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A precision medicine approach to metabolic therapy for breast cancer in mice

Increasing evidence highlights approaches targeting metabolism as potential adjuvants to cancer therapy. Sodium-glucose transport protein 2 (SGLT2) inhibitors are the newest class of antihyperglycemic drugs. To our knowledge, SGLT2 inhibitors have not been applied in the neoadjuvant setting as a precision medicine approach for this devastating disease. Here, we treat lean breast tumor-bearing mice with the SGLT2 inhibitor dapagliflozin as monotherapy and in combination with paclitaxel chemotherapy. We show that dapagliflozin enhances the efficacy of paclitaxel, reducing tumor glucose uptake and prolonging survival. Further, the ability of dapagliflozin to enhance the efficacy of chemotherapy correlates with its effect to reduce circulating insulin in some but not all breast tumors. Our data suggest a genetic signature for breast tumors more likely to respond to dapagliflozin in combination with paclitaxel. In the current study, tumors driven by mutations upstream of canonical insulin signaling pathways responded to this combined treatment, whereas tumors driven by mutations downstream of canonical insulin signaling did not. These data demonstrate that dapagliflozin enhances the response to chemotherapy in mice with breast cancer and suggest that patients with driver mutations upstream of canonical insulin signaling may be most likely to benefit from this neoadjuvant approach.


INTRODUCTION
Breast cancer is the second leading cause of cancer deaths in women, with one in eight women in the U.S. developing this disease over her lifetime. The incidence of breast cancer is projected to increase more than 50% between 2011 and 2030 (1). This troubling prediction is attributable in part to rising rates of obesity, which accelerates the appearance and progression of breast cancer in postmenopausal women (2,3). Similarly, primary tumor appearance (4)(5)(6), volume (5,(7)(8)(9), and metastasis (7)(8)(9)(10)(11) increase with a high-calorie diet in implanted, genetic, and chemicallyinduced mouse models of breast cancer.
Metabolism-targeting neoadjuvant approaches have gained increasing popularity in breast cancer treatment. The biguanide metformin, an inhibitor of gluconeogenesis (12)(13)(14) and the most prescribed antidiabetes drug worldwide (15), has been by far the most popular antihyperglycemic approach used for cancer. At supra-pharmacologic doses -several orders of magnitude higher than in human patients -metformin is capable of reducing breast cancer cell proliferation (16)(17)(18)(19), and slowing tumor growth in vivo in rodents (20)(21)(22). Metformin is safe and shows good target engagement in women with breast cancer. Studies regarding its efficacy, however, have been mixed, with most (23)(24)(25)(26)(27)(28)(29), but not all, adjuvant and neoadjuvant studies (30)(31)(32) failing to observe differences in tumor cell proliferation markers or in outcomes in human patients.
Preclinical data highlight the newest class of diabetes drug, SGLT2 inhibitors, as a potentially attractive alternative to metformin therapy for breast cancer. As metformin acts primarily to inhibit gluconeogenesis, its ability to lower blood glucose and insulin concentrations is likely minimal under postprandial conditions in individuals without diabetes. SGLT2 inhibitors, by contrast, cause glycosuria and, therefore, glucose wasting under both fasting and postprandial conditions. We recently demonstrated that the SGLT2 inhibitor dapagliflozin slows breast tumor growth when administered as monotherapy in obese mice, and that its anticancer effect was reliant upon its ability to reverse fasting hyperinsulinemia (33). A direct effect of SGLT2 inhibition in tumors cannot be ruled out, because data from the Human Protein Atlas (https://www.proteinatlas.org/about/licence) indicate that breast tumors do express SGLT2, albeit at low levels (34). However, considering data demonstrating that chronically infusing insulin to abrogate the insulin-lowering effects of dapagliflozin fully abrogated its beneficial effects, it is most likely that the tumor suppressive effects of dapagliflozin are likely attributable to its ability to reduce insulin-stimulated tumor glucose uptake (33). However, to our knowledge, SGLT2 inhibitors have never been administered in combination with chemotherapy in in vivo breast cancer models. This is a point of substantial clinical importance because breast cancer treatment regimens are effective, but not unanimously so: response rates are particularly low in estrogen receptor-positive breast cancer (35). Thus, new neoadjuvant approaches are critical to improve the efficacy of standard-of-care breast cancer treatment.
In addition, at a time when precision medicine approaches are gaining increasing popularity in selecting targeted therapies for patients. Such approaches, however, have largely failed to investigate and target metabolism. In this study, we aimed to generate a genetic signature for breast tumors responsive to dapagliflozin as an adjunct to chemotherapy. In vivo studies in seven murine models of breast cancer with different driver mutations demonstrated a genetic signature of those tumors that responded to dapagliflozin: in tumors with driver mutations upstream of the insulin receptor, dapagliflozin improved the efficacy of paclitaxel, whereas in tumors with driver mutations downstream of the insulin receptor, dapagliflozin was ineffective. These data predict that tumor genetics may be utilized to design metabolism-targeting neoadjuvant treatments for patients with hyperinsulinemia.

