Ejaculatory Function Following Stereotactic Body Radiation Therapy for Prostate Cancer.

BACKGROUND: Ejaculatory dysfunction is an important male quality of life issue which has not yet been studied in the setting of prostate stereotactic body radiation therapy (SBRT). AIM: The purpose of this study is to evaluate ejaculatory function following SBRT for prostate cancer. METHODS: Two hundred and thirty-one patients on a prospective quality of life study with baseline ejaculatory capacity treated with prostate SBRT from 2013 to 2019 were included in this analysis. Ejaculation was assessed via the Ejaculation Scale (ES-8) from the Male Sexual Health Questionnaire. Patients completed the questionnaire at 1, 3, 6, 9, 12, 18, and 24 months post-SBRT. Elderly patients (Age > 70) and those who received hormonal therapy were excluded from analysis. Patients were treated to 35-36.25 Gy in 5 fractions delivered with the CyberKnife Radiosurgical System (Accuray). OUTCOMES: Ejaculatory function was assessed by ES-8 scores (range 4-40) with lower values representing increased interference or annoyance. RESULTS: Median age at the time of treatment was 65 years. Median follow up was 24 months (IQR 19-24.5 months). 64.5% of patients had ED at baseline (SHIM < 22). The 2-year anejaculation rate was 15%. Mean composite ES-8 scores showed a decline in the first month following treatment then stabilized: 30.4 (start of treatment); 26.5 (1 month); 27.6 (3 month); 27.0 (6 month); 26.2 (9 month); 25.4 (12 month); 25.0 (18 month) and 25.4 (24 month). White race, higher pre-treatment SHIM (≥22), and higher ES-8 (≥31) at treatment start were significantly associated with a decreased probability of a clinically significant decline. Patient-reported ejaculate volume was significantly reduced at all time points post-SBRT. Ejaculatory discomfort peaked at 1 month and 9 months post-SBRT. Prior to treatment, 8.0% of men reported that they were very to extremely bothered by their ejaculatory dysfunction. The number of patients reporting this concern increased to 14.4% at one year and dropped to 11% at 24-months post-SBRT. CLINICAL TRANSLATION: Patients undergoing prostate SBRT may experience meaningful changes in ejaculatory function and should be counseled on the trajectory of these side effects. STRENGTHS & LIMITATIONS: This was a retrospective analysis of a prospectively maintained database. Subjective questionnaire responses captured limited aspects of ejaculatory function in this cohort. CONCLUSION: The high incidence of moderate to extreme bother in ejaculatory function before and after SBRT suggests a need for novel approaches to improving ejaculation. Sholklapper T, Creswell M, Cantalino J, et al. Ejaculatory Function Following Stereotactic Body Radiation Therapy for Prostate Cancer. J Sex Med 2022;19:771-780.

PD-L1 Glycosylation and Its Impact on Binding to Clinical Antibodies.

Immune checkpoint inhibitors, including PD-L1/PD-1, are key regulators of the immune response and promising targets in cancer immunotherapy. N-glycosylation of PD-L1 affects its interaction with PD-1, but little is known about the distribution of glycoforms at its four NXS/T sequons. We optimized LC-MS/MS methods using collision energy modulation for the site-specific resolution of specific glycan motifs. We demonstrate that PD-L1 on the surface of breast cancer cell line carries mostly complex glycans with a high proportion of polyLacNAc structures at the N219 sequon. Contrary to the full-length protein, the secreted form of PD-L1 expressed in breast MDA-MB-231 or HEK293 cells demonstrated minimum N219 occupancy and low contribution of the polyLacNAc structures. Molecular modeling of PD-L1/PD-1 interaction with N-glycans suggests that glycans at the N219 site of PD-L1 and N74 and N116 of PD-1 may be involved in glycan-glycan interactions, but the impact of this potential interaction on the protein function remains at this point unknown. The interaction of PD-L1 with clinical antibodies is also affected by glycosylation. In conclusion, PD-L1 expressed in the MDA-MB-231 breast cancer cell line carries polyLacNAc glycans mostly at the N219 sequon, which displays the highest variability in occupancy and is most likely to influence the interaction with PD-1.

Distinguishing Physiological Ureter Uptake From an Involved Lymph Node in Staging Prostate-Specific Membrane Antigen (PSMA) Scans: Implications for Radiation Planning.

