Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer.

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. Significance: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.

As a staple food and dense source of nutrients, milk and alternatives play an important role in nutrient adequacy. The aims of this study were to quantify the consumption of milk and alternatives within Canadian self-selected diets and determine their contribution to intakes of nutrients and energy. First, 24-h dietary recalls from the 2015 Canadian Community Health Survey—Nutrition were used to assess 1-d food and nutrient intakes among Canadian adults ≥19 y (n = 13,616). Foods were classified as milk and alternatives according to the 2007 Canada’s Food Guide. Descriptive statistics were used to calculate daily servings of milk and alternatives by different age groups and demographic characteristics. Population ratios were used to discern their contribution to total intakes of nutrients and energy. Mean daily servings (±SE) were highest for milk (0.60 ± 0.02) and cheese (0.42 ± 0.01), intermediate for frozen dairy (0.16 ± 0.01) and yoghurt (0.14 ± 0.01), and lowest for soy and other dairy (20% to total intakes of calcium (52.62 ± 0.46%), vitamin D (38.53 ± 0.78%), saturated fat (28.84 ± 0.51%), vitamin B12 (27.73 ± 0.57%), vitamin A (26.16 ± 0.58%), phosphorus (24.76 ± 0.35%), and riboflavin (24.43 ± 0.37%), of which milk was the top source. Milk and alternatives contribute substantially to nutrient intakes and thus warrant further attention in terms of mitigating nutrient inadequacy among the Canadian population.

The 2019 Canada’s Food Guide (CFG) emphasizes consumption of plant protein with implications for protein adequacy and nutrient intakes, yet a baseline with which to compare future dietary trends that may result from its adoption is not available. The objectives were to assess usual protein intake, inadequacy, and the contribution of animal- and plant-based foods to intake of protein, nutrients, and energy in Canada. Twenty-four-hour dietary recalls from the 2015 Canadian Community Health Survey – Nutrition were used to assess dietary intake among adults (n = 13 616). The National Cancer Institute method was used to estimate usual protein intake and inadequacy. Population ratios were used to determine the contribution of animal- and plant-based foods to intake of protein, nutrients, and energy. Usual protein intake averaged 79.47 ± 0.70 g/d; inadequacy was highest for females ≥71 y (9.76 ± 2.04%). Top protein contributors were red and processed meat (21.6 ± 0.55%), poultry and eggs (20.1 ± 0.81%), cereals, grains, and breads (19.5 ± 0.31%), and dairy (16.7 ± 0.38%). Dairy contributed most to calcium (53.4 ± 0.61%), vitamin D (38.7 ± 1.01%), but also saturated fat (40.6 ± 0.69%), whereas cereals, grains, and breads contributed most to iron (46.5 ± 0.57%) and vegetables and fruit to potassium (32 ± 0.45%). Given that animal sources contributed overwhelmingly to protein intake in 2015, dietary shifts towards plant protein needed to meet the 2019 CFG recommendations may pose a challenge, particularly for populations most at risk of inadequacy. Novelty: Older adults and females are most at risk of not meeting protein recommendations. Animal sources contribute two-thirds of the protein consumed by Canadian adults.