Dapagliflozin reduces insulin more than other common diabetes drugs
Our previous studies have shown plasma insulin concentrations to be a predictor of tumor growth rates in colon (36) and breast cancer (33). Therefore, we first aimed to determine which commonly prescribed antihyperglycemic medications would most significantly reduce both fasting and postprandial plasma insulin concentrations. The biguanide metformin, the glucagonlike peptide-1 agonist semaglutide, the thiazolidinedione pioglitazone, and the sulfonylurea glipizide all showed some ability to lower plasma glucose concentrations. The SGLT2 inhibitor dapagliflozin, however, lowered plasma insulin more substantially than any of the other agents in healthy mice. Moreover, dapagliflozin was the only agent of the five tested to lower both fasting and postprandial plasma insulin ( Figure 1A-B). For this reason, we selected dapagliflozin for future studies in murine models of breast cancer.

Dapagliflozin slows tumor growth in lean and obese MMTV-PyMT mice
In previous work, we have shown that dapagliflozin slowed E0771 breast cancer growth when applied as monotherapy in obese mice (29). Here, we examined for the first time whether dapagliflozin would slow tumor growth in lean MMTV-PyMT mice, a murine breast cancer model driven by the polyoma virus middle T antigen and commonly expressing an Erbb2 mutation. We found that, as expected, SGLT2 inhibition caused glucose wasting in urine, suppressing weight gain without causing ketosis (Supplementary Figure 1A Figure 1D). These reductions in tumor glucose uptake correlated with a profound effect of metabolic manipulation on tumor growth: whereas both glucose uptake and tumor growth were accelerated in Western diet fed mice as compared to chow fed, dapagliflozin slowed tumor growth in both lean and obese animals ( Figure 2F).

Dapagliflozin slows 4T1 tumor growth in lean and obese mice
Next, we studied a second murine tumor model, 4T1 subcutaneous breast cancer, which is driven by a p53 mutation (34,35). BALB/c mice treated with Western diet did become overweight (Supplementary Figure 2A), and both hyperglycemic and hyperinsulinemic but not ketotic ( Figure 3A Figure 2D). Taken together, these data argue against a direct effect of dapagliflozin to inhibit, or adipose-derived fatty acids to promote, breast cancer growth.

Dapagliflozin enhances efficacy of chemotherapy in mice with breast cancer
Although our and others' preclinical data demonstrating an effect of SGLT2 inhibitors to slow tumor growth are promising, dapagliflozin should and will never be administered as monotherapy for breast cancer. We next examined the impact of dapagliflozin in mice treated with standard-of-care paclitaxel. Both paclitaxel and dapagliflozin reduced tumor glucose uptake and improved survival in both MMTV-PyMT and 4T1 breast cancer models. However, dapagliflozin and paclitaxel showed synergistic effects against breast cancer: the two drugs showed additive effects to reduce tumor glucose uptake and improve survival in MMTV-PyMT and 4T1 tumor-bearing mice ( Figure 4A-D).

