Endocrine Therapy-resistant Cancer Research Articles

MiR-484 suppresses endocrine therapy-resistant cells by inhibiting KLF4-induced cancer stem cells in estrogen receptor-positive cancers.

Endocrine therapy (mainly anti-estrogen therapy) is the mainstay of treatment for estrogen receptor (ER) positive breast cancer (BCa). However, approximately one-third of BCa patients who receive endocrine therapy may develop resistance. The detailed mechanism is still unclear. MCF7 and T-47D cells were treated with ERα antagonist tamoxifen for 2months until they became tamoxifen-resistant. qPCR was used to detect the stem markers like CD44, OCT4 and SOX2. Flow cytometry and sphere formation were performed to test the stemness. Cell growth and invasiveness were measured by MTS assay, xenograft mouse model, and invasion assay. We found that tamoxifen resistant BCa cells acquired certain malignant phenotypes, such as higher expression of KLF4, stemness and enhanced invasiveness. Furthermore, miR-484 was found to act as a tumor suppressor and directly downregulated KLF4. KLF4-induced cancer stem cell (CSCs) contributes to anti-ER therapy resistant and is a potential target in endocrine therapy-resistant cancers.

Abstract P2-11-06: Assessment of ESR1 genomic aberrations and their role in endocrine therapy resistance in breast cancer

Abstract Background: Endocrine therapy (ET) is an effective treatment of estrogen receptor positive (ER+) breast cancer (BC). However, not all ER+ cancers respond to ET and many eventually acquire resistance. Genomic aberrations in ESR1 have been reported to play a role in resistance to treatment. ESR1 mutations (ESRMs), reported in 10-50% of metastatic or recurrent BCs treated with aromatase inhibitors (AIs), can lead to constitutive activation and reduced sensitivity to ET. The incidence and clinical implications of ESR1 amplification (ESRA) is not well-established. A variety of structural rearrangements involving ESR1 have been reported in primary BCs, with some more strongly associated with tamoxifen and AI resistance. This study aimed to establish a rapid, reliable and cost-effective method to screen and monitor ESR1 genomic aberrations in clinical BC tissue samples and relate these to the 1st line and subsequent ETs patients received. Patients: • Cohort A - 20 post-menopausal women (PMW) with ER+ BC who had acquired resistance to AIs and received subsequent lines of ET. Previous NGS data were available for these patients. • Cohort (B) - 425 ER+ BC patients, with paired matched tissue samples from the primary and progressive/recurrent cancer on 1st line ET; sites included local recurrence (25%), nodal recurrence (29%), distant recurrence (3%) and primary progression on neoadjuvant ET (44%). Median follow-up 10 years. All patients received 2nd line ET, 14% developed a further recurrence on 2nd line ET. Methods: ESRMs were assessed by allele-specific real-time quantitative (rt-qPCR) and digital droplet PCR (ddPCR) assays, a novel fluorometric in situ mutation detection (ISMD) approaches and AmpliSeq targeted sequencing. ESRA and ESR1 fusions were detected by targeted sequencing and validated using FISH and custom ligation assays for commonly-reported fusion proteins (currently ESR1-e6>YAP1 and ESR1-e6>PCDH11X), respectively. Results: Results from ddPCR and ISMD were consistent with NGS findings in cohort A. There was expansion of D538G mutant clones with acquired resistance in 5/20 patients (25%). ESR1 copy number gain was seen in 11/20 patients (55%) in resistant samples. Gene amplification was confirmed by FISH in 6, corresponding to those with the highest gain from the NGS data. In cohort B, recurrent/resistant samples (including 2nd recurrences) with matched primaries are currently being screened for ESR1 genomic aberrations using all methods allowing for a comprehensive comparison. This will allow full characterisation of mutations, copy number changes and gene fusions in the largest cohort of ET resistant cancers to date. Results will be interpreted in the context of 1st line ET (51% Tamoxifen, 34% non-steroidal AI, 8% exemestane, 7% other ET) and 2nd line ET in the 14% of patients who developed a 2nd recurrence. ESR1 genomic aberrations have been identified in 38% of samples to date, with specific aberrations associated with particular ETs. Discussion: • A reliable, robust and cost-effective methodology for the detection and quantitation of ESR1 aberrations in clinical BC samples has been developed and compared with NGS and targeted sequencing approaches. • This method would allow rapid screening for key aberrations with the potential to inform selection of 2nd line therapy. • Multiplexing of fluorometric assays may enable in situ clonality analysis that allows visualisation of multiple genomic driver aberrations simultaneously. • In the largest cohort of patients with resistance to ET to date, there is a high incidence of ESR1 genomic aberrations. These are associated with specific ETs. Analysis between these changes and response, disease-free and overall breast cancer-specific survival on 2nd line ET is currently ongoing. Citation Format: Carlos Martinez-Perez, Charlene Kay, James Meehan, Mark Gray, Rebecca Swan, Lorna Renshaw, Jane Keys, Andrew H Sims, Olga Oikonomidou, J Michael Dixon, Arran K Turnbull. Assessment of ESR1 genomic aberrations and their role in endocrine therapy resistance in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-06.

