Leveraging Expertise from Community Resources to Improve the Role of the Pharmacist in HIV Testing and Counseling.

The human immunodeficiency virus (HIV) epidemic continues to be a major global public health issue. Moreover, disparities continue to persist in HIV among racial and ethnic minority populations, with the highest rates of new diagnoses in Black/African American and Hispanic/Latino men who have sex with men in the United States. Pharmacists are one of the most accessible and trusted health care professionals. Therefore, it is imperative that student pharmacists are educated on culturally-competent HIV testing and risk behaviors counseling. This study describes the development of a partnership between a pharmacy school and a community-based organization to offer an HIV counseling and testing training program to help develop skills in delivering HIV testing services. The HIV counseling and testing training program contains learning modules that provide a wide array of in-depth information about HIV patient care in the community. The partnership allows for the enjoyment of a myriad of benefits for students, the pharmacy program, the community-based organization, and the public health of the community-at-large. Students feel more prepared and comfortable working with patients in discussing HIV transmission risk factors and test results as a result of this training. Such partnerships support the pharmacist's role in the public health arena. A successful and durable relationship between a community partner and a school of pharmacy is a feasible strategy for pharmacy progress in public health.

Using Biochemistry to Educate Students on the Causal Link between Social Epigenetics and Health Disparities.

BACKGROUND: While pharmacy education standards require students to recognize social determinants of health (SDOH), there is an opportunity to improve how this is taught in the curriculum. One innovative approach is to educate student pharmacists in a biochemistry course through the integration of topics like epigenetics using SDOH as the framework. INNOVATION: A 50-minute educational activity was used to supplement material on the regulation of gene expression, in which epigenetic changes are driven by SDOH. It provided students with a biochemical basis to explain some health disparities, rather than viewing them exclusively as social obstacles to health. The activity employed a mini-lecture, a short video, as well as both small and large group discussion. A reflective paper was used to assess students' understanding of the topic, and the role of the pharmacist in helping patients prevent diseases caused by epigenetic changes due to social determinants of health. FINDINGS: A post-activity survey showed that the activity increased students' perception of knowledge about SDOH, as well as the effect of epigenetic changes on health outcomes. Furthermore, this activity increased students' awareness about the role that SDOH play in epigenetic changes and challenged students to understand the role that society plays in health outcomes. CONCLUSIONS: The preventable nature of health inequities creates an opportunity to integrate public health into pharmacy education. The integration of epigenetics and SDOH gives the student an opportunity to provide a mechanistic link between social inequities and biochemical processes.

Developing a Co-Curricular Activity to Introduce Student Pharmacists to the Cultural Proficiency Continuum Framework.

BACKGROUND: As the United States becomes more culturally diverse, health professionals must be able to demonstrate competency in caring for a multitude of diverse patients. The cultural proficiency continuum has proven to be an effective framework to assess where individuals and institutions are on the continuum of cultural sensitivity and competence in educational settings. INNOVATION: A co-curricular activity was developed as an exercise in self-awareness to allow first year pharmacy students the opportunity to explore potential biases by evaluating comfort in both social and patient care settings. The 90-minute activity employed a lecture, followed by both small and large group discussions and a debriefing session. FINDINGS: Student survey responses showed their appreciation of this framework and its application to patient-centered care. Student self-rated knowledge increased by 3 points on a 10-point scale after completion of the activity. Students agreed that their level of cultural awareness would lead them to respond appropriately in cross-cultural situations, and that the provision of care is dependent on approaches that are culturally proficient. CONCLUSION: This activity dismantles the misconception of cultural competence as an attainable finite skill, but instead presents it as an ongoing process of self-awareness. The co-curricular activity offers an easy to implement model of education that could potentially fit the needs of pharmacy programs searching for ideas to teach cultural competency and social determinants of health, while circumventing the need to affect curricular structure.

If cultural sensitivity is not enough to reduce health disparities, what will pharmacy education do next?