Dapagliflozin does not cause fatigue, weight loss, or neuropathy
In considering dapagliflozin as a potential neoadjuvant for future study in humans, it is crucial to examine the possibility that it may enhance existing side effects of paclitaxel chemotherapy. To that end, we assessed the impact of dapagliflozin on anorexia and on neuropathy, both common adverse effects of chemotherapy treatment. Encouragingly, we found that dapagliflozin did not cause fatigue as indicated by spontaneous activity, reduce food intake, or alter thermal latency as monotherapy or when added to chemotherapy in either MMTV-PyMT or 4T1 tumor-bearing mice ( Figure 5A-H). This demonstrates that dapagliflozin might hold promise as a safe and effective adjuvant to standard-of-care chemotherapy in breast cancer.

Driver mutations predict the response to dapagliflozin in breast cancer
Precision medicine is an increasingly attractive arena. To determine whether certain tumor drivers may be more likely than others to respond to dapagliflozin, we examined the impact of this agent on survival in obese mice with tumors driven by five additional mutation profiles (Supplementary Table 2). We observed a genetic signature of tumors responding to SGLT2 inhibition. While dapagliflozin did not synergize with paclitaxel in mice with p53-driven M6, MYC-driven M158 tumors, or MEK1-driven EpH4 1424 tumors with Brca1-driven tumors, it prolonged survival in mice with Pten-driven EMT6 tumors and HRAS-driven Ac711 tumors ( Figure 6A-E), in addition to ERBB2-driven MMTV-PyMT, and p53 and Pik3ca-driven 4T1 tumors ( Figure 4B, Figure 4D). Taken together, these data suggest that tumors with mutations upstream of canonical insulin signaling responded to dapagliflozin as an adjuvant to chemotherapy, whereas tumors driven by mutations downstream of canonical insulin signaling did not ( Figure 7). These data highlight opportunities for the development of precision medicine approaches to utilize metabolic adjunct therapies for breast cancer informed by tumor genetics.