Prostate-specific membrane antigen (PSMA) imaging has become a mainstay diagnostic tool in staging unfavorable primary prostate cancer (PC) and identifying sites of recurrence in previously treated PC. One of the biggest pitfalls of PSMA imaging is rapid radionucleotide excretion in the urine via the​ kidneys, ureters, and bladder.​ The positron-emission tomography (PET) images obtained show increased radiotracer activity in these structures, which can occlude or even mimic true malignant disease. We describe the diagnostic challenges encountered in differentiating benign versus malignant disease with PSMA scans. A 78-year-old male presented ​to our outpatient radiation oncology office ​with high-risk prostate cancer. His medical history was significant for ulcerative colitis (UC). Magnetic resonance imaging (MRI) revealed an enlarged prostate and a Prostate Imaging Reporting and Data System (PI-RADS) class 4 lesion. A subsequent transperineal biopsy confirmed unilateral Gleason 8 adenocarcinoma. A PSMA PET scan was read as increased uptake in the right prostate and a left external iliac node. The patient, having been initially informed of a positive lymph node metastasis, sought a second opinion,​resulting in​​ ​a CT urogram that revealed physiologic ureteral uptake. We were thus able to avoid lymph node radiation and morbidity to the surrounding bowel, already chronically inflamed with ulcerative colitis. This study ​demonstrates the ​potential for misinterpretation of PSMA uptake in the ureter as lymph node metastases. We discuss how peri-uretic activity can hinder accurate visualization of pelvic lymph node metastases. This study highlights the need for careful image interpretation of PSMA uptake patterns in order to avoid diagnostic errors and unnecessary radiation to ​at-risk​​ ​organs in prostate cancer management.

Prognostic utility of biopsy-based PTEN and ERG status on biochemical progression and overall survival after SBRT for localized prostate cancer.

Introduction/background: Phosphatase and tensin homolog (PTEN) genomic deletions and transmembrane protease, serine 2/v-ets avian erthyroblastosis virus E26 oncogene homolog (ERG) rearrangements are two of the most common genetic abnormalities associated with prostate cancer. Prior studies have demonstrated these alterations portend worse clinical outcomes. Our objective is to evaluate the impact of biopsy-determined PTEN losses and TMPRSS2-ERG fusion on biochemical progression-free survival (bPFS) and overall survival (OS) in patients who receive SBRT for localized prostate cancer. Methods/materials: Patients received SBRT for localized prostate cancer on a prospective quality-of-life (QoL) and cancer outcomes study. For each patient, the single biopsy core with the highest grade/volume of cancer was evaluated for PTEN and ERG abnormalities. Differences in baseline patient and disease characteristics between groups were analyzed using ANOVA for age and χ2 for categorical groupings. bPFS and OS were calculated using the Kaplan Meier (KM) method with Log-Rank test comparison between groups. Predictors of bPFS and OS were identified using the Cox proportional hazards method. For all analyses, p <0.05 was considered statistically significant. Results: Ninety-nine consecutive patients were included in the analysis with a median follow-up of 72 months. A statistically significant improvement in bPFS (p = 0.018) was observed for wild type ERG patients with an estimated 5-year bPFS of 94.1% vs. 72.4%. Regarding PTEN mutational status, significant improvements in were observed in both bPFS (p = 0.006) and OS (p < 0.001), with estimated 5-year bPFS rates of 91.0% vs. 67.9% and 5-year OS rates of 96.4% vs. 79.4%. When including both ERG and PTEN mutational status in the analysis, there were statistically significant differences in both bPFS (p = 0.011) and OS (p < 0.001). The estimated 5-year bPFS rates were 100%, 76.6%, 72.9%, and 63.8% for patients with ERG+/PTEN+, ERG-/PTEN+, ERG+/PTEN-, and ERG-/PTEN- phenotypes respectively. The estimated 5-year OS rates were 93.9%, 100%, 80.0%, and 78.7% for patients with ERG+/PTEN+, ERG-/PTEN+, ERG+/PTEN-, and ERG-/PTEN- phenotypes respectively. Conclusion: ERG rearrangements and PTEN deletions detected on biopsy samples are associated with poorer oncologic outcomes in prostate cancer patients treated with SBRT and merit further study in a dedicated prospective trial.

Bothersome Hot Flashes Following Neoadjuvant Androgen Deprivation Therapy and Stereotactic Body Radiotherapy for Localized Prostate Cancer.