ESR1 mutations are frequent in newly diagnosed metastatic and loco-regional recurrence of endocrine-treated breast cancer and carry worse prognosis

BackgroundEmerging mutations in the ESR1 gene that encodes for the estrogen receptor (ER) are associated with resistance to endocrine therapy. ESR1 mutations rarely exist in primary tumors (~ 1%) but are relatively common (10–50%) in metastatic, endocrine therapy-resistant cancers and are associated with a shorter progression-free survival. Little is known about the incidence and clinical implication of these mutations in early recurrence events, such as local recurrences or newly diagnosed metastatic disease.MethodsWe collected 130 archival tumor samples from 103 breast cancer patients treated with endocrine therapy prior to their local/metastatic recurrence. The cohort consisted of 41 patients having at least 1 sample from local/loco-regional recurrence and 62 patients with metastatic disease (of whom 41 newly diagnosed and 28 with advanced disease). The 5 most common ESR1 hotspot mutations (D538G, L536R, Y537S/N/C) were analyzed either by targeted sequencing or by droplet digital PCR. Progression-free survival (PFS), disease-free survival (DFS), and distant recurrence-free survival (DRFS) were statistically tested by Kaplan-Meier analysis.ResultsThe prevalence of ESR1 mutations was 5/41 (12%) in newly diagnosed metastatic patients and 5/28 (18%) for advanced metastases, detected at allele frequency > 1%. All mutations in advanced metastases were detected in patients previously treated with both tamoxifen (TAM) and aromatase inhibitors (AI). However, in newly diagnosed metastatic patients, 4/5 mutations occurred in patients treated with TAM alone. PFS on AI treatment in metastatic patients was significantly shorter for ESR1 mutation carriers (p = 0.017). In the local recurrence cohort, ESR1 mutations were identified in 15/41 (36%) patients but only 4/41 (10%) were detected at allele frequency > 1%. Again, most mutations (3/4) were detected under TAM monotherapy. Notably, 1 patient developed ESR1 mutation while on neoadjuvant endocrine therapy. DFS and DRFS were significantly shorter (p = 0.04 and p = 0.017, respectively) in patients that had ESR1 mutations (> 1%) in their loco-regional recurrence tumor.ConclusionsClinically relevant ESR1 mutations are prevalent in newly diagnosed metastatic and local recurrence of endocrine-treated breast cancer. Since local recurrences are amenable to curative therapy, these mutations may inform the selection of subsequent endocrine therapies.

FKBPL and its peptide derivatives inhibit endocrine therapy resistant cancer stem cells and breast cancer metastasis by downregulating DLL4 and Notch4

BackgroundOptimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease.MethodsIn this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett’s multiple comparison test.ResultsWe demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4.ConclusionThis study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.

Efficacy of SERD/SERM Hybrid-CDK4/6 Inhibitor Combinations in Models of Endocrine Therapy-Resistant Breast Cancer.