INTRODUCTION: Social determinants greatly impact health, and evidence suggests that health disparities still exist between patient populations. The pharmacist's role in public health continues to evolve; therefore, the deliberate integration of public health into pharmacy education is essential. Pharmacy graduates must be able to recognize social determinants of health in order to help reduce disparities and inequities in access to quality care. PERSPECTIVE: Cultural sensitivity training has been one of the most commonly used vehicles to educate students about health disparities. Whereas cultural sensitivity is of critical importance, it could unintentionally perpetuate the idea of culture as the sole construct to predict health outcomes. Consequently, it is important that we approach this societal problem from a public health perspective by addressing not just culture, but all other social determinants of health. IMPLICATIONS: As disparities in health outcomes continue to widen for some patient groups, it is imperative for pharmacy programs to explore novel instructional approaches to teach health disparities. A paradigm shift from awareness to action and advocacy must involve educating students on the multifaceted effects of social determinants when designing interventions to help reduce health disparities.

Enoxacin and Epigallocatechin Gallate (EGCG) Act Synergistically to Inhibit the Growth of Cervical Cancer Cells in Culture.

Cervical cancer is a major cause of death in females worldwide. While survival rates have historically improved, there remains a continuous need to identify novel molecules that are effective against this disease. Here, we show that enoxacin, a drug most commonly used to treat a broad array of bacterial infections, is able to inhibit growth of the cervical cancer cells. Furthermore, our data show that epigallocatechin gallate (EGCG), a plant bioactive compound abundant in green tea, and known for its antioxidant effects, similarly functions as an antiproliferative agent. Most importantly, we provide evidence that EGCG functions synergistically against cancer cell proliferation in combined treatment with enoxacin. These data collectively suggest that enoxacin and EGCG may be useful treatment options for cases of cervical cancer.

Comparison of tamoxifen and letrozole response in mammary preneoplasia of ER and aromatase overexpressing mice defines an immune-associated gene signature linked to tamoxifen resistance.

Response to breast cancer chemoprevention can depend upon host genetic makeup and initiating events leading up to preneoplasia. Increased expression of aromatase and estrogen receptor (ER) is found in conjunction with breast cancer. To investigate response or resistance to endocrine therapy, mice with targeted overexpression of Esr1 or CYP19A1 to mammary epithelial cells were employed, representing two direct pathophysiological interventions in estrogen pathway signaling. Both Esr1 and CYP19A1 overexpressing mice responded to letrozole with reduced hyperplastic alveolar nodule prevalence and decreased mammary epithelial cell proliferation. CYP19A1 overexpressing mice were tamoxifen sensitive but Esr1 overexpressing mice were tamoxifen resistant. Increased ER expression occurred with tamoxifen resistance but no consistent changes in progesterone receptor, pSTAT3, pSTAT5, cyclin D1 or cyclin E levels in association with response or resistance were found. RNA-sequencing (RNA-seq) was employed to seek a transcriptome predictive of tamoxifen resistance using these models and a second tamoxifen-resistant model, BRCA1 deficient/Trp53 haploinsufficient mice. Sixty-eight genes associated with immune system processing were upregulated in tamoxifen-resistant Esr1- and Brca1-deficient mice, whereas genes related to aromatic compound metabolic process were upregulated in tamoxifen-sensitive CYP19A1 mice. Interferon regulatory factor 7 was identified as a key transcription factor regulating these 68 immune processing genes. Two loci encoding novel transcripts with high homology to human immunoglobulin lambda-like polypeptide 1 were uniquely upregulated in the tamoxifen-resistant models. Letrozole proved to be a successful alternative to tamoxifen. Further study of transcriptional changes associated with tamoxifen resistance including immune-related genes could expand our mechanistic understanding and lead to biomarkers predictive of escape or response to endocrine therapies.

