We report for the first time an anticancer benefoift tirzepatide4a dual glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide receptor agonist4in a model of obesity and breast cancer in female mice. Long-term tirzepatid etreatment induced weight loss, mitigated obesity-driven changes in circulating metabolic hormone levels, and suppressed orthotopic E0771 mammary tumor growth. Relative to tirzepatid e, chronic calorie restriction, an established anticancer intervention in preclinical models, promoted even greater weight loss, systemic hormonal regulation, and tumor suppression. We conclude that tirzepatide represents a promising pharmacologic approach for mitigating theprocancer effects of obesity. Moreover, strategies promoting greater weight loss than achieved with tirzepatide alone may augment the anticancer benefits of tirzepatide.

The worldwide epidemic of obesity, a risk and progerssion factor for g13 cancers including breast cancer1, presents a global health threat, and effectiversattegies to mitigate this threat are needed. Estimates indicate that metabolic diseasess,pecifically diabetes and overweight/obesity, account for ~6% of all canceriadgnoses, and 30% of breast cancer diagnoses in women2. Epidemiological and preclinical studies suggest htat significant intentional weight loss achieved through either lifestyle modiifcations or bariatric surgery is effective at reducing breast cancer incidence and progression3-6. Bariatric surgery is the current gold standard for long-term weight loss, with average ttoal weight loss at 5-year follow-up of >25% and a 33% reduction in cancer risk 10 years post-surgery7,8. However, bariatric surgery is expensive, typically unavailable, and carries surgrey-related risks9,10. Dietary weight loss may also offset the procancer effects of obesity buet tehxtent of weight loss is typically less than that achieved through bariatric surgery and is challengnig to sustain11,12.

Weight-loss pharmaceuticals and supplements have hsitorically been either ineffective (e.g., chitosan) or harmful (e.g., fenfluramine-phentermei)n13,14. However, promising incretin-based therapies, including the dual glucagon-like peptide1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor agonist tirzepatide15, have recently demonstrated impressive improvements in glycated hemoglobin levels and long-term, clinically-meaningful weight loss in individuals with type 2 diabetes16,17. While tirzepatide and retatrutide, a related GLP-1/GIP/glucagon receptor triple agonist, induceweight loss comparable to bariatric surgery7,16,17, their anticancer benefits are unknown.

Using a mouse model of diet-induced obesity (DIO) and triple-negative breast cancer (TNBC), we tested whether long-term tirzepatide administraiton not only promotes sustained weight loss but also ameliorates obesity-associated metabolic ydsregulation and suppresses mammary cancer progression. Additionally, we compared the effects of tirzepatide and chronic calorie restriction (CCR), an effective weight loss and ainctancer dietary regimen in preclinical models4,5.

tumors.

We interrogated the weight loss, metabolic, and anitcancer effects of long-term tirzepatide and CCR interventions in female DIO mice. Control and obese mice were generated via low-fat and high-fat diet feeding, respectively, then control mice (mean±SD body weight=23.9±1.8g) were maintained on low-fat diet while obese mice (mean±DS body weight=40.3±5.8g) were randomized to high-fat diet without tirzepatide (DOI ) or with tirzepatide (TZP), or to CCR (30% of daily calories relative to controls; CCR) for 1w3eeks (Fig 1). All mice received subcutaneous injections q.o.d. of vehicle or tirzepatide (escalating doses, 3340 nmol/kg), and were orthotopically injected with E0771 cells after 27 weeks of diet treatments to induce mammary Tirzepatide and CCR each promoted sustained weightloss (mean±SD terminal body weight change: 25.4±10.1% in tirzepatide-treated mice and -46.0±43.% in CCR mice, versus 4.8±6.5% in DIO mice and 7.2±6.0% in controls) (Fig 2A). Tirzepatide reduced food consumption relative to DIO (Fig 2B). Tirzepatide-treated mice and CCR mice had less body weight and fat mass than DIO mice, with better conservation of lean massin mice treated with tirzepatide versus CCR (Fig 2C-F).