BACKGROUND: Androgen deprivation therapy (ADT) improves local cancer control in unfavorable localized prostate cancer treated with radiotherapy. ADT is known to cause hormonally related symptoms that resolve with testosterone recovery. Hot flashes are particularly burdensome. This study sought to evaluate the timeline of hot flashes following short-course ADT and stereotactic body radiotherapy (SBRT) as well as its relationship with testosterone recovery. METHODS: Institutional IRB approval was obtained for this retrospective review of prospectively collected data (IRB#: 2009-510). ADT was initiated three months prior to the start of SBRT. Hot flashes were self-reported via question 13a of the Expanded Prostate Index Composite (EPIC)-26 prior to ADT initiation, the first day of robotic SBRT, and at each follow-up (one, three, six, nine, 12, 18, 24, and 36 months). The responses were grouped into three relevant categories (no problem, very small-small problem, and moderate-big problem). Scores were transformed to a 0-100 scale with higher scores reflecting less bother. Testosterone levels were measured at each follow-up. RESULTS: From 2007 to 2010, 122 localized prostate cancer patients (nine low-, 64 intermediate-, and 49 high-risk according to the D'Amico classification) at a median age of 72 years (range 54.5-88.3) were treated with short course ADT (three to six months) and SBRT (35-36.25 Gy) at Georgetown University Hospital. Thirty-two percent were Black and 27% were obese. Seventy-seven percent of patients received three months of ADT. At baseline, 2% of men experienced hot flashes that were a "moderate to big problem" and that proportion peaked at the start of SBRT (45%) before returning to baseline (2%) nine months post-SBRT with a cumulative incidence of 52.4%. The median baseline EPIC-26 hot flash score of 94 declined to 50 at the start of SBRT but this returned to baseline (92) by six months post SBRT. These changes were both statistically and clinically significant (MID = 9.5083, p<0.01). Testosterone recovery (> 230 ng/dL) occurred in approximately 70% of patients by 12 months post SBRT. Resolution of hot flashes correlated with testosterone recovery. CONCLUSION: Bothersome hot flashes occur in greater than 50% of men treated with neoadjuvant ADT. Resolution of hot flashes occurs in the majority of patients within one year after treatment. Reassurance of the temporary nature of hot flashes may assist in reducing patient anxiety. Measuring testosterone levels at follow-up visits may allow for anticipatory counseling that may limit the associated bother.

Impact of neoadjuvant relugolix on patient-reported sexual function and bother.

Introduction: Sexual function following local treatment for prostate cancer is an important quality of life concern. Relugolix is a novel oral GnRH receptor antagonist used in combination with radiation therapy in the treatment of unfavorable prostate cancer. It has been shown to achieve rapid and profound testosterone suppression. As a result, these very low testosterone levels may impact both sexual functioning and perceptions. This prospective study sought to assess neoadjuvant relugolix-induced sexual dysfunction prior to stereotactic body radiation therapy (SBRT). Methods: Between March 2021 and September 2023, 87 patients with localized prostate cancer were treated with neoadjuvant relugolix followed by SBRT per an institutional protocol. Sexual function and bother were assessed via the sexual domain of the validated Expanded Prostate Index Composite (EPIC-26) survey. Responses were collected for each patient at pre-treatment baseline and after several months of relugolix. A Utilization of Sexual Medications/Devices questionnaire was administered at the same time points to assess erectile aid usage. Results: The median age was 72 years and 43% of patients were non-white. The median baseline Sexual Health Inventory for Men (SHIM) score was 13 and 41.7% of patients utilized sexual aids prior to relugolix. Patients initiated relugolix at a median of 4.5 months (2-14 months) prior to SBRT. 95% and 87% of patients achieved effective castration (≤ 50 ng/dL) and profound castration (< 20 ng/dl) at SBRT initiation, respectively. Ability to have an erection, ability to reach orgasm, quality of erections, frequency of erections, and overall sexual function significantly declined following relugolix. There was a non- significant increase in sexual bother. Discussion: In concordance with known side effects of androgen deprivation therapy (ADT), neoadjuvant relugolix was associated with a significant decline in self-reported sexual function. However, patients indicated only a minimal and non-significant increase in bother. Future investigations should compare outcomes while on relugolix directly to GnRH agonist-induced sexual dysfunction.

Early biochemical outcomes following neoadjuvant/adjuvant relugolix with stereotactic body radiation therapy for intermediate to high risk prostate cancer.

