Wnt5a suppresses tumor formation and redirects tumor phenotype in MMTV-Wnt1 tumors.

Wnt5a is a non-canonical signaling Wnt that has been implicated in tumor suppression. We previously showed that loss of Wnt5a in MMTV-PyVmT tumors resulted in a switch in tumor phenotype resulting in tumors with increased basal phenotype and high Wnt/β-catenin signaling. The object of this study was to test the hypothesis that Wnt5a can act to inhibit tumors formed by activation of Wnt/β-catenin signaling. To this end, we characterized tumor and non-tumor mammary tissue from MMTV-Wnt1 and double transgenic MMTV-Wnt1;MMTV-Wnt5a mice. Wnt5a containing mice demonstrated fewer tumors with increased latency when compared to MMTV-Wnt1 controls. Expression of markers for basal-like tumors was down-regulated in the tumors that formed in the presence of Wnt5a indicating a phenotypic switch. Reduced canonical Wnt signaling was detected in double transgenic tumors as a decrease in active β-catenin protein and a decrease in Axin2 mRNA transcript levels. In non-tumor tissues, over-expression of Wnt5a in MMTV-Wnt1 mammary glands resulted in attenuation of phenotypes normally observed in MMTV-Wnt1 glands including hyperbranching and increased progenitor and basal cell populations. Even though Wnt5a could antagonize Wnt/β-catenin signaling in primary mammary epithelial cells in culture, reduced Wnt/β-catenin signaling was not detected in non-tumor MMTV-Wnt1;Wnt5a tissue in vivo. The data demonstrate that Wnt5a suppresses tumor formation and promotes a phenotypic shift in MMTV-Wnt1 tumors

Wnt5a inhibits Wnt/β-catenin signaling in E-cadherin negative wild type and MMTV-Wnt1 cells.

<p>(A) <i>Immunostaining for E-cadherin and α-smooth muscle actin</i>. Sections from MMTV-Wnt1 mammary glands were stained with both anti- E-cadherin (Ecad, green) and anti- α-smooth muscle actin (αSMA, red) antibodies. The merged image of both red and green channels is shown (left) as well as separate channels for Ecad (middle) and αSMA (right). No overlap between Ecad and αSMA was detected indicating Ecad is not expressed in the basal myoepithelial cells. (B–C) <i>Immunostaining for E-cadherin on sorted cells</i>. Total (B) and E-cadherin negative (C) PMECs were plated overnight and then fixed and stained with anti-E-cadherin antibody (green). Nuclei were counterstained with DAPI (blue). Sorted cells demonstrated little to no E-cadherin staining. (D) <i>Western blot analysis of β-catenin protein in isolated E-Cadherin-negative cells</i>. Western blot analysis using protein lysates prepared from E-Cadherin-negative cells that were isolated from wild type mammary glands and treated with parental or Wnt5a conditioned media for 24 hours. Keratin 14 was used as the protein loading control. The average active β-catenin/β-catenin ratios are shown for cells isolated from 2 separate mice. Active β-catenin was reduced after treatment with Wnt5a conditioned media an average of 70% in five separate experiments. (E) <i>Quantitative RT-PCR of Axin2, a Wnt/β-catenin target gene, in wild type E-Cadherin-negative cells</i>. Expression of Axin2 mRNA in E-Cadherin-negative cells after treatment with parental or Wnt5a conditioned media as determined by quantitative RT-PCR (n = 3 separate experiments). Data are shown as tables obtained using REST software. <i>Axin2</i>, a Wnt/β-catenin target gene, was down-regulated after treatment with Wnt5a conditioned media. Expression is the fold difference in Wnt5a treated versus parental media treated after normalization. Gapdh mRNA levels were used as normalization controls. (F) <i>Quantitative RT-PCR of Axin2 in E-Cadherin-negative cells from MMTV-Wnt1 mice</i>. Expression of Axin2 mRNA in E-Cadherin-negative cells after treatment with parental or Wnt5a conditioned media as determined by quantitative RT-PCR (n = 2 separate experiments). Data are shown as tables obtained using REST software. <i>Axin2</i>, a Wnt/β-catenin target gene, was down-regulated after treatment with Wnt5a conditioned media. Expression is the fold difference in Wnt5a treated versus parental media treated after normalization to Gapdh.</p

Wnt5a expressing tumors demonstrate a decrease in markers of the basal tumor subtype.

<p>(A) Western blot using protein lysates isolated from the epithelium of MMTV-Wnt1 and MMTV-1;MMTV-Wnt5a mammary tumors. The expression of molecular markers of basal and luminal tumor subtypes were compared. Keratin 6 and Keratin 5 were strongly down-regulated in Wnt5a expressing tumors. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B–D) <i>Immunostaining for K6</i>. Sections from MMTV-Wnt1 (B) and MMTV-1;MMTV-Wnt5a (C) tumors were stained with anti-Keratin 6 antibody using immunofluorescence (K6 = green; nuclei = blue). The percentage of cells expressing K6 was determined and graphed (D). Values are means +/− standard error (n = 6 MMTV-Wnt1, 3 fields per tumor; n = 5 MMTV-Wnt1;MMTV-Wnt5a, 3 fields per tumor). MMTV-Wnt1;MMTV-Wnt5a tumors demonstrated a significant decrease in K6-expressing cells as measured by T-test (* = p<0.05).</p

List of quantitative RT-PCR primers.