Endocrine therapy, using tamoxifen or an aromatase inhibitor, remains first-line therapy for the management of estrogen receptor (ESR1)-positive breast cancer. However, ESR1 mutations or other ligand-independent ESR1 activation mechanisms limit the duration of response. The clinical efficacy of fulvestrant, a selective estrogen receptor downregulator (SERD) that competitively inhibits agonist binding to ESR1 and triggers receptor downregulation, has confirmed that ESR1 frequently remains engaged in endocrine therapy-resistant cancers. We evaluated the activity of a new class of selective estrogen receptor modulators (SERM)/SERD hybrids (SSH) that downregulate ESR1 in relevant models of endocrine-resistant breast cancer. Building on the observation that concurrent inhibition of ESR1 and the cyclin-dependent kinases 4 and 6 (CDK4/6) significantly increased progression-free survival in advanced patients, we explored the activity of different SERD- or SSH-CDK4/6 inhibitor combinations in models of endocrine therapy-resistant ESR1(+) breast cancer. SERDs, SSHs, and the CDK4/6 inhibitor palbociclib were evaluated as single agents or in combination in established cellular and animal models of endocrine therapy-resistant ESR1(+) breast cancer. The combination of palbociclib with a SERD or an SSH was shown to effectively inhibit the growth of MCF7 cell or ESR1-mutant patient-derived tumor xenografts. In tamoxifen-resistant MCF7 xenografts, the palbociclib/SERD or SSH combination resulted in an increased duration of response as compared with either drug alone. A SERD- or SSH-palbociclib combination has therapeutic potential in breast tumors resistant to endocrine therapies or those expressing ESR1 mutations. See related commentary by DeMichele and Chodosh, p. 4999.

Identification of a 1‐cM region of common deletion on 13q14 associated with human prostate cancer

Frequent allelic losses on chromosome arm 13q are observed in carcinomas of the head and neck, breast, ovary, and pituitary gland. We analyzed 59 primary prostate tumors (stage B, 18 patients; C, 12 patients; D1, 4 patients; and endocrine therapy-resistant cancer death, 25 patients), as well as 18 metastatic tissues from 14 of the 25 cancer death patients for loss of heterozygosity (LOH) using 35 microsatellite markers on chromosome arm 13q. Of the 59 primary tumors, 31 (53%) showed LOH involving at least one locus. Detailed deletion mapping identified a distinct commonly deleted region in the 1-cM interval flanked by D13S153 and D13S273 on 13q14 and this region overlapped a part of the RB1 gene. Paired DNAs were available from both primary and metastatic tumors in the 14 cases of cancer death; among those pairs, we detected LOH on 13q in seven (50%) primary tumors, and in all metastatic foci (P = 0.0029). Moreover, the regions lost in metastatic tissues were more extensive than those seen in the corresponding primary tumors. These results suggest that inactivation of a putative tumor suppressor gene(s) including the RB1 gene on 13q14 plays an important role in human prostate cancer. Genes Chromosomes Cancer 24:183–190, 1999. © 1999 Wiley-Liss, Inc.

Identification of a 1-cM region of common deletion on 13q14 associated with human prostate cancer

Frequent allelic losses on chromosome arm 13q are observed in carcinomas of the head and neck, breast, ovary, and pituitary gland. We analyzed 59 primary prostate tumors (stage B, 18 patients; C, 12 patients; D1, 4 patients; and endocrine therapy-resistant cancer death, 25 patients), as well as 18 metastatic tissues from 14 of the 25 cancer death patients for loss of heterozygosity (LOH) using 35 microsatellite markers on chromosome arm 13q. Of the 59 primary tumors, 31 (53%) showed LOH involving at least one locus. Detailed deletion mapping identified a distinct commonly deleted region in the I-cM interval flanked by D13S153 and D13S273 on 13q14 and this region overlapped a part of the RB1 gene. Paired DNAs were available from both primary and metastatic tumors in the 14 cases of cancer death; among those pairs, we detected LOH on 13q in seven (50%) primary tumors, and in all metastatic foci (P = 0.0029). Moreover, the regions lost in metastatic tissues were more extensive than those seen in the corresponding primary tumors. These results suggest that inactivation of a putative tumor suppressor gene(s) including the RB1 gene on 13q14 plays an important role in human prostate cancer.

Codon 877 mutation in the androgen receptor gene in advanced prostate cancer: relation to antiandrogen withdrawal syndrome.

