Collateral-resistance to estrogen and HER-activated growth is associated with modified AKT, ERα, and cell-cycle signaling in a breast cancer model.

Aim: A model of progressively endocrine-resistant breast cancer was investigated to identify changes that can occur in signaling pathways after endocrine manipulation. Methods: The MCF7 breast cancer model is sensitive to estrogens and anti-estrogens while variant lines previously derived from wild-type MCF7 are either relatively 17ß-estradiol (E2)-insensitive (LCC1) or fully resistant to estrogen and anti-estrogens (LCC9). Results: In LCC1 and LCC9 cell lines, loss of estrogen sensitivity was accompanied by loss of growth response to transforming growth factor alpha (TGFα), heregulin-beta and pertuzumab. LCC1 and LCC9 cells had enhanced AKT phosphorylation relative to MCF7 which was reflected in downstream activation of phospho-mechanistic target of rapamycin (mTOR), phospho-S6, and phospho-estrogen receptor alpha Ser167 [ERα(Ser167)]. Both AKT2 and AKT3 were phosphorylated in the resistant cell lines, but small interfering RNA (siRNA) knockdown suggested that all three AKT isoforms contributed to growth response. ERα(Ser118) phosphorylation was increased by E2 and TGFα in MCF7, by E2 only in LCC1, but by neither in LCC9 cells. Multiple alterations in E2-mediated cell cycle control were identified in the endocrine-resistant cell lines including increased expression of MYC, cyclin A1, cyclin D1, cyclin-dependent kinase 1 (CDK1), CDK2, and hyperphosphorylated retinoblastoma protein (ppRb), whereas p21 and p27 were reduced. Estrogen modulated expression of these regulators in MCF7 and LCC1 cells but not in LCC9 cells. Seliciclib inhibited CDK2 activation in MCF7 cells but not in resistant variants; in all lines, it reduced ppRb, increased p53 associated responses including p21, p53 up-regulated modulator of apoptosis (PUMA), and p53 apoptosis-inducing protein 1 (p53AIP1), inhibited growth, and produced G2/M block and apoptosis. Conclusions: Multiple changes occur with progression of endocrine resistance in this model with AKT activation contributing to E2 insensitivity and loss of ERα(Ser118) phosphorylation being associated with full resistance. Cell cycle regulation is modified in endocrine-resistant breast cancer cells, and seliciclib is effective in both endocrine-sensitive and resistant diseases.

Integrin αvß6-specific therapy for pancreatic cancer developed from foot-and-mouth-disease virus.

Goals of investigation: The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained at <5% for decades because no effective therapies have been identified. Integrin αvß6 is overexpressed in most PDAC and represents a promising therapeutic target. Thus, we attempted to develop an αvß6-specific peptide-drug conjugate (PDC) for therapy of PDAC. Methodology: We conjugated the DNA-binding pyrrolobenzodiazepine (PBD)-based payload SG3249 (tesirine) to an αvß6-specific 20mer peptide from the VP1 coat protein of foot-and-mouth-disease virus (FMDV) (forming conjugate SG3299) or to a non-targeting peptide (forming conjugate SG3511). PDCs were tested for specificity and toxicity on αvß6-negative versus-positive PDAC cells, patient-derived cell lines from tumor xenografts, and on two different in vivo models of PDAC. Immunohistochemical analyses were performed to establish therapeutic mechanism. Results: The αvß6-targeted PDC SG3299 was significantly more toxic (up to 78-fold) for αvß6-expressing versus αvß6-negative PDAC cell lines in vitro, and achieved significantly higher toxicity at equal dose than the non-targeted PDC SG3511 (up to 15-fold better). Moreover, SG3299 eliminated established (100mm3) Capan-1 PDAC human xenografts, extending the lifespan of mice significantly (P=0.005). Immunohistochemistry revealed SG3299 induced DNA damage and apoptosis (increased γH2AX and cleaved caspase 3, respectively) associated with significant reductions in proliferation (Ki67), ß6 expression and PDAC tumour growth. Conclusions: The FMDV-peptide drug conjugate SG3299 showed αvß6-selectivity in vitro and in vivo and can specifically eliminate αvß6-positive cancers, providing a promising new molecular- specific therapy for pancreatic cancer.

The integrin αvß6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy.

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvß6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of ß6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10-8 ). In two separate cohorts, we showed that over 80% of PDACs expressed αvß6 protein and that paired metastases retained αvß6 expression. In vitro, integrin αvß6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvß6-positive human PDAC xenografts and transgenic mice bearing αvß6-positive PDAC with the αvß6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p < 0.0001) and increased survival (log-rank test, p < 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase-3) and suppression of the pro-tumourigenic microenvironment (suppression of TGFß signalling, fewer αSMA-positive myofibroblasts, decreased blood vessel density). These data show that αvß6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

PHLDA1 Mediates Drug Resistance in Receptor Tyrosine Kinase-Driven Cancer.

Development of resistance causes failure of drugs targeting receptor tyrosine kinase (RTK) networks and represents a critical challenge for precision medicine. Here, we show that PHLDA1 downregulation is critical to acquisition and maintenance of drug resistance in RTK-driven cancer. Using fibroblast growth factor receptor (FGFR) inhibition in endometrial cancer cells, we identify an Akt-driven compensatory mechanism underpinned by downregulation of PHLDA1. We demonstrate broad clinical relevance of our findings, showing that PHLDA1 downregulation also occurs in response to RTK-targeted therapy in breast and renal cancer patients, as well as following trastuzumab treatment in HER2+ breast cancer cells. Crucially, knockdown of PHLDA1 alone was sufficient to confer de novo resistance to RTK inhibitors and induction of PHLDA1 expression re-sensitized drug-resistant cancer cells to targeted therapies, identifying PHLDA1 as a biomarker for drug response and highlighting the potential of PHLDA1 reactivation as a means of circumventing drug resistance.

Therapeutic targeting of integrin αvß6 in breast cancer.

BACKGROUND: Integrin αvß6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvß6 has yet to be elucidated in breast cancer. METHODS: Protein expression of integrin subunit beta6 (ß6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of ß6 in breast cell lines, the role of αvß6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student's t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni's Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided. RESULTS: High expression of either the mRNA or protein for the integrin subunit ß6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of ß6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts. CONCLUSIONS: Targeting αvß6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.