Human Pancreatic Cancer-Associated Stellate Cells Remain Activated after in vivo Chemoradiation.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive fibrotic reaction or desmoplasia and complex involvement of the surrounding tumor microenvironment. Pancreatic stellate cells are a key mediator of the pancreatic matrix and they promote progression and invasion of pancreatic cancer by increasing cell proliferation and offering protection against therapeutic interventions. Our study utilizes human tumor-derived pancreatic stellate cells (HTPSCs) isolated from fine needle aspirates of pancreatic cancer tissue from patients with locally advanced, unresectable pancreatic adenocarcinoma before and after treatment with full-dose gemcitabine plus concurrent hypo-fractionated stereotactic radiosurgery. We show that HTPSCs survive in vivo chemotherapy and radiotherapy treatment and display a more activated phenotype post-therapy. These data support the idea that stellate cells play an essential role in supporting and promoting pancreatic cancer and further research is needed to develop novel treatments targeting the pancreatic tumor microenvironment.

The CDK4/6 inhibitor PD0332991 reverses epithelial dysplasia associated with abnormal activation of the cyclin-CDK-Rb pathway.

Loss of normal growth control is a hallmark of cancer progression. Therefore, understanding the early mechanisms of normal growth regulation and the changes that occur during preneoplasia may provide insights of both diagnostic and therapeutic importance. Models of dysplasia that help elucidate the mechanisms responsible for disease progression are useful in highlighting potential targets for prevention. An important strategy in cancer prevention treatment programs is to reduce hyperplasia and dysplasia. This study identified abnormal upregulation of cell cycle-related proteins cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, and phosphorylated retinoblastoma protein (pRb) as mechanisms responsible for maintenance of hyperplasia and dysplasia following downregulation of the initiating viral oncoprotein Simian virus 40 (SV40) T antigen. Significantly, p53 was not required for successful reversal of hyperplasia and dysplasia. Ligand-induced activation of retinoid X receptor and PPARγ agonists attenuated cyclin D1 and CDK6 but not CDK4 or phosphorylated pRb upregulation with limited reversal of hyperplasia and dysplasia. PD0332991, an orally available CDK4/6 inhibitor, was able to prevent upregulation of cyclin D1 and CDK6 as well as CDK4 and phosphorylated pRb and this correlated with a more profound reversal of hyperplasia and dysplasia. In summary, the study distinguished CDK4 and phosphorylated pRb as targets for chemoprevention regimens targeting reversal of hyperplasia and dysplasia.

Association of Over-Expressed Estrogen Receptor Alpha with Development of Tamoxifen Resistant Hyperplasia and Adenocarcinomas in Genetically Engineered Mice.

BACKGROUND: Estrogen receptor alpha (ERα) and cyclin D1 are frequently co-expressed in human breast cancer. Some, but not all, studies link tamoxifen resistance to co-expression of cyclin D1 and ERα. In mice over-expression of either cyclin D1 or ERα in mammary epithelial cells is sufficient to induce mammary hyperplasia. Cyclin D1 over-expression in mice leads to mammary adenocarcinoma associated with activated estrogen signaling pathways. ERα over-expression in mice leads to mammary hyperplasia and cancer. Significantly, disease development in these mice is abrogated by loss of cyclin D1. METHODS: Genetically engineered mouse models were used to determine whether or not ERα over-expression demonstrated cooperativity with cyclin D1 over-expression in cancer development, reaction to the chemical carcinogen DMBA, or tamoxifen response. RESULTS: Adding ERα over-expression to cyclin D1 over-expression increased the prevalence of hyperplasia but not cancer. Single dose DMBA exposure did not increase cancer prevalence in any of the genotypes although cyclin D1 over-expressing mice demonstrated a significant increase in hyperplasia. Tamoxifen treatment was initiated at both young and older ages to test for genotype-specific differences in response. Although normal ductal structures regressed in all genotypes at both younger and older ages, tamoxifen did not significantly reduce the prevalence of either hyperplasia or cancer in any of the genotypes. All of the cancers that developed were hormone receptor positive, including those that developed on tamoxifen, and all showed expression of nuclear-localized cyclin D1. In summary, development of tamoxifen resistant hyperplasia and cancer was associated with expression of ERα and cyclin D1. CONCLUSION: These preclinical models will be useful to test strategies for overcoming tamoxifen resistance, perhaps by simultaneously targeting cell cycle regulatory pathways associated with cyclin D1.