Introduction: Injectable GnRH receptor agonists have been shown to improve cancer control when combined with radiotherapy. Prostate SBRT offers an abbreviated treatment course with comparable efficacy to conventionally fractionated radiotherapy. Relugolix is a new oral GnRH receptor antagonist which achieves rapid, sustained testosterone suppression. This prospective study sought to evaluate early testosterone suppression and PSA response following relugolix and SBRT for intermediate to high prostate cancer. Methods: Relugolix was initiated at least 2 months prior to SBRT. Interventions to improve adherence were not utilized. PSA and total testosterone levels were obtained prior to and 1-4 months post SBRT. Profound castration was defined as serum testosterone ≤ 20 ng/dL. Early PSA nadir was defined as the lowest PSA value within 4 months of completion of SBRT. Per prior trials, we examined the percentage of patients who achieved PSA level of ≤ 0.5 ng/mL and ≤ 0.2 ng/mL during the first 4 months post SBRT. Results: Between July 2021 and January 2023, 52 men were treated at Georgetown with relugolix (4-6 months) and SBRT (36.25-40 Gy in 5 fractions) per an institutional protocol (IRB 12-1775). Median age was 71 years. 26.9% of patients were African American and 28.8% were obese (BMI ≥30 kg/m2). The median pretreatment PSA was 9.1 ng/ml. 67% of patients were ≥ Grade Group 3. 44 patients were intermediate- and 8 were high-risk. Patients initiated relugolix at a median of 3.6 months prior to SBRT with a median duration of 6.2 total months. 92.3% of patients achieved profound castration during relugolix treatment. Poor drug adherence was observed in 2 patients. A third patient chose to discontinue relugolix due to side effects. By post-SBRT month 4, 87.2% and 74.4% of patients achieved PSA levels ≤ 0.5 ng/ml and ≤ 0.2 ng/ml, respectively. Discussion: Relugolix combined with SBRT allows for high rates of profound castration with low early PSA nadirs. We observed a 96% testosterone suppresion rate without the utilization of scheduled cues/reminders. This finding supports the notion that patients with localized prostate cancer can consistently and successfully follow an oral ADT protocol without daily reminders. Given relugolix's potential benefits over injectable GnRH receptor agonists, its usage may be preferred in specific patient populations (fear of needles, prior cardiovascular events). Future studies should focus on boundaries to adherence in specific underserved populations.

Intensity modulated radiation therapy with stereotactic body radiation therapy boost for unfavorable prostate cancer: five-year outcomes.

Purpose: Intensity-modulated radiation therapy (IMRT) with brachytherapy boost for unfavorable prostate cancer has been shown to improve biochemical relapse-free survival compared to IMRT alone. Stereotactic body radiation therapy (SBRT) is a less-invasive alternative to brachytherapy. Early outcomes utilizing SBRT boost suggest low rates of high-grade toxicity with a maintained patient-reported quality of life. Here, we report the 5-year progression-free survival (PFS) and prostate cancer-specific survival (PCSS) of patients treated with IMRT plus SBRT boost. Materials and methods: Between 2008 and 2020, 255 patients with unfavorable prostate cancer were treated with robotic SBRT (19.5 Gy in three fractions) followed by fiducial-guided IMRT (45-50.4 Gy) according to an institutional protocol. For the first year, the patient's PSA level was monitored every 3 months, biannually for 2 years, and annually thereafter. Failure was defined as nadir + 2 ng/mL or a rising PSA with imaging suggestive of recurrence. Detection of recurrence also included digital rectal examination and imaging studies, such as MRI, CT, PET/CT, and/or bone scans. PFS and PCSS were calculated using the Kaplan-Meier method. Results: The median follow-up period was 71 months. According to the NCCN risk classification, 5% (13/255) of the patients had favorable intermediate-risk disease, 23% (57/255) had unfavorable intermediate-risk disease, 40% (102/255) had high-risk disease, and 32% (83/255) had very high-risk disease. Androgen deprivation therapy was administered to 80% (204/255) of the patients. Elective pelvic lymph node IMRT was performed in 28 (10%) patients. The PFS for all patients at 5 years was 81% (favorable intermediate risk, 91%; unfavorable intermediate risk, 89%; high-risk, 78%; and very-high risk, 72%). The PCSS for all patients at 5 years was 97% (favorable intermediate risk, 100%; unfavorable intermediate risk, 100%; high risk, 100%; and very high risk, 89%). Conclusion: The incidence of failure following IMRT plus SBRT for unfavorable prostate cancer remains low at 5 years.

Association of baseline self-reported fatigue with overall survival after stereotactic body radiation therapy for localized prostate cancer.