<p>List of quantitative RT-PCR primers.</p

Wnt5a does not affect early development in MMTV-Wnt1 mammary glands.

<p>(A–D) <i>Whole mount staining, 8 weeks</i>. Mammary glands from 8 week old MMTV-Wnt1 (A, B) and MMTV-Wnt1;MMTV-Wnt5a (C, D) mice were stained with carmine (n = 2 each). Delay in ductal extension through the fat pad was not detected and terminal end buds (arrow, EB) were intact in Wnt5a containing glands. (E–G) <i>Whole mount staining, 6 months</i>. Mammary glands from 6-month-old, virgin MMTV-Wnt1 (E) and MMTV-Wnt1;MMTV-Wnt5a (F) mice were stained with carmine. Quantification of the area of mammary epithelium in images from the carmine stained glands (G) demonstrated a significant decrease in MMTV-Wnt1;MMTV-Wnt5a ducts compared to MMTV-Wnt1 controls. Values are shown as means +/− standard error (n = 8 MMTV-Wnt1; n = 6 MMTV-Wnt1;MMTV-Wnt5a, T-test *<i>p</i>-value = 0.05). (H–J) <i>Ki-67 staining</i>. Sections from MMTV-Wnt1 (H) and MMTVWnt1;MMTV-Wnt5a (I) mammary glands were immunostained using an anti-Ki-67 antibody (Ki-67, green; nuclei, blue). No statistical difference in the percentage of Ki-67 positive cells was observed (J). All values are shown as means +/− standard error (n = 6 MMTV-Wnt1, 3 fields per mammary gland; n = 5 MMTV-Wnt1;MMTV-Wnt5a, 3 fields per mammary gland).</p

Wnt5a expressing tumors have less Wnt/β-catenin signaling than MMTV-Wnt1 tumors.

<p>(A) <i>Quantitative RT-PCR of Wnt/</i>β<i>-catenin target genes</i>. Expression of <i>Axin2</i> mRNA in MMTV-Wnt1 versus MMTV-Wnt1;MMTV-Wnt5a tumors as determined by quantitative RT-PCR (n = 5 MMTV-Wnt1, n = 5 MMTV-Wnt1;MMTV-Wnt5a). Data are shown as tables obtained using REST software. <i>Axin2</i> mRNA was significantly down-regulated in MMTV-Wnt1;MMTV-Wnt5a tumors. (B) <i>Western blot for β-catenin protein</i>. Protein lysates were prepared from MMTV-Wnt1 and MMTV-Wnt1;MMTV-Wnt5a tumors. β-catenin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading controls. The ratio of active β-catenin to β-catenin as determined by densitometic analysis is shown. MMTV-Wnt1;MMTV-Wnt5a tumors displayed decreased levels of active β-catenin compared to controls.</p

Rationale and design of a multisite randomized clinical trial examining an integrated behavioral treatment for veterans with co-occurring chronic pain and opioid use disorder: The pain and opioids integrated treatment in veterans (POSITIVE) trial.

BackgroundChronic pain and opioid use disorder (OUD) individually represent a risk to health and well-being. Concerningly, there is evidence that they are frequently co-morbid. While few treatments exist that simultaneously target both conditions, preliminary work has supported the feasibility of an integrated behavioral treatment targeting pain interference and opioid misuse. This treatment combined Acceptance and Commitment Therapy (ACT) and Mindfulness-Based Relapse Prevention (ACT+MBRP). This paper describes the protocol for the adequately powered efficacy study of this integrated treatment.MethodsA multisite randomized controlled trial will examine the efficacy of ACT+MBRP in comparison to a parallel education control condition, focusing on opioid safety and pain education. Participants include veterans (n&nbsp;=&nbsp;160; 21-75&nbsp;years old) recruited from three Veterans Administration (VA) Healthcare Systems with chronic pain who are on a stable dose of buprenorphine. Both conditions include twelve weekly 90&nbsp;min group sessions delivered via telehealth. Primary outcomes include pain interference (Patient Reported Outcome Measurement Information System - Pain Interference) and hazardous opioid use (Current Opioid Misuse Measure), which will be examined at the end of the active treatment phase and through 12&nbsp;months post-intervention. Secondary analyses will evaluate outcomes including pain intensity, depression, pain-related fear, and substance use, as well as treatment mechanisms.ConclusionThis study will determine the efficacy of an integrated behavioral treatment program for pain interference and hazardous opioid use among veterans with chronic pain and OUD who are prescribed buprenorphine, addressing a critical need for more integrated treatments for chronic pain and OUD.Trial registrationClinicalTrials.gov Identifier: NCT04648228