Signal transducer and activator of transcription 5 as a key signaling pathway in normal mammary gland developmental biology and breast cancer.

STAT5 consists of two proteins, STAT5A/B, that impact mammary cell differentiation, proliferation, and survival. In normal development, STAT5 expression and activity are regulated by prolactin signaling with JAK2/ELF5, EGF signaling networks that include c-Src, and growth hormone, insulin growth factor, estrogen, and progesterone signaling pathways. In cancer, erythropoietin signaling can also regulate STAT5. Activation levels are influenced by AKT, caveolin, PIKE-A, Pak1, c-Myb, Brk, beta-integrin, dystroglycan, other STATs, and STAT pathway molecules JAK1, Shp2, and SOCS. TGF-ß and PTPN9 can downregulate prolactin- and EGF-mediated STAT5 activation, respectively. IGF, AKT, RANKL, cyclin D1, BCL6, and HSP90A lie downstream of STAT5.

Comparison of increased aromatase versus ERα in the generation of mammary hyperplasia and cancer.

Factors associated with increased estrogen synthesis increase breast cancer risk. Increased aromatase and estrogen receptor α (ERα) in both normal epithelium and ductal carcinoma in situ lesions are found in conjunction with breast cancer, leading to the idea that altered estrogen signaling pathways predispose the mammary gland to cancer development. Here, we developed a transgenic mouse that conditionally expresses aromatase in the mammary gland, and used it along with a deregulated ERα expression model to investigate the molecular pathways involved in the development of mammary gland preneoplasia and carcinoma. Both increased ERα and aromatase expression led to the development of preneoplasia, but increased preneoplasia, in addition to carcinoma, was found in aromatase overexpressing mice. Increased prevalence of mammary pathologic changes in mice expressing aromatase correlated with increased cyclin E and cyclin-dependent kinase 2 expression. Gain of both ERα and aromatase increased expression of ERα and progesterone receptor, but aromatase produced a higher increase than ERα, accompanied by higher levels of downstream target genes Ccnd1, Myc, and Tnfsf11. In summary, whereas gain of both ERα and aromatase activate abnormal growth pathways in the mammary gland, aromatase induced a wider range of abnormalities that was associated with a higher prevalence of mammary preneoplasia and cancer progression.

Assessing estrogen signaling aberrations in breast cancer risk using genetically engineered mouse models.

Aberrations in estrogen signaling increase breast cancer risk. Molecular mechanisms that impact breast cancer initiation, promotion, and progression can be investigated using genetically engineered mouse models. Increasing estrogen receptor alpha (ERα) expression levels twofold is sufficient to initiate and promote breast cancer progression. Initiation and promotion can be increased by p53 haploinsufficiency and by coexpressing the nuclear coactivators amplified in breast cancer 1 (AIB1) or the splice variant AIB1Δ3. Progression to invasive cancer is found with coexpression of these nuclear coactivators as well as following a single dose of 7,12-dimethylbenz(a)anthracene. Loss of signal transducer and activator of transcription 5a reduces the prevalence of initiation and promotion but does not protect from invasive cancer development. Cyclin D1 loss completely interrupts mammary epithelial proliferation and survival when ERα is overexpressed. Loss of breast cancer gene 1 increases estrogen signaling and cooperates with ERα overexpression in initiation, promotion, and progression of mammary cancer.

Altered AIB1 or AIB1Δ3 expression impacts ERα effects on mammary gland stromal and epithelial content.