Introduction: Stereotactic Body Radiation Therapy (SBRT) has emerged as a definitive therapy for localized prostate cancer (PCa). However, more data is needed to predict patient prognosis to help guide which patients will benefit most from treatment. The FACIT-Fatigue (FACIT-F) is a well validated, widely used survey for assessing fatigue. However, the role of fatigue in predicting PCa survival has yet to be studied. Herein, we investigate the role of FACIT-F as a baseline predictor for overall survival (OS) in patients undergoing SBRT for localized PCa. Methods: A retrospective review was conducted of 1358 patients who received SBRT monotherapy between January 2008 to April 2021 at an academic, tertiary referral center. FACIT-F scores (range 0 to 52) were summed for patients who answered all 13-items on the survey. FACIT-F total scores of ≥35 represented severe fatigue. Patients receiving androgen deprivation therapy were excluded. Differences in fatigue groups were evaluated using chi-squared tests. OS rates were determined using the Kaplan-Meier method and predictors of OS were evaluated using Cox proportional hazard method. Results: Baseline full FACIT-F scores and survival data was available for 891 patients. 5-year OS was 87.6% and 95.2%, respectively, for the severely fatigued and non-fatigued groups. Chi-squared analysis of fatigue groups showed no significant difference in the following categories: D'Amico risk group, age, ethnicity, grade group, T-stage, or PSA density. Severe fatigue was associated with a significant decrease in OS (hazard ratio 2.76; 95%CI 1.55 - 4.89). The Cox proportional hazard model revealed that age and FACIT-F were both statistically significant (p <0.05). Conclusion: Baseline FACIT-F scores are significantly associated with OS. Higher FACIT-F scores, representing less fatigued patients, are associated with an overall survival benefit. These results indicate that the FACIT-F survey could serve as an additional metric for clinicians in determining prognostic factors for patients undergoing SBRT.

Patient-Reported Financial Burden Following Stereotactic Body Radiation Therapy for Localized Prostate Cancer.

Introduction and Objectives: In patients with localized prostate cancer, 5-fraction, stereotactic body radiation therapy (SBRT) has been found to offer comparable oncologic outcomes and potential for improved treatment compliance compared to conventional, 40-plus fraction radiation therapy (RT). Recent studies of oncologic patient experiences have highlighted both the impact of therapy-associated financial toxicity (FT) on treatment adherence and health-related quality of life (HRQOL). Methods: A cross-sectional assessment of FT after SBRT was performed using the 12-item COST questionnaire. The total questionnaire score (range 0-44) was used to evaluate the FT grade (0-3), with a higher COST value representing lower grade. The patient zip code was used to approximate the distance from the index hospital. Univariate and multivariate analyses of the average COST score (0-4) are performed. Results: The response rate was 57.5% (332 of 575 consented patients) with 90.7%, 8.2%, and 1.1% experiencing grade 0, 1, and 2 FT, respectively, with no grade 3. Unemployment or disability, non-white race, low income, and concurrent hormonal therapy were associated with a statistically significant worse FT (lower COST value) on univariate and multivariate analyses (p < 0.05). Education level and insurance status significant were evaluated on univariate analysis only. There was a non-statistically significant difference in age, marital status, time since treatment, and distance from the index hospital. Conclusions: SBRT was associated with low FT. However, statistically significant socioeconomic disparities in FT remain despite ultra-hypofractionated treatment.

BCL2 and CASP8 regulation by NF-kappaB differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells.

Resistance to endocrine therapies remains a major problem in the management of estrogen receptor-alpha (ER)-positive breast cancer. We show that inhibition of NF-kappaB (p65/RELA), either by overexpression of a mutant IkappaB (IkappaBSR) or a small-molecule inhibitor of NF-kappaB (parthenolide; IC(50)=500 nM in tamoxifen-resistant cells), synergistically restores sensitivity to 4-hydroxytamoxifen (4HT) in resistant MCF7/RR and MCF7/LCC9 cells and further sensitizes MCF-7 and MCF7/LCC1 control cells to 4HT. These effects are independent of changes in either cell cycle distribution or in the level of autophagy measured by inhibition of p62/SQSTM1 expression and cleavage of LC3. NF-kappaB inhibition restores the ability of 4HT to decrease BCL2 expression, increase mitochondrial membrane permeability, and induce a caspase-dependent apoptotic cell death in resistant cells. Each of these effects is reversed by a caspase 8 (CASP8)-specific inhibitor that blocks enzyme-substrate binding. Thus, increased activation of NF-kappaB can alter sensitivity to tamoxifen by modulating CASP8 activity, with consequent effects on BCL2 expression, mitochondrial function, and apoptosis. These data provide significant new insights into how molecular signaling affects antiestrogen responsiveness and strongly suggest that a combination of parthenolide and tamoxifen may offer a novel therapeutic approach to the management of some ER-positive breast cancers.