The growth of prostate cancer is androgen responsive, and androgen receptor (AR) is thought to play an important role in the development of this cancer. Recently, some reports demonstrated that AR gene mutations were detected in human prostate cancer tissues. We have previously reported that one of eight endocrine therapy-resistant prostate cancer cases showed AR gene mutation [Suzuki et al: J Steroid Biochem Mol Biol 46:759-765, 1993]. To further investigate structural abnormality of the AR in a large number of human prostate cancers, exons B-H encoding DNA-and hormone-binding domains were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products and direct sequencing. Tissues surgically removed from 30 cases of stage B or C prostate cancer and from 22 cases of endocrine therapy-resistant cancers obtained at autopsy were used in the study. Three out of 22 cancer death cases (14%) revealed AR gene mutations, one of which contained two different mutations-exon D in cancerous prostate and exon H in metastatic tissues. In the other two cases, AR gene mutations in exon H were found in metastatic tissues. All three cases in metastatic tissues showed the same mutation at codon 877 (877Thr-->Ala). In stage B or C cancer tissues and the other cancer death samples, no AR mutation was detected. The mutation in exon H was identical to that reported in a human prostate cancer cell line, LNCaP. These results indicate that AR gene mutation scarcely occurs in the early stage of prostate cancer and that the mutation is found in relation to endocrine therapy resistance. Two patients with an AR gene mutation at codon 877 revealed a remarkable fall in prostate-specific antigen after withdrawal of antiandrogen. Data on the other case were not available. These results indicate that a codon 877 mutation in the AR gene in advanced prostate cancer evokes the antiandrogen withdrawal syndrome. To our knowledge, this report is the first description of relationship between an AR mutation at codon 877 and the antiandrogen withdrawal syndrome.

Localization of a tumor suppressor gene associated with progression of human prostate cancer within a 1.2 Mb region of 8p22-p21.3.

Human prostate cancers frequently show loss of heterozygosity at loci on the short arm of chromosome 8. In order to take a step toward isolation of the putative tumor suppressor gene(s) on 8p via positional cloning, we performed high-resolution deletion mapping in 46 prostate cancers (stage B, 20 cases; stage C, 8 cases; endocrine therapy-resistant cancer death, 18 cases) using new 12 restriction fragment length polymorphism markers for this chromosomal region. Allelic losses were observed in 25 of the 44 cases (57%) that were informative with at least one locus. Detailed deletion mapping defined a 1.2 Mb commonly deleted region at 8p22-p21.3 flanked by markers cMSR-32 and C18-1051. A second region of common deletion was identified between C18-1312 and C18-494 at 8p21-8p11.22, suggesting that at least two tumor suppressor genes associated with prostate cancer are present on chromosome arm 8p. Allelic losses on 8p were observed more frequently in the cancer death cases (14/17, 82%) than in early-stage tumors (11/27, 40%; P < 0.01, Fisher's exact test). In two out of 7 patients for whom DNA was available from metastatic cancers as well as from normal tissues and primary tumors, the primary cancer foci had no detectable abnormality of 8p, but the metastatic tumors showed loss of heterozygosity. These results suggest that inactivation of tumor suppressor genes on 8p plays an important role in the progression of prostate cancer.

Androgen receptor gene mutations in human prostate cancer

To investigate the structural abnormality of the androgen receptor (AR) in human prostate cancers, exons B-H encoding DNA- and hormone-binding domains were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products using originally designed oligoprimers. Tissues from 7 cases of untreated stage B prostate cancer surgically removed and from 8 cases of endocrine therapy-resistant cancers obtained at autopsy were used in the study. Two different mutations were identified in exons D and H in the different cancer foci of the same cancer death patient. One mutation in exon D (at codon 701, Leu to His) was detected in the prostate, and the other in exon H (at codon 877, Thr to Ala) was found in metastatic tissues. In untreated cancer tissues and the other autopsy samples, no mutations were detected. The mutation in exon H was identical to that reported in LNCaP cells. These results indicate that AR gene mutations occur in relation to endocrine therapy-resistance, although the mutation was found in 1 out of 8 resistant cases (12.5%) at autopsy.