DISCUSSION
Despite the undisputed epidemiologic link between excess weight and numerous cancers, including breast cancer, clinical trials examining the effect of metabolic therapy (almost exclusively, metformin) against cancer have been under-pursued and underwhelming. This may be at least in part to metformin's underwhelming effect to reduce plasma insulin concentrations by less than 20% (37, 38) ( Figure 1B). Given its higher potential efficacy, in this study, we examined the impact of dapagliflozin as an adjunct in combination with chemotherapy. We treated mice with breast cancer driven by a complement of mutations with dapagliflozin in combination with paclitaxel chemotherapy.
Importantly, without enhancing adverse effects of paclitaxel, dapagliflozin improved the efficacy of paclitaxel to promote neuropathy, but it improved the efficacy of chemotherapy to slow tumor growth in murine breast cancer models with mutations in pathways upstream, but not downstream, of canonical insulin signaling ( Figure 7). These data suggest that precision medicine approaches to neoadjuvant treatment are of pressing importance. Indeed, a recent meta-analysis hinted at a differing effect of obesity on outcomes based on cancer subtype: Lohmann and colleagues observed a larger magnitude of the deleterious effect of excess body weight in triple-negative and hormone receptor-positive, HER2-negative breast cancer as compared to hormone receptorpositive, HER2-positive breast cancer (39). However, these data stop short of suggesting specific breast cancer driver mutations that may be more or less responsive to therapies aiming to reduce circulating insulin concentrations. Therefore, in this study we aimed to determine a genetic signature for responsiveness to dapagliflozin in mice with breast cancer.
The primary advance of the current study is its comprehensive assessment of which genetic drivers of breast cancer may be most responsive to SGLT2 inhibitors and perhaps other insulin-lowering agents. Our data demonstrate a genetic signature for tumors in which dapagliflozin improved the response to chemotherapy: E0771, 4T1, EMT6, and Ac711 tumors are driven by proteins upstream of PI3K/Akt, key mediators of insulin signaling in health (40) and disease (41,42). Dapagliflozin Further emphasizing the physiologic relevance of dapagliflozin's inhibition of the insulin signaling pathway, matching plasma insulin concentrations in dapagliflozin-treated 4T1 and MMTV-PyMT tumor-bearing mice to concentrations measured in untreated controls abrogated the beneficial effect of dapagliflozin to enhance the efficacy of chemotherapy in these animals. While we cannot completely rule out a direct effect of dapagliflozin to reduce tumor glucose uptake independently of insulin, two data sets argue against this interpretation. First, we observed no difference in tumor growth rates between untreated controls and mice treated with dapagliflozin plus insulin ( Figures   2F, 3D). This suggests that adding insulin undermines the efficacy of the SGLT2 inhibitor in slowing tumor growth. If the SGLT2 inhibitor were acting directly to reduce tumor glucose uptake, insulin infusion would not be expected to fully abrogate the effect of dapagliflozin. Second, the mutational signature of tumors responsive to dapagliflozin -that is, those with mutations downstream of canonical insulin signaling -argues against a direct effect of dapagliflozin on the tumor. If the "direct action" hypothesis were correct, one would predict a similar effect of SGLT2 inhibition to slow tumor growth regardless of whether the driver mutation is related to insulin and/or glucose metabolism.
An additional key point in this study is the efficacy of dapagliflozin in lean animals. Although both canagliflozin (33,43,44), dapagliflozin (33), empagliflozin (45), and impragliflozin (46)  Relatedly, fasting hyperinsulinemia can occur in those without diabetes and even without impaired glucose tolerance (47) and is rapidly on the rise in the U.S. and worldwide. Between the National Health and Nutrition Examination Surveys conducted between 1988-1994 and 1999-2002, for instance, the age-adjusted incidence of fasting hyperinsulinemia increased by 35% (48). In the last two decades, the prevalence of hyperinsulinemia even in those without diabetes has increased, while the prevalence of diabetes also increased by close to 50% in the same time period (49). These data emphasize that there is a critical need to understand therapies that might work for both those with diabetes and those with hyperinsulinemia without diabetes.
In summary, we demonstrate here that the SGLT2 inhibitor dapagliflozin improves the efficacy of chemotherapy to slow breast tumor growth in a tumor driver-dependent manner. We reveal that mice with breast cancer driven by mutations upstream of the PI3K/Akt insulin signaling pathway were responsive to dapagliflozin, while those driven by mutations downstream of PI3K/Akt or in pathways with other driver mutations did not. These data support the development of precision medicine, insulin-lowering approaches to breast cancer in hyperinsulinemic patients, with or without diabetes, and position SGLT2 inhibitors as an attractive target to fill this niche. All mouse studies were approved by the Yale University Animal Care and Use Committee. To determine the appropriate diabetes drug to lower both fasting and postprandial plasma insulin concentrations, female C57bl/6J mice were fed a high fat, high carbohydrate Western diet (Research Diets D12492; 60% calories from fat, 20% protein, 20% carbohydrate; 5.21 kcal/g in combination with 5% sucrose drinking water) for four weeks, after which two weeks of antihyperglycemic drug treatment were initiated. Mice were randomized to receive either vehicle (regular water and daily intraperitoneal injections of PBS), metformin (1 mg/ml in drinking water; approximate daily dose 200 mg/kg), dapagliflozin (0.0125 mg/ml, approximate daily dose 2.5 mg/kg), semaglutide (50 nmol/kg/day by subcutaneous injection), pioglitazone (10 mg/kg/day by intraperitoneal injection), or glipizide (0.5 mg/ml; approximate daily dose 100 mg/kg). After two weeks, blood glucose and plasma insulin were measured in the same mice after fasting for 4 and 16 hrs.