Treatment Interruptions During Stereotactic Body Radiotherapy for Prostate Cancer.

BACKGROUND: During the course of radiation treatment for prostate cancer, patients may have unintentional interruptions in their treatment course due to a wide variety of factors. Stereotactic body radiation therapy (SBRT) decreases the number of treatments compared to conventionally fractionated radiation; hence, it has the potential to decrease treatment delays and non-completion. This study sought to determine the incidence of treatment delay and characterize the etiology and length in a large cohort of men treated with SBRT for their prostate cancer. METHODS: One thousand three hundred and thirty-six patients treated with SBRT from 2008 to 2021 at the Georgetown University Hospital for prostate cancer were included in this retrospective study. A treatment delay was defined as a patient requiring longer than 14 days to complete 5 fractions of SBRT. Non-completion was defined as patients treated with less than 5 fractions. In the patients who experienced delays, chart review was performed to characterize the length and etiology of each delay. Multivariate analysis was performed via binary logistic regression modeling on PSPP. RESULTS: All individuals in the cohort eventually completed the planned 5-fraction regimen. Thirty-three patients experienced a treatment delay. Median length of time to complete treatment was 11 days (range 5-155 days). In patients who experienced a delay, nearly half (45.5%) experienced only a one-day delay. The most common reason for a delay was a technical issue (48.5%), including the machine maintenance, fiducial misalignment, or inadequate pretreatment bowel preparation. Other reasons included unplanned breaks due to acute side effects (21.2%), logistical issues (18.2%), non-treatment related health issues (9.1%), and inclement weather (3.0%). There were no significant sociodemographic, oncologic, or treatment variables that predicted treatment interruption on multivariate analysis. CONCLUSIONS: The incidence of treatment interruptions in patients undergoing SBRT for their prostate cancer was low. Most treatment delays were short.

Human X-box binding protein-1 confers both estrogen independence and antiestrogen resistance in breast cancer cell lines.

Human X-box binding protein-1 (XBP1) is an alternatively spliced transcription factor that participates in the unfolded protein response (UPR), a stress-signaling pathway that allows cells to survive the accumulation of unfolded proteins in the endoplasmic reticulum lumen. We have previously demonstrated that XBP1 expression is increased in antiestrogen-resistant breast cancer cell lines and is coexpressed with estrogen receptor alpha (ER) in breast tumors. The purpose of this study is to investigate the role of XBP1 and the UPR in estrogen and antiestrogen responsiveness in breast cancer. Overexpression of spliced XBP1 [XBP1(S)] in ER-positive breast cancer cells leads to estrogen-independent growth and reduced sensitivity to growth inhibition induced by the antiestrogens Tamoxifen and Faslodex in a manner independent of functional p53. Data from gene expression microarray analyses imply that XBP1(S) acts through regulation of the expression of ER, the antiapoptotic gene BCL2, and several other genes associated with control of the cell cycle and apoptosis. Testing this hypothesis, we show that overexpression of XBP1(S) prevents cell cycle arrest and antiestrogen-induced cell death through the mitochondrial apoptotic pathway. XBP1 and/or the UPR may be a useful molecular target for the development of novel predictive and therapeutic strategies in breast cancer.

Autophagy and unfolded protein response (UPR) regulate mammary gland involution by restraining apoptosis-driven irreversible changes.

The postnatal mammary gland undergoes repeated cycles of proliferation and cell death, most notably when the fully differentiated (lactating) gland dedifferentiates to a prelactation state. Accumulation of milk proteins in the secretory epithelium creates the stress signal that triggers this process (involution). How this stress is perceived, and the cellular processes that are subsequently activated, remain unclear. We now report that Unfolded Protein Response (UPR), autophagy, and apoptosis related genes cluster separately during lactation and involution in the mouse mammary gland. Time-course experiments in rodents show that autophagy and UPR signaling are tightly co-regulated at the transition from reversible to irreversible involution. Inhibition of autophagy by chloroquine or genetic deletion of one ATG7 allele enhanced progression of mammary involution into the irreversible phase, as characterized by an early/precocious induction of apoptosis. These are the first preclinical in vivo data in support of a clinical trial testing an autophagy inhibitor for prevention of intraductal breast malignancy progression to invasive breast cancer. In marked contrast, stimulation of autophagy by low dose tunicamycin treatment reduced apoptosis and extended the reversible phase of involution by sustaining the secretory epithelium. Autophagy stimulators could be used short-term to promote lactation in women experiencing difficulties or irregularities in nursing. Taken together, these data indicate that UPR and autophagy play a key role in regulating the balance between cell survival and apoptosis during normal mammary gland regression.