Amplified in breast cancer 1 (AIB1) (also known as steroid receptor coactivator-3) is a nuclear receptor coactivator enhancing estrogen receptor (ER)α and progesterone receptor (PR)-dependent transcription in breast cancer. The splice variant AIB1Δ3 demonstrates increased ability to promote ERα and PR-dependent transcription. Both are implicated in breast cancer risk and antihormone resistance. Conditional transgenic mice tested the in vivo impact of AIB1Δ3 overexpression compared with AIB1 on histological features of increased breast cancer risk and growth response to estrogen and progesterone in the mammary gland. Combining expression of either AIB1 or AIB1Δ3 with ERα overexpression, we investigated in vivo cooperativity. AIB1 and AIB1Δ3 overexpression equivalently increased the prevalence of hyperplastic alveolar nodules but not ductal hyperplasia or collagen content. When AIB1 or AIB1Δ3 overexpression was combined with ERα, both stromal collagen content and ductal hyperplasia prevalence were significantly increased and adenocarcinomas appeared. Overexpression of AIB1Δ3, especially combined with overexpressed ERα, led to an abnormal response to estrogen and progesterone with significant increases in stromal collagen content and development of a multilayered mammary epithelium. AIB1Δ3 overexpression was associated with a significant increase in PR expression and PR downstream signaling genes. AIB1 overexpression produced less marked growth abnormalities and no significant change in PR expression. In summary, AIB1Δ3 overexpression was more potent than AIB1 overexpression in increasing stromal collagen content, inducing abnormal mammary epithelial growth, altering PR expression levels, and mediating the response to estrogen and progesterone. Combining ERα overexpression with either AIB1 or AIB1Δ3 overexpression augmented abnormal growth responses in both epithelial and stromal compartments.

Deregulated estrogen receptor alpha and p53 heterozygosity collaborate in the development of mammary hyperplasia.

Both increased estrogen receptor alpha (ER(alpha)) expression and germline disruption of one p53 allele increase breast cancer risk in women. Genetically engineered mouse models of deregulated ER(alpha) expression and p53 haploinsufficiency were used to investigate similarities and differences of each genetic lesion alone and in combination on mammary preneoplasia development. Each genetic lesion independently and in combination led to development of age-dependent preneoplasia, but the highest prevalence was found in compound mice with increased ER(alpha) expression coupled with p53 heterozygosity. All genetic lesions were associated with extracellular signal-regulated kinase 1/2 activation; however, only p53 heterozygous and compound mice showed increased levels of phosphorylated AKT and decreased p27 expression. The highest levels of cell proliferation were found in compound mice, but increased levels were also found with either increased ER(alpha) expression or p53 heterozygosity. Mice with increased ER(alpha) expression showed predicted higher levels of nuclear-localized ER(alpha), but this was attenuated in compound mice in association with a relative increase in Src phosphorylation. Parity protection was limited to p53 heterozygous mice and not found in mice with increased ER(alpha) alone. In summary, increased and deregulated ER(alpha) collaborates with p53 heterozygosity in increasing the risk of mammary preneoplasia development.

BRCA1 deficient mouse models to study pathogenesis and therapy of triple negative breast cancer.

Genetically engineered mice along with allograft and xenograft models can be used to effectively model triple negative breast cancer both for studies of pathophysiology as well as preclinical prevention and therapeutic drug studies. In this review eight distinct genetically engineered mouse models of BRCA1 deficiency are discussed in relationship to the generation of triple negative mammary cancer. Allograft models derived from some of these genetically engineered mice are considered and xenograft models derived from breast cancers that developed from BRCA1 mutation are presented. Examples of the use of genetically engineered, allograft and xenografts models for preventive and therapeutic studies are presented.

Suppression of aromatase in human breast cells by a cyclooxygenase-2 inhibitor and its analog involves multiple mechanisms independent of cyclooxygenase-2 inhibition.

