nu serum. Then, the cells were rinsed with PBS with 0.1 mM CaCl₂ and 1 mM MgCl₂ (PBS/CM) and fixed with 2% paraformaldehyde in PBS/CM for 30 min. After fixation, cells were washed three times with PBS/CM and permeabilized with IF buffer (PBS/CM with 0.1% Triton-X100 and 0.2% bovine serum albumin) for 10 min. Then, cells were quenched with 50 mM NH₄Cl in PBS/CM for 10 min, rinsed and incubated with anti-BNIP3 or anti-LAMP1 antibodies for 1 h at room temperature. Cells were washed with IF buffer and incubated with fluorescent secondary antibodies (Molecular Probes) for 30 min. After washing, cells were incubated with Hoechst-33258 for nuclear staining, rinsed and mounted with ProLong Gold anti-fade (Molecular Probes). Images were acquired with a Zeiss LSM510 Meta confocal microscope system and analyzed with Zeiss LSM Browser (3,5,0,359; Zeiss).

Mitochondrial activity studies

We stained cell lines with MitoTracker Orange (CMTMRos; cat #M7510, Invitrogen, Inc.) to measure mitochondrial activity, as the stain’s accumulation is dependent upon an intact mitochondrial membrane potential. Fibroblasts and cancer cells were cultured for 2 d in DMEM supplemented with 10% Nu serum. Then, after 2 d, the cells were then incubated with pre-warmed MitoTracker staining solution (diluted in serum-free DMEM to a final concentration of 25 nM) for 10 min at 37°C. All subsequent steps were performed in the dark. Cells were washed in PBS, trypsinized and re-suspended in 300 μL of Binding Buffer (BD Biosciences). Cells were then analyzed by FACS. Data analysis was performed using FlowJo 8.8 software.

Animal studies

All studied animals were maintained in a pathogen-free environment/barrier facility at the Kimmel Cancer Center at Thomas Jefferson University under National Institutes of Health (NIH) guidelines. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC). hTERT-BJ1 fibroblasts (3 × 10⁵ cells) with tumor cells (MDA-MB-231; 1 × 10⁶ cells) in 100 μl of sterile PBS were co-injected into the flanks of athymic NCr nude mice (NCRNU; Taconic Farms; 6–8 wk of age). Mice were then sacrificed at 31 d post-injection; tumors were excised to determine their weight.