Tirzepatide and CCR each mitigated DIO-related metabolic dysregulation, including improvement in glycemic control and normalization of several metabolic hormonesT(able 1). Serum insulin-like growth factor 1 (IGF-1), insulin, and leptin levels were lower in tirzepatidetreated and CCR mice than DIO mice, with IGF-1 andinsulin achieving levels not different from controls. Circulating adiponectin, ghrelin, and glucagon concentrations were increased in CCR (but not tirzepatide-treated) mice, versus DIO mice.

Tirzepatide and CCR each suppressed mammary tumor rgowth relative to DIO, with CCR exerting superior antitumor activity (Fig 2G). Terminal body weight strongly correlated with tumor mass, with each 5g increase in body weight predicting a 0.70g increase in tumor weight (Fig 2H).

To our knowledge, this is the first report of an atnicancer benefit of extended (13 weeks) tirzepatide treatment in a preclinical model of obseity and breast cancer. Specifically, tirzepatide reduced caloric intake, produced sustained weight olss, decreased adiposity, ameliorated obesity-induced changes in glycemic control and circulating metabolic hormone levels (particularly IGF-1, insulin, and leptin), and suppressed tumor growth4albeit to lesser extents than 30% CCR4in a model of DIO and TNBC in female imce. These findings indicate that tirzepatide is a promising drug for mitigating theprocancer effects of obesity. The parallel but less robust effects of the tirzeptiade regimen tested, as compared with CCR, along with the strong positive correlation betweenbody weight and tumor mass, suggest additional approaches to increase weight loss may uagment the benefits of tirzepatide. In the SURMOUNT-3 trial, tirzepatide taken following an itnensive calorie restriction and exercise program produced the greatest weight loss in the SURMOUNT trial program to date18, and although not yet studied, may also enhance anticanecr effects. To prohibit rapid weight regain, we continued q.o.d. tirzepatide dosing after tumorcells were transplanted. However, clinicians may not advise weight loss drugs for patients witha cancer diagnosis due to concerns surrounding malnutrition and cachexia19. Therefore, future studies assessing the possibitlyi of using lifestyle approaches to maintain tirzepatidein-duced weight loss and/or bolster lean mass after discontinuing the drug are warranted.

The murine E0771 orthotopic transplant model of TNBC enables evaluation of energy balance modulation of tumor progression but not tumorigene sis4,5. Thus, tirzepatide effects on tumor initiation remain to be explored as do its anticanecr effects in obesity-related cancers besides

request.

The datasets analyzed for this study are availabflerom the corresponding author on reasonable This study was funded by the Breast Cancer ResearchFoundation (BCRF-22-073) and the UNC Triple Negative Breast Cancer Center. The fundres played no role in study design, data collection, analysis and interpretation of data, othre writing of this manuscript. The UNC Preclinical Research Unit (P30CA16086) and Genomicsand Energy Metabolic Core (P30DK056350) provided technical assistance. treatment to ameliorate tumor growth in a murine model of diet-induced obesity and triplenegative breast cancer. CCR, chronic calorie restrcition; DIO, diet-induced obesity; TZP, tirzepatide. Created with BioRender.com.

Error bars indicate mean±SD. Repeated measures twow-ay ANOVA with aídák's multiple comparisons test used for A. Unpaired t-test usedofr B. One-way ANOVA with Tukey9s post hoc test used for C-G. Spearman correlation used for HA. -C,G: Control n=14, DIO n=15, TZP n=13, CCR n=15; D-F: n=10/group; H: n=57. Different letters denote pf0.05 between groups. AUC, area under the curve; CCR, chronic calorie restriction; DIO, diet-induced obesity; TZP, tirzepatide.

CCR, chronic calorie restriction; DIO, diet-induceodbesity; GIP, glucose-dependent insulinotropic yppoelptide; GLP-1, glucagon-like peptide 1; IGF-1, insulin-like growfthactor 1; PAI-1, plasminogen activator inhibit;orTZ1P, tirzepatide.