Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1.

This corrects the article DOI: 10.1038/onc.2016.383.

Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1.

Fibroblasts within the mammary tumor microenvironment are active participants in carcinogenesis mediating both tumor initiation and progression. Our group has previously demonstrated that genetic loss of phosphatase and tensin homolog (PTEN) in mammary fibroblasts induces an oncogenic secretome that remodels the extracellular milieu accelerating ErbB2-driven mammary tumor progression. While these prior studies highlighted a tumor suppressive role for stromal PTEN, how the adjacent normal epithelium transforms in response to PTEN loss was not previously addressed. To identify these early events, we have evaluated both phenotypic and genetic changes within the pre-neoplastic mammary epithelium of mice with and without stromal PTEN expression. We report that fibroblast-specific PTEN deletion greatly restricts mammary ductal elongation and induces aberrant alveolar side-branching. These mice concomitantly exhibit an expansion of the mammary epithelial stem cell (MaSC) enriched basal/myoepithelial population and an increase in in vitro stem cell activity. Further analysis revealed that NOTCH signaling, specifically through NOTCH3, is diminished in these cells. Mechanistically, JAGGED-1, a transmembrane ligand for the NOTCH receptor, is downregulated in the PTEN-null fibroblasts leading to a loss in the paracrine activation of NOTCH signaling from the surrounding stroma. Reintroduction of JAGGED-1 expression within the PTEN-null fibroblasts was sufficient to abrogate the observed increase in colony forming activity implying a direct role for stromal JAGGED-1 in regulation of MaSC properties. Importantly, breast cancer patients whose tumors express both low stromal JAG1 and low stromal PTEN exhibit a shorter time to recurrence than those whose tumors express low levels of either alone suggesting similar stromal signaling in advanced disease. Combined, these results unveil a novel stromal PTEN-to-JAGGED-1 axis in maintaining the MaSC niche, and subsequently inhibiting breast cancer initiation and disease progression.

The effects of acutely administered cocaine on responding maintained by a progressive-ratio schedule of food presentation.

Lever pressing in rats (N=5) was reinforced under a progressive-ratio (PR) schedule of food presentation, in which the number of responses required increased exponentially. The session was terminated when 1 h passed without completion of the scheduled ratio. Doses of cocaine (5.6-42.0 mg/kg; one subject received a dose of 56.0 mg/kg) as well as saline were administered i.p. prior to the session. Under non-drug conditions, breakpoints were typically less than 100, and substantial responding usually occurred only during about the first 10 min of the session. The rate of responding usually increased over the first 2-8 reinforcers and then decreased for the last few reinforcers obtained. For four of five rats, breakpoint, overall rate of response, and session duration were first increased above control and vehicle levels by increasing doses of cocaine. Larger doses produced smaller increases, no effect, or decreases. Cocaine, in the range of doses near the apex of the breakpoint dose-effect functions, suppressed rates of responding at the small ratios present at the beginning of the session. It is suggested that cocaine increases low rates of response if: (1). rates are low due to extinction; and (2). the stimuli present are those present when the response is reinforced.

Antagonism of delta(2)-opioid receptors by naltrindole-5'-isothiocyanate attenuates heroin self-administration but not antinociception in rats.

delta-Opioid receptors have been implicated in reinforcement processes and antagonists are available that produce long-lasting and selective antagonism of delta-opioid receptors in vivo. This experiment assessed the contribution of delta-opioid receptors to the antinociceptive and reinforcing properties of heroin. The effects of the irreversible delta-antagonist naltrindole-5'-isothiocyanate (5'-NTII) were evaluated on heroin self-administration and hot-plate antinociception in rats. 5'-NTII (10 nmol i.c.v.) shifted the dose-response curve for heroin self-administration downward, increasing the A(50) values on the ascending and descending limbs by approximately 0.5 log units and decreasing the maximum by 33%. 5'-NTII (40 nmol i.c.v.) shifted both limbs of the heroin self-administration dose-effect curve 1.2 log units to the right and decreased the maximum by 90%. Heroin self-administration gradually returned to baseline levels over 7 or 17 days after administration of 10 or 40 nmol 5'-NTII, respectively. 5'-NTII (40 nmol i.c.v.) decreased the self-administration of 0.17 mg/infusion cocaine by 40% while having no effect on responding maintained by 0.33 or 0.67 mg/infusion. 5'-NTII attenuated the antinociceptive effects of deltorphin (delta(2)) in a dose-dependent manner while having no effect on antinociception elicited after i.c. v. administration of [D-Pen(2),D-Pen(5)]-enkephalin (delta(1)) or [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (mu). In addition, the antinociceptive effects of heroin were not significantly affected by 5'-NTII (40 nmol i.c.v.). Therefore, 5'-NTII can attenuate the reinforcing effects of heroin at doses that do not affect its antinociceptive effects. Long-acting delta(2)-opioid antagonists may be beneficial in the treatment of heroin dependence or as adjuncts to reduce the abuse liability of opioid analgesics.