Study Design
For the tumor studies, C57bl/6J, BALB/c, FVB, and MMTV-PyMT mice were purchased from Jackson Laboratories at 7 weeks of age. Upon arrival, a randomly selected subset was given a Western diet, while the remaining mice were given regular chow (ENVIGO-Teklad 2018; 18% calories from fat, 24% protein, 58% carbohydrate; 3.10 kcal/g) and regular water. Water intake was monitored twice weekly for each cage of five mice by weighing water bottles. Two weeks after arrival, mice were injected with tumor cells into the right mammary fat pad as shown in Supplementary Table 1. All mice -whether they had spontaneous or orthotopic tumors -were monitored twice weekly until they developed palpable tumors, and daily thereafter. Chemotherapy (intraperitoneal paclitaxel, 15 mg/kg twice weekly) was initiated when tumors reached 300 mm 3 , measured using calipers. A subset of mice in both the chow and Western diet fed groups was randomized to receive dapagliflozin in drinking water beginning the day of chemotherapy induction, with the dapagliflozin concentration adjusted for the calculated daily water intake so that each mouse would receive approximately 2.5 mg/kg/day. A subset of otherwise untreated, tumor-bearing mice was subjected to acute dapagliflozin treatment: two doses of dapagliflozin (2.5 mg/kg per dose), each a day apart, by oral gavage. Terminal studies were performed 6 hr after the second dose.
The infusion rates were selected to match plasma insulin concentrations in 4 hr fasted dapagliflozin-treated mice to those measured in the diet-matched controls: 18 mU/kg/hr in chow fed mice, and 40 mU/kg/hr in Western diet fed mice. Mice with 4T1 and MMTV-PyMT tumors were monitored, and tumor size measured by a blinded investigator, twice weekly. Body weight was measured weekly. Food intake and activity were measured in Columbus Lab Animal Monitoring System metabolic cages and averaged over three days during week 3 of treatment.
Thermal algesia was assessed by placing mice on a hot plate (IITC Life Sciences) at 52°C. Thermal latency was defined as the time elapsed before the mouse withdrew its back paw(s), jumped, or scaled the wall of the hot plate enclosure. Mice with Ac711, EMT6, Eph4 1424, and M158 tumors were monitored daily to determine survival. Failure to right was considered a surrogate marker for mortality.

Flux Studies
One week prior to terminal isotope tracer studies, mice underwent surgery under isoflurane anesthesia to place a catheter in the right jugular vein. Mice were allowed a week of post-surgical recovery, during which they received daily intraperitoneal non-steroidal anti-inflammatory (Carprofen, 5 mg/kg per day) providing analgesic coverage for the first 72 hr after surgery.
Terminal studies were performed following a 6 hr fast. Animals were placed in restrainers and their tails secured lightly using tape, allowing them some space to move around. After 2 hr to acclimate to the restrainer, a 1.0 mCi bolus of [2-14 C] 2-deoxyglucose was administered through the catheter. Blood was obtained from the tail vein every 10 min following 2-deoxyglucose injection, and blood glucose concentrations were measured using a handheld glucometer. 60 min after radioisotope tracer injection, mice were euthanized using IV Euthasol (50 mg/kg). Tumors were freeze-clamped in tongs prechilled in liquid nitrogen and stored at -80°C pending further analysis.
Glucose uptake was measured as described previously (50,51). . BOHB concentrations were measured by GC/MS run in chemical ionization mode, with peak areas for 12 C BOHB compared to peak areas for 13 C4 BOHB at a known concentration.

Statistical Analysis
Survival analyses were performed using the Mantel-Cox logrank test, adjusting for multiple comparisons. Tumor size, fluxes, and biochemical parameters were compared by ANOVA with Tukey's multiple comparisons test. The mean±S.E.M. are shown.