Seventy percent of breast cancers express estrogen receptor (ER) and most of these are sensitive to ER inhibition. However, many such tumors become refractory to inhibition of estrogen action in the metastatic setting for unknown reasons. We conducted a comprehensive genetic analysis of two independent cohorts of metastatic ER+ breast tumors and identified mutations in the ligand binding domain (LBD) of ESR1 in 14/80 cases. These included highly recurrent mutations p.Tyr537Ser/Asn and p.Asp538Gly. Molecular dynamics simulations suggest the Tyr537Ser and Asp538Gly structures lead to hydrogen bonding of the mutant amino acid with Asp351, thus favoring the receptor’s agonist conformation. Consistent with this model, mutant receptors drive ER-dependent transcription and proliferation in the absence of hormone and reduce the efficacy of ER antagonists. These data implicate LBD mutant forms of ER in mediating clinical resistance to hormonal therapy and suggest that more potent ER antagonists may have significant therapeutic benefit.

SUMMARY In PTEN-mutated tumors, we show that PI3Kα activity is suppressed and PI3K signaling is driven by PI3Kβ. A selective inhibitor of PI3Kβ inhibits the Akt/mTOR pathway in these tumors but not in those driven by receptor tyrosine kinases. However, inhibition of PI3Kβ only transiently inhibits Akt/mTOR signaling because it relieves feedback inhibition of IGF1R and other receptors and thus causes activation of PI3Kα and a rebound in downstream signaling. This rebound is suppressed and tumor growth inhibition enhanced with combined inhibition of PI3Kα and PI3Kβ. In PTEN deficient models of prostate cancer, this effective inhibition of PI3K causes marked activation of androgen receptor activity. Combined inhibition of both PI3K isoforms and androgen receptor results in major tumor regressions.

The effects of selective PI3K and AKT inhibitors were compared in human tumor cell lines in which the pathway is dysregulated. Both caused inhibition of AKT, relief of feedback inhibition of RTKs, and growth arrest. However, only the PI3K inhibitors caused rapid induction of cell death. In seeking a mechanism for this phenomenon, we found that PI3K inhibition, but not AKT inhibition, causes rapid inhibition of wild type RAS and of RAF/MEK/ERK signaling. Inhibition of RAS-ERK signaling is transient, rebounding a few hours after drug addition, and is required for rapid induction of apoptosis. Combined MEK and AKT inhibition also promotes cell death and in murine models of HER2+ cancer, either pulsatile PI3K inhibition or combined MEK and AKT inhibition causes tumor regressions. We conclude that PI3K is upstream of RAS and AKT and that pulsatile inhibition of both pathways is sufficient for effective antitumor activity.

I . Male hooded Lister rats given a diet containing 40 mg zinc/kg were described as Znadequate. Other rats were subsequently given diets containing less than I m g Zn/kg. After a period of approximately 5 d these animals ceased to grow and were described as Zn-deficient.2. Zn-deficient rats offered ad lib. a Zn-deficient diet containing 200 g egg albumenjkg ateonly 55 "/o of the weight eaten by Zn-adequate rats given a similar diet supplemented with Zn.The intake of the deficient rats increased when the metabolizable energy content of the diet was decreased and also when the environmental temperature was lowered. 3-Zn-deficient rats offered Zn-deficient diets containing 200 g egg albumen/kg showed a high day-to-day variability of intake. When the albumen content was raised to 400g/kg, neither the mean food intake of the rats nor the variability of food intake changed, but with diets containing only 50 g albumen/kg the quantity eaten increased and the variability of food intake decreased. Results obtained when the low-protein diet was supplemented with essential and non-essential amino acids indicated that increased variability of intake was associated with the essential amino acid content of the diet. The effect on variability of intake was greatest when the supplements contained methionine, phenylalanine, threonine and tryptophan; addition of this group of amino acids to a Zn-supplemented, low-protein diet produced the largest increase in the growth rate of Zn-adequate rats.4. When the food intake of the rats was examined for periods of z h throughout the day, the Zn-deficient rats were found to eat on fewer occasions than the control rats. IIowever, in those periods when the Zn-deficient rats did eat, the quantities eaten in z h showed the same distribution of weights as did those for the Zn-adequate rats.5. There were significant relationships hetween food intake and plasma Zn concentration; the most significant was the negative correlation between food intake in 24 h and plasma Zn concentration at the end of the 24 h period.6. Zn deficiency resulted in a failure of growth in the young rat and therefore in a reduction in its rate of energy expenditure but did not appear to cause directly a loss of appetite. It is suggested that cyclical patterns of food intake associated with Zn deficiency in young rats resulted from the slow but effective control of food intake by the energy balance of the animals.