Preclinical Evaluation of Fatty Acid Synthase and EGFR Inhibition in Triple Negative Breast Cancer

Triple Negative Breast Cancer (TNBC) is the molecular subtype associated with the highest relapse rates and worse outcome. Patients lack targeted therapy and are generally treated with cytotoxic chemotherapy. There is a need to develop novel therapeutic strategies based on TNBC biology. Although EGFR overexpression is observed in 50-70% of TNBC, its inhibition alone or in combination with chemotherapy has resulted in minimal clinical benefit. The enzyme fatty acid synthase (FASN) has been shown to be a key therapeutic target in several human carcinomas. We found FASN is expressed in all TNBC primary tumor samples tested. We then showed therapeutic benefit of FASN inhibition in sensitive and chemoresistant TNBC preclinical models, as well as strong synergistic anti-tumor effect of FASN inhibitors in combination with cetuximab (an EGFR inhibitor). Our findings provide a rationale for further investigation of FASN inhibitors in combination with anti-EGFR signaling agents in TNBC. Abstract Purpose: Triple Negative Breast Cancer (TNBC) lacks an approved targeted therapy. Despite initial good response to chemotherapy, 30% of the patients relapse within 5 years after treatment. EGFR overexpression is a common marker in TNBC, and its expression has been correlated with poor outcome. Inhibition of Fatty Acid Synthase (FASN) activity leads to apoptosis of human carcinoma cells overexpressing FASN. We tested the hypothesis that blocking FASN in combination with anti-EGFR signaling agents would be an effective antitumor strategy in sensitive and chemoresistant TNBC. Experimental Design: Several TNBC cell lines and 29 primary tumors were included to determine whether FASN is a potential target in TNBC. Doxorubicin-Research. resistant TNBC cell lines (231 DXR and HCC DXR ) have been developed and characterized in our laboratory. Cellular and molecular interactions of anti-FASN compounds (EGCG and C75) with cetuximab were analyzed. In vivo tumor growth inhibition was evaluated after cetuximab, EGCG or the combination in TNBC orthoxenograft models. Results: TNBC cell lines showed overexpression of FASN enzyme and its inhibiton correlated to FASN levels. FASN staining was observed in all of the 29 TNBC tumor samples. In vitro , EGCG and C75 plus cetuximab showed strong synergism in sensitive and chemoresistant cells. In vivo , the combination of EGCG with cetuximab displayed strong antitumor activity against the sensitive and chemoresistant TNBC orthoxenografts, without signs of toxicity. Conclusions: Our results show that the simultaneous blockade of FASN and EGFR is effective in preclinical models of sensitive and chemoresistant TNBC. FASN inhibitors shows strong synergism cetuximab sensitive resistant combination settings in all models.


Statement of Translational Relevance
Triple Negative Breast Cancer (TNBC) is the molecular subtype associated with the highest relapse rates and worse outcome. Patients lack targeted therapy and are generally treated with cytotoxic chemotherapy. There is a need to develop novel therapeutic strategies based on TNBC biology. Although EGFR overexpression is observed in 50-70% of TNBC, its inhibition alone or in combination with chemotherapy has resulted in minimal clinical benefit. The enzyme fatty acid synthase (FASN) has been shown to be a key therapeutic target in several human carcinomas. We found FASN is expressed in all TNBC primary tumor samples tested. We then showed therapeutic benefit of FASN inhibition in sensitive and chemoresistant TNBC preclinical models, as well as strong synergistic anti-tumor effect of FASN inhibitors in combination with cetuximab (an EGFR inhibitor). Our findings provide a rationale for further investigation of FASN inhibitors in combination with anti-EGFR signaling agents in TNBC.

Introduction
Breast cancer is the most prevalent and deadly cancer type in western women (1). Triple Negative Breast Cancer (TNBC) represents 15%-20% of the patients diagnosed with breast carcinomas and is characterized by the lack of expression of estrogen and progesterone receptors (ER/PR) and no amplification of the HER2 oncogene (2). TNBC runs an aggressive course and has a poor prognosis. As TNBC lacks a validated targeted therapy, patients are treated mainly with cytotoxic chemotherapy (anthracyclines and taxanes). Even though TNBC patients show a good initial response to chemotherapy, mesenchymal like (ML) (~20%) (8). Other molecular subtypes are also represented within TNBC subtype, because TNBC is such a heterogeneous disease. However, between 50-70% of TNBC have been shown to express the epidermal growth factor receptor (EGFR) (7,9), and it expression has been associated with poor prognosis (10).
While EGFR inhibition has been considered a promising approach for TNBC, minimum benefit has been observed in the clinical settings alone or in combination with chemotherapy (11,12).
Alteration of metabolism is one of the hallmarks of cancer, providing advantages to cancer cells in the tumor environment (13,14). Fatty acid synthase (FASN), the major enzyme capable of de novo synthesis of fatty acids, has been described to be overexpressed in several carcinomas, such as breast, prostate and colon among others (15). This enzyme is not expressed in normal tissue, except in lipogenic tissues where its expression is tightly regulated by diet. In cancer, however, cells overexpress FASN enzyme to synthesize almost all their fatty-acids de novo (14,16). We and others have shown that blocking FASN activity has anticancer activity via the apoptotic pathway in vitro and in vivo (17)(18)(19), by disrupting lipid membrane synthesis, modification of protein palmitoylation and deregulating oncogenic signaling pathways (20, 21).
The purpose of this study was to evaluate FASN as a target or co-target in sensitive-and chemoresistant-TNBC. First, we evaluated FASN expression in a panel of TNBC cell lines and in TNBC primary tumor samples. In vitro, C75 and EGCG (FASN inhibitors) were tested for anti-tumor efficacy in combination with chemotherapy and with cetuximab (an EGFR inhibitor). In vivo orthoxenografts of two different sensitive and resistant TNBC models were performed to test the anti-tumor effect of EGCG in combination with cetuximab. FASN