Ventral pallidal extracellular fluid levels of dopamine, serotonin, gamma amino butyric acid, and glutamate during cocaine self-administration in rats.

RATIONALE: Dopamine innervation of the nucleus accumbens is thought to have a major role in the biological processes underlying cocaine self-administration. Recent data suggest that dopamine innervation of the ventral pallidum (VP) may also play an important role. OBJECTIVES: This experiment was initiated to assess extracellular fluid levels of dopamine (DA), serotonin (5-HT), gamma-aminobutyric acid (GABA), and glutamate (Glu) in the VP of rats self-administering cocaine using in vivo microdialysis. METHODS: Rats were implanted with intravenous jugular catheters and a microdialysis probe guide cannula into the VP and trained to self-administer (SA) three different doses of cocaine during each daily session. Other rats (yoked rats) were surgically prepared in identical fashion and received vehicle infusions during microdialysis sessions when the SA rat to whom they were yoked produced cocaine infusions. When stable baselines of self-administration were obtained, microdialysates were collected during two consecutive daily self-administration sessions. Neurotransmitter levels were measured using HPLC with electrochemical (DA and 5-HT) or fluorescence detection (GABA and Glu). RESULTS: In SA rats, extracellular fluid levels of DA [DA]e and 5-HT [5-HT]e were elevated throughout the session and levels of Glu [Glu]e showed small increases at a few isolated time points during the session. The increases in [DA]e and 15-HT]e were dose-dependent. Extracellular fluid levels of GABA [GABA]e were unchanged, as were levels of all four neurotransmitters in the yoked rats. CONCLUSIONS: These data support a potential role for DA and 5-HT innervations of the VP in intravenous cocaine self-administration.

Toward a mathematical description of dose-effect functions for self-administered drugs in laboratory animal models.

RATIONALE: The interpretation of dose-effect functions for self-administered drugs remains elusive. Since, for self-administered drugs, the amount of drug in an animal depends on its behavior, a mathematical theory of drug self-administration must include terms relevant to receptor theory, as well as a description of how an organism's behavior affects the amount of drug in the animal over time. OBJECTIVE: A theory was constructed in which the ability of a dose to maintain responding was described in terms of receptor theory and the function relating rate of responding to amount of drug self-administered. The main predictions of the theory were that: 1) there should be no ascending limb for drugs self-administered under ratio schedules, 2) running rate of response should not change as a function of dose and, 3) pause duration should be an exponential function of dose. RESULTS: Low doses of cocaine were either self-administered at high rates, or not at all. Run rates, though somewhat variable, did not change as an orderly function of dose. Pause duration could be well described by an exponential function. CONCLUSIONS: The theory provides an acceptable, though no doubt preliminary, description of drug self-administration.

Effects of beta-funaltrexamine on dose-effect curves for heroin self-administration in rats: comparison with alteration of [3H]DAMGO binding to rat brain sections.

These studies were undertaken to determine the effects of mu-opioid receptor depletion through irreversible alkylation on the dose-effect curve for heroin self-administration. Heroin maintained responding in rats with an inverted U-shaped dose-effect curve and administration of 10 nmol of beta-funaltrexamine i.c.v. (beta-FNA) significantly increased the ED50 on the ascending limb from 1.9 to 5.3 micrograms/infusion, and from 24.3 to 211.8 micrograms/infusion on the descending limb. Administration of saline i.c.v. produced no effect on heroin self-administration. Administration of 40 nmol of beta-FNA increased the ED50S from 5.1 to 33.9 and from 14.4 to 502.8 micrograms/infusion on the ascending and descending portions of heroin's dose-effect curve, respectively. beta-FNA (40 nmol, i.c.v.) had no effect on cocaine self-administration. [3H]DAMGO binding density was decreased in the caudate and nucleus accumbens by 29 or 54% 24 h after administration of 10 or 40 nmol of beta-FNA i.c.v., respectively. The effects of beta-FNA on heroin self-administration were completely overcome by increasing the dose of heroin however, as the shape and slope of the self-administration dose-effect curve was not different when higher doses of heroin were made available for self-administration compared to control data or saline administration. Therefore, there appear to be spare mu-opioid receptors for heroin for the production of its reinforcing effects in rats. Furthermore, the self-administration dose-effect curves returned to control values prior to the return of [3H]DAMGO binding, further suggesting that the full complement of mu-opioid receptors is not necessary for heroin to produce its reinforcing effects. These findings support the existence of spare mu-opioid receptors for heroin in maintaining self-administration in rats.

RTI-113 administration reduces cocaine self-administration at high occupancy of dopamine transporter.