Integrated molecular analysis of Tamoxifen-resistant invasive lobular breast cancer cells identifies MAPK and GRM/mGluR signaling as therapeutic vulnerabilities.

Invasive lobular breast cancer (ILC) is an understudied malignancy with distinct clinical, pathological, and molecular features that distinguish it from the more common invasive ductal carcinoma (IDC). Mounting evidence suggests that estrogen receptor-alpha positive (ER+) ILC has a poor response to Tamoxifen (TAM), but the mechanistic drivers of this are undefined. In the current work, we comprehensively characterize the SUM44/LCCTam ILC cell model system through integrated analysis of gene expression, copy number, and mutation, with the goal of identifying actionable alterations relevant to clinical ILC that can be co-targeted along with ER to improve treatment outcomes. We show that TAM has several distinct effects on the transcriptome of LCCTam cells, that this resistant cell model has acquired copy number alterations and mutations that impinge on MAPK and metabotropic glutamate receptor (GRM/mGluR) signaling networks, and that pharmacological inhibition of either improves or restores the growth-inhibitory actions of endocrine therapy.

The A4396G polymorphism in interferon regulatory factor 1 is frequently expressed in breast cancer cell lines.

Loss or mutation of known tumor suppressor genes accounts for a small proportion of all breast cancers. We have recently shown that interferon regulatory factor 1 (IRF1) is a putative tumor suppressor gene in breast cancer. We now report that the A4396G single nucleotide polymorphism in the IRF1 gene is more frequent in human breast cancer cell lines than in the general population (P = 0.01). Furthermore, A4396G is more frequently expressed in African American (black) than in European ancestry (white) subjects (n = 70 subjects; P < or = 0.001), leading to a significant difference in genotype distribution between these populations (P = 0.002).

Effects of In Utero Exposure to Ethinyl Estradiol on Tamoxifen Resistance and Breast Cancer Recurrence in a Preclinical Model.

Background: Responses to endocrine therapies vary among patients with estrogen receptor (ER+) breast cancer. We studied whether in utero exposure to endocrine-disrupting compounds might explain these variations. Methods: We describe a novel ER+ breast cancer model to study de novo and acquired tamoxifen (TAM) resistance. Pregnant Sprague Dawley rats were exposed to 0 or 0.1 ppm ethinyl estradiol (EE2), and the response of 9,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors to 15 mg/kg TAM, with (n = 17 tumors in the controls and n = 20 tumors in EE2 offspring) or without 1.2 g/kg valproic acid and 5 mg/kg hydralazine (n = 24 tumors in the controls and n = 32 tumors in EE2 offspring) in the female offspring, was assessed. One-sided Chi2 tests were used to calculate P values. Comparisons of differentially expressed genes between mammary tumors in in utero EE2-exposed and control rats, and between anti-estrogen-resistant LCC9 and -sensitive LCC1 human breast cancer cells, were also performed. Results: In our preclinical model, 54.2% of mammary tumors in the control rats exhibited a complete response to TAM, of which 23.1% acquired resistance with continued anti-estrogen treatment and recurred. Mammary tumors in the EE2 offspring were statistically significantly less likely to respond to TAM (P = .047) and recur (P = .007). In the EE2 offspring, but not in controls, adding valproic acid and hydralazine to TAM prevented recurrence (P < .001). Three downregulated and hypermethylated genes (KLF4, LGALS3, MICB) and one upregulated gene (ETV4) were identified in EE2 tumors and LCC9 breast cancer cells, and valproic acid and hydralazine normalized the altered expression of all four genes. Conclusions: Resistance to TAM may be preprogrammed by in utero exposure to high estrogen levels and mediated through reversible epigenetic alterations in genes associated with epithelial-mesenchymal transition and tumor immune responses.

Estrogen receptor alpha positive breast tumors and breast cancer cell lines share similarities in their transcriptome data structures.