Western blot analysis of cell and tumor lysates
Parental and resistant TNBC cells were synchronized by starvation in serum-deprived medium (0.5% FBS) for 24 hours. Cells were lysed in ice-cold lysis buffer (Cell Signaling Technology, Inc.) with 100 μg/mL PMSF by vortexing every 5min for 30 min. Frozen tumors were first ground (Dounce) in ice-cold lysis buffer and then vortexed as described above. Equal amounts of protein were heated in LDS Sample Buffer with Sample Reducing Agent (Invitrogen) for 10 min at 70°C, electrophoresed on SDS-polyacrylamide gel (SDS-PAGE), and transferred onto nitrocellulose membranes. Blots were incubated for 1h in blocking buffer (5% powdered-skim milk in Phosphate-buffered saline 0.05% Tween (PBS-T)) and incubated overnight at 4°C with the appropriate primary antibodies diluted in blocking buffer. Specific horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated for 1h at room temperature. The immune complexes were detected using a chemiluminescent HPR substrate [Super Signal West Femto (Thermo Scientific Inc.) or Immobilon Western (Millipore)]. β-actin (Santa Cruz Biotechnology Inc.) was used as a control of protein loading. Western blot analyses were repeated at least three times and representative results are shown.

Cell viability assays
Parental and resistant cells were plated in 96-well plates at a cell density of 4 x 10 3 cells per well in their corresponding growth medium. After  in combination with a serial of concentrations of (i) doxorubicin for 48 h or (ii) cetuximab for 4 days. Same treatments were assessed in monotherapy. Following treatment, cell viability was measured using the standard colorimetric MTT assay as previously described (19). Combinatorial effects were evaluated using the Interaction index (Ix) = ∑( % CPI drug alone) / % CPI combination. Ix <1, synergism; Ix =1, additivism and Ix >1, antagonism.

Quantitative real-time PCR analysis
Cells were PBS washed, and then 1mL of Qiazol (Qiagen) was added. Total-RNA was Institute (University of Utah, Salt Lake City, Utah, USA) and at the Oklahoma Medial Research Foundation (ORMF, Oklahoma, USA). All protocols and experiments involving animals, including care and handling, were conducted in accordance with guidelines on animal care and use established by institutional guidelines. All mice were maintained in a specific pathogen-free facility with controlled light/dark cycle, temperature, and humidity. All surgery was performed under inhaled isoflurane anesthesia; burprenorphine was administered as analgesic to mice after surgery. All efforts were made to minimize pain and distress.

Statistical analysis
Data were analyzed by Student's t-test when comparing two groups or ANOVA using a Bonferrony post-hoc test when comparing more than 2 groups. Non-Parametric analysis by Kruskal Wallis was used when data did not follow normal distribution. Statistical significant levels were p < 0.05 (denoted as *), p < 0.01 (denoted as **) and p < 0.001 (denoted as ***). P-value is shown in results when significance is reached (p < 0.05).
All data are means ± standard error of the mean (SEM). All observations were confirmed by at least three independent experiments.

Triple negative breast cancer cell lines express fatty acid synthase (FASN) enzyme and are sensitive to FASN inhibitors
We screened several TNBC cell lines for FASN, EGFR and downstream protein expression and activation status (    Fig. S3). Interestingly, no changes in sensitivity to FASN inhibitors alone (C75 or EGCG) were observed between parental and doxorubicin resistant cell lines ( Supplementary Fig. S4).