Previous studies have demonstrated that cyclooxygenase-2 (COX-2) inhibitor NS-398 decrease aromatase activity at the transcript level in breast cancer cells. However, N-Methyl NS-398, which does not have COX-2 inhibitory activity but has very similar structure to NS-398, decreases aromatase activity and transcription in MCF-7 and MDA-MB-231 breast cells to the same extent as NS-398. This suggests that NS-398 decrease aromatase expression in breast cancer cells via other mechanism(s). Further investigations find that both compounds only decrease aromatase activity stimulated by forskolin/phorbol ester at the transcript level in both breast cancer cell lines and in breast stromal cells from patients. They do not affect aromatase expression and activity stimulated by dexamethasone. Both compounds also suppress MCF-7 cell proliferation stimulated by testosterone. Aromatase inhibition studies using placental microsomes demonstrate that the compounds show only weak direct aromatase inhibition. These results suggest that NS-398 and its N-methyl analog suppress aromatase expression and activity with multiple mechanisms.

Evaluation of synthetic isoflavones on cell proliferation, estrogen receptor binding affinity, and apoptosis in human breast cancer cells.

Natural isoflavones have demonstrated numerous pharmacological activities in breast cancer cells, including antiproliferative activities and binding affinities for estrogen receptors (ERs). Chemical modifications on the isoflavone ring system have been prepared and explored for the development of new therapeutics for hormone-dependent breast cancer. The antiproliferative actions of the synthesized isoflavones on MCF-7 and MDA-MB-231 breast cancer cells were examined, as well as cytotoxicity, interaction with estrogen receptors, and proapoptotic activity. The compounds were screened in the absence and in the presence of estradiol to evaluate whether or not estradiol could rescue cell proliferation on MCF-7 cells. Several compounds were able to inhibit cell proliferation in a dose-dependent manner, and compounds containing the bulky 7-phenylmethoxy substituent resulted in cell toxicity not only in MCF-7 cells but also in MDA-MB-231 cells. Selected synthetic isoflavones were able to bind to estrogen receptor with low affinity. Apoptotic pathways were also activated by these compounds in breast cancer cells. The majority of the compounds can bind to both ERs with low affinity, and their effects on hormone-independent breast cancer cells suggest that their ability to inhibit cell growth in breast cancer cells is not exclusively mediated by ERs. Thus, the synthetic trisubstituted isoflavones act on multiple signaling pathways leading to activation of mechanisms of cell-death and ultimately affecting breast cancer cell survival.

Aromatase and COX in breast cancer: enzyme inhibitors and beyond.

Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Complex regulation of aromatase expression in human tissues involves alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Additionally, nonsteroidal anti-inflammatory drugs (NSAIDs) and COX selective inhibitors can suppress CYP19 gene expression and decrease aromatase activity. Our current hypothesis is that pharmacological regulation of aromatase and/or cyclooxygenases can act locally to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer. Two pharmacological approaches are being developed, one involving mRNA silencing by selective short interfering RNAs (siRNA) molecules and the second utilizing small molecule drug design. In the first approach, short interfering RNAs were designed against either human aromatase mRNA or human COX-2 mRNA. Treatment of breast cancer cells with siAROMs completely masked the aromatase enzyme activity. Treatment with COX-2 siRNAs decreased the expression of COX-2 mRNA; furthermore, the siCOX-2-mediated decrease also resulted in suppression of CYP19 mRNA. The small molecule drug design approach focuses on the synthesis and biological evaluation of a novel series of sulfonanilide analogs derived from the COX-2 selective inhibitors. The compounds suppress aromatase enzyme activity in SK-BR-3 breast cancer cells in a dose and time-dependent manner, and structure activity analysis does not find a correlation between aromatase suppression and COX inhibition. Real-time PCR analysis demonstrates that the sulfonanilide analogs decrease aromatase gene transcription in breast cells. Thus, these results suggest that the siRNAs and novel sulfonanilides targeting aromatase expression may be valuable tools for selective regulation of aromatase in breast cancer.