Established human breast cancer cell lines are widely used as experimental models in breast cancer research. While these cell lines and their variants share many phenotypic characteristics with human breast tumors, the extent to which they reflect the underlying molecular biology of breast cancer remains controversial. We explored this issue using a probabilistic rather than heuristic approach. Data from gene expression microarrays were used to compare the global structures of the transcriptomes of three estrogen receptor alpha positive (ER+) human breast cancer cell lines (MCF-7, T47D, ZR-75-1) and 13 human breast tumors (11 ER+; 2 ER-). Linear representations of the respective data structures were obtained by deriving those top principal components (PCs) required to capture > or =80% of the cumulative variance for each data set (M PCs). We then identified those genes most highly correlated with the M PCs (Pearson's correlation coefficient r > or =0.800) and identified a group of 36 genes commonly correlated with both the cell line (M = 5 PCs) and tumor (M = 6 PCs) data structures. All 36 common genes were correlated with PC1 from the breast tumor data: 21/36 genes were correlated with PC1, 14/36 genes correlated with PC2, and 1/36 genes correlated with PC3 from the cell line data. Genes important in defining the data structures include NFkappaB p65, IGFBP-6, ornithine decarboxylase-1, and paxillin. When data from MDA-MB-435 xenografts (ER-) were included in the analysis, we were unable to find any common genes between these xenografts and the breast tumors. These data clearly imply that MCF-7, T47D, and ZR-75-1 cells and ER+ breast tumors share substantial global similarities in the structures of their respective transcriptomes, and that these cell lines are good models in which to identify molecular events that are likely to be important in some ER+ human breast cancers.

The nuclear factor kappa B inhibitor parthenolide restores ICI 182,780 (Faslodex; fulvestrant)-induced apoptosis in antiestrogen-resistant breast cancer cells.

The molecular mechanisms underlying the acquisition of resistance to the antiestrogen Faslodex are poorly understood, although enhanced expression and activity of nuclear factor kappaB (NFkappaB) have been implicated as a critical element of this phenotype. The purpose of this study was to elucidate the mechanism by which NFkappaB up-regulation contributes to Faslodex resistance and to determine whether pharmacologic inhibition of NFkappaB by the small molecule parthenolide could restore Faslodex-mediated suppression of cell growth. Basal expression of multiple NFkappaB-related molecules in MCF7-derived LCC1 (antiestrogen-sensitive) and LCC9 (antiestrogen-resistant) breast cancer cells was determined, and cells were treated with Faslodex or parthenolide. The effect of these drugs either singly or in combination was assessed by cell proliferation, estrogen receptor (ER)-dependent transcriptional activation, cell cycle analysis, and apoptosis assays. Expression of the p65 NFkappaB subunit and the upstream NFkappaB regulator IkappaB kinase gamma/NFkappaB essential modulator were increased in the resistant MCF7/LCC9 cells (P=0.001 and 0.04, respectively). Whereas MCF7/LCC9 cells were unresponsive to Faslodex alone, parthenolide effectively inhibited MCF7/LCC9 cell proliferation and the combination of Faslodex and parthenolide resulted in a 4-fold synergistic reduction in cell growth (P=0.03). This corresponded to a restoration of Faslodex-induced apoptosis (P=0.001), with no observable changes in ER-dependent transcription or cell cycle phase distribution. Because parthenolide has shown safety in Phase I clinical trials, these findings have direct clinical relevance and provide support for the design of clinical studies combining antiestrogens and parthenolide in ER-positive breast cancer.

Endoplasmic Reticulum Stress Protein GRP78 Modulates Lipid Metabolism to Control Drug Sensitivity and Antitumor Immunity in Breast Cancer.

The unfolded protein response is an endoplasmic reticulum stress pathway mediated by the protein chaperone glucose regulated-protein 78 (GRP78). Metabolic analysis of breast cancer cells shows that GRP78 silencing increases the intracellular concentrations of essential polyunsaturated fats, including linoleic acid. Accumulation of fatty acids is due to an inhibition of mitochondrial fatty acid transport, resulting in a reduction of fatty acid oxidation. These data suggest a novel role of GRP78-mediating cellular metabolism. We validated the effect of GRP78-regulated metabolite changes by treating tumor-bearing mice with tamoxifen and/or linoleic acid. Tumors treated with linoleic acid plus tamoxifen exhibited reduced tumor area and tumor weight. Inhibition of either GRP78 or linoleic acid treatment increased MCP-1 serum levels, decreased CD47 expression, and increased macrophage infiltration, suggesting a novel role for GRP78 in regulating innate immunity. GRP78 control of fatty acid oxidation may represent a new homeostatic function for GRP78. Cancer Res; 76(19); 5657-70. ©2016 AACR.