FASN inhibition resensitizes doxorubicin resistant models to chemotherapy
Resistant cell lines 231DXR and HCCDXR showed similar FASN expression levels and FASN inhibitor sensitivity when compared to parental cells ( Fig. 1C and Supplementary Figure S4). Therefore, we tested the effects of EGCG and C75 in combination with doxorubicin in parental and doxorubicin-resistant models. The results of CPI ratios induced by the mono treatments versus the dual treatments are shown in Table 1  Several studies reported that EGFR is a common receptor expressed in TNBC (23)(24).
In vitro, EGFR activation is observed in almost all TNBC cell lines (Fig 1A), and 70% of the TNBC tumors evaluated in this study were positively stained for EGFR protein ( Fig 1B). Furthermore, we have shown that the expression of EGFR is either maintained or increased in our doxorubicin-resistant models ( Fig 1C). Therefore, the effect of combining cetuximab and the FASN inhibitors C75 and EGCG was studied.
Control animals achieved a relative volume growth of 3.3±0.33, whereas EGCG reduced it to 3.0±0.27 and cetuximab to 2.5±0.29. Interestingly, dual FASN and EGFR blockade showed significantly higher tumor growth inhibition (2.0±0.07) compared to the control (p <0.01) or EGCG alone (p<0.05, Fig. 2A). Under the same schedule, 231DXR xenograft showed a similar relative volume growth as the sensitive model weight, no significant changes on food and fluid intake or body weight after treatment were identified (Fig. 2F).

Dual blockade of FASN and EGFR changes EGFR downstream activated proteins
We have shown that EGCG combined with cetuximab was synergic in several cellular and animal models of TNBC. Therefore, we planned to study the effect of the EGCG plus cetuximab on FASN, EGFR, and downstream protein expression and activation, alone or in combination, in sensitive (231 and HCC) and resistant (231DXR and HCCDXR) cells (Fig. 3).
Cetuximab as a single agent induced a decrease in total EGFR in 231, 231DXR and HCCDXR cellular models. On the other hand, the same treatment increased total EGFR in HCC. While cetuximab treatment decreased p-EGFR levels in HCC and HCCDXR, 231 and 231DXR showed overactivation of p-EGFR after 72h of treatment. Previously described studies suggest that this activation may be likely to receptor homodimerization and autophosphorylation, and does not activate downstream proteins (22). Protein levels of AKT, ERK1/2 were maintained after cetuximab treatment in all cellular models. Regarding p-AKT activation levels, lower levels were observed compared to control in 231DXR and HCCDXR after 72h of treatment. 231 and HCC, however, showed similar or increased p-AKT levels compared to control after 72h of treatment respectively. Cetuximab treatment induced apoptosis in 231 and 231DXR after 72h, while cleaved PARP is observed already after 48h in HCCDXR. Total PARP increases after cetuximab treatment in HCC, but cleaved PARP was not observed. In summary, the combination of cetuximab and EGCG enhances the inhibitory effect observed in the EGFR pathway by single treatments, with a general decreasing of EGFR, p-EGFR and p-AKT levels in all cellular models. The activation of ERK1/2 observed after EGCG treatment was decreased or inhibited after combination treatment.

FASN and EGFR are expressed in TNBC primary tumor samples
To further validate FASN as a potential target in TNBC,