RTI-113 [3beta-(4-chlorophenyl)tropan-2beta carboxylic acid phenyl ester hydrochloride], one of many phenyltropanes potent at and selective for DAT, inhibited self-administration of cocaine in rat at doses that did not alter responding maintained by food. The doses that inhibited cocaine intake produced significant levels of occupancy of DAT.

Dose-effect functions for cocaine self-administration: effects of schedule and dosing procedure.

Research related to determining how procedural variables can alter dose-effect functions for cocaine self-administration is limited. Toward clarifying the role of procedural variables, responding was maintained in rats under either variable-interval (VI) or fixed-ratio (FR) schedules of cocaine infusion. In addition to free-operant FR schedules, discrete-trial FR schedules were evaluated. The dose-effect functions were obtained by either substituting a dose for the usual daily dose, instituting a particular dose for several sessions, or making all doses available within a session. Dose-effect functions for response rate (or number of trials with infusions for the discrete-trial FR) were bitonic for the VI and discrete-trial FR schedules but tended to be strictly decreasing for the free-operant FR schedules. Responding was maintained under FR schedules by a low dose (0.083 mg/infusion) if the dose was substituted for a higher daily dose but not when made available daily. Rate of response was higher under ratio schedules at 0.17 mg/infusion when this dose occurred within the context of other higher doses within a session than when the dose was simply substituted for a higher daily dose. These data indicate that procedural variables can alter dose-response curves for cocaine self-administration.

Within-session determination of dose-response curves for heroin self-administration in rats: Comparison with between-session determination and effects of naltrexone.

A procedure was employed in the present study to obtain dose-response curves for heroin self-administration within each experimental session. The data generated using this procedure were compared to dose-response data obtained using between-session dose manipulations. The dose of heroin (18, 30, 60 or 100 micrograms/kg/inf) was varied across 4-hourly segments separated by a 20-min time-out period during which heroin was not available. The within-session dose-response procedure yielded data similar to those obtained using between-session dose manipulations when the order of dose presentation was increasing or random. However, the dose-response curve for total drug-intake was flat when the doses were presented in decreasing order. Further analysis of the dose-response curves in the within-session procedure demonstrated that the rate of heroin intake increased in the third and fourth hourly components compared to the first component, suggesting acute tolerance to the reinforcing and/or rate-suppressive effects of heroin. Furthermore, using a random order of dose presentation, administration of 3.0 mg/kg of naltrexone prior to the session shifted the dose-response curve for heroin self-administration 5-fold to the right in the within-session procedure. The data indicate that the within-session dose-response procedure can be used to investigate the pharmacology of heroin self-administration in rodents.

Behavioral tolerance to cocaine in squirrel monkeys: acute and chronic effects on complex operant behavior.

Four food-deprived squirrel monkeys were trained to emit complex sequences of responses. The sequences involved pressing lighted response keys in orders dictated by colors that illuminated the keys, and ranged in length from two to five responses. Appropriate completion of these behavioral chains could be followed by food presentation. Acute administration of a range of doses (0.1-1.7 mg/kg) of cocaine hydrochloride produced dose-related decreases in the rate of completing chains and in accuracy of performance during chains. There was little evidence that the drug's effects on overall accuracy were related to the length of the chain. Three of the monkeys were exposed to daily administration of a large dose of cocaine, first after daily sessions and then prior to sessions. Daily postsession administration did not alter the dose-effect curves, but daily presession injection did, indicating the development of behavioral or "contingent" tolerance. In all cases, tolerance was accompanied by an increase in reinforcement frequency relative to the frequency observed following acute administration. Omission of the daily dose during presession drug administration resulted in performance near original control levels indicating essentially no withdrawal effect. The findings illustrate the importance of behavioral factors in the development of tolerance to cocaine in a primate.

Cocaine tolerance: acute versus chronic effects as dependent upon fixed-ratio size.

The effects of cocaine on operant behavior were studied by examining fixed-ratio value as a factor in the development of tolerance. Pigeons pecked a response key under a three-component multiple schedule, with each bird being exposed to fixed-ratio values that were categorized as small, medium, or large. Administered acutely, cocaine (1.0 to 10.0 mg/kg) produced dose-related decreases in overall rate of responding. Responding maintained by the largest ratio was decreased by lower doses than those required to reduce rates of responding maintained by the other two ratio schedules. Following repeated daily administration of 5.6 mg/kg of cocaine, dose-effect functions (obtained from sessions during the chronic regimen by making substitutions for the daily dose) indicated tolerance under the smaller ratios, but no tolerance or less tolerance under the largest ratio. Thus, whether tolerance developed, and the degree to which it developed, depended on the ratio value. The results are partially consistent with the notion that tolerance to drug effects on schedule-controlled behavior will develop if drug administration initially reduces reinforcement frequency, but they indicate that reinforcement loss alone is not a sufficient condition for the generation of tolerance under such conditions. The findings suggest that amount of responding required for reinforcement, or "effort," may contribute to the development of tolerance to effects of cocaine.