Discussion
Triple Negative Breast Cancer (TNBC) lacks an approved targeted therapy, and it is mainly treated with a combination of anthracyclines and taxanes (25). The high rate of relapse after treatment urges for the development of new targeted therapies for these patients. TNBC breast cancer is well known for its heterogeneity (8). Here we show that After four decades, doxorubicin is still the most used and effective chemotherapy agent in the clinic for the treatment of breast cancer (28). For this reason, two doxorubicinresistant models representative of the two major molecular subtypes in TNBC, 231DXR (ML) and HCCDXR (BL) were developed in our laboratory. Both models showed significant resistance when exposed to high doses of doxorubicin compared to the parental cell lines. Several mechanisms have been described for doxorubicin resistance in the last few years (28,29), some of them regarding changes in the EGFR pathway.
HCCDXR showed overexpression of EGFR and overactivation of AKT and ERK1/2 when compared to its parental cell line. Overexpression of EGFR in breast cancer patients after chemotherapy has been already described to be related with higher local recurrence, metastasis and so poor outcome (30,31). On the other hand, AKT and ERK1/2 have been shown to be overactivated and also linked to chemoresistance in several cancers (32,33). Interestingly, similar levels of FASN were observed in parental cell lines compared to their resistant derivate (DXR). Recent studies pointed out that FASN activity plays an important role in drug resistance because it allows fast synthesis of new phospholipids for membrane remodeling and plasticity (34,35). Here, we performed combinatorial experiments with doxorubicin and the FASN inhibitors C75 or EGCG in our sensitive and resistant cell lines to determine whether FASN inhibition may be useful to overcome doxorubicin resistance in TNBC. Although the EGCG antitumor effects may be due to binding probably with other cellular targets than FASN (36), we and others have previously identified EGCG as an inhibitor of FASN activity, able to induce apoptosis in several tumor cell lines and also to reduce the size of mammary tumors in animal models (17)(18)(19)37,38). Our results showed that the Interaction index for both combinations was improved in both resistant models (231DXR and HCCDXR) compared to the parental lines. Interestingly, C75 and EGCG showed same CPI between parental or DXR derivate when treated alone, suggesting Several studies have shown that EGFR is overexpressed in TNBC (22,23). Our results showed a high percentage of immunostaining positivity for EGFR in the core biospies from TNBC patients, in concordance with previous findings. The anti-EGFR compound cetuximab showed low levels of CPI in vitro in all cell lines except for HCCDXR, which showed significantly increased CPI and apoptosis compared to the parental cell line. In vivo, while none, low or moderate antitumor effect was observed in 231DXR, 231 and HCC xenografts, respectively, cetuximab treatment obtained almost complete tumor shrinkage after two cycles of treatment in HCCDXR. These results are consistent with the EGFR overexpression in HCCDXR cell line, probably as an adaptation to overcome doxorubicin-induced apoptosis. Paradoxically, this mechanism of chemoresistance could sensitize the cell line to anti-EGFR treatments. This is in agreement with the results of a phase II study combining cetuximab with cisplatin in metastatic TNBC, where the overall response rate was doubled and the progression free survival increased from 1.5 to 3.7 months when compared to cisplatin monotherapy (41).
The common overexpression of EGFR in TNBC and its relationship with cancer progression after therapy, together with FASN expression in all TNBC and in DXR models, provided the rationale to test combined FASN inhibitors and EGFR blockade in this these settings. We found that simultaneous treatment of cetuximab and EGCG or C75 resulted in strong synergism in both parental and resistant models. Strong cooperative growth inhibition between FASN and EGFR have been also observed in vitro for ovarian cancer cell lines (42). The synergism between FASN and EGFR may be due to the mechanisms involving FASN blockade that results in blockage of lipid synthesis, lipid raft destabilization, and further EGFR degradation (42). Furthermore, EGCG has been described to be closely associated with destabilization and inhibition of EGFR receptor and downstream signaling pathways (43,44). In addition, Bollu et al. recently found that FASN-dependent palmitoylation of EGFR is required for EGFR dimerization and kinase activation (45). The same authors, in a previous study, described that FASN-dependent palmitoylation also activates mitochondrial EGFR and promotes cell survival, while its inhibition leads to apoptosis (46). Furthermore, phosphorylation and activation of FASN by direct interaction with members of the EGFR family have already been described (46).
These in vitro results together with TNBC patient sample study, supported the evaluation of the antitumor efficacy of cetuximab in combination with the FASN inhibitor EGCG in doxo-sensitive and -resistant TNBC orthoxenograftx in vivo. We and others have demonstrated that EGCG displays anti-tumor activity without affecting food intake or weight loss, which are common side effects of other FASN inhibitors (37,38).
We have previously shown that EGCG produces apoptosis in vitro and in vivo in breast cancer (19,37,38). Here, EGCG showed strong CPI and high apoptosis in vitro, and moderate anti-tumour activity in vivo. EGFR inactivation and downregulation is observed in all cell lines after EGCG treatment in vitro, consistent with previous studies (43,44,48). At the same time, slightly decreases in FASN levels were observed in all cell lines, suggesting that the EGFR pathway could be implicated in FASN expression regulation in TNBC (38,49). Furthermore, p-AKT decreased its activation levels, while p-MAPK increased considerably after EGCG treatment, maybe due to a negative crosstalk between these two proteins (33,42,38).
Here, we clearly validate our in vitro results by showing that the combination of EGCG with the EGFR inhibitor cetuximab results in synergistic tumor growth reduction in both doxorubicin-sensitive and resistant xenograft models. Tumor analysis showed decreased EGFR and p-EGFR in the cetuximab and combination settings in all models. However, this event was not observed in the EGCG treatment in vivo possibly due to the unstable nature of EGCG in serum (50). Nevertheless, increased apoptosis was observed in the combination settings in all models compared to the single treatments, even after only one cycle in the HCC and HCCDXR xenograft. In vitro study interactions also reveal that p-ERK1/2 levels decreased in the combination settings in the HCC and HCCDXR cell lines, and reduced the loop effect in p-ERK1/2 in the combination treatment in 231 and 231DXR.
In summary, we found FASN is expressed in all TNBC primary tumor samples tested.
We then showed therapeutic benefit of FASN inhibition in sensitive and chemoresistant TNBC preclinical models, as well as strong synergistic anti-tumor effect of FASN inhibitors in combination with cetuximab. Our findings provide a rationale for further investigation of FASN inhibitors in combination with anti-EGFR signaling agents in TNBC and TNBC who progressed after chemotherapy.