Pooled ctDNA analysis of MONALEESA phase III advanced breast cancer trials.

BACKGROUND: The phase III MONALEESA trials tested the efficacy and safety of the cyclin-dependent kinase (CDK)4/6 inhibitor ribociclib with different endocrine therapy partners as first- or second-line treatment of hormone receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer (ABC). Using the largest pooled biomarker dataset of the CDK4/6 inhibitor ribociclib in ABC to date, we identified potential biomarkers of response to ribociclib. PATIENTS AND METHODS: Baseline circulating tumour DNA from patients in the MONALEESA trials was assessed using next-generation sequencing. An analysis of correlation between gene alteration status and progression-free survival (PFS) was carried out to identify potential biomarkers of response to ribociclib. RESULTS: Multiple frequently altered genes were identified. Alterations in ERBB2, FAT3, FRS2, MDM2, SFRP1, and ZNF217 were associated with a greater PFS benefit with ribociclib versus placebo. Patients with high tumour mutational burden (TMB) and with ANO1, CDKN2A/2B/2C, and RB1 alterations exhibited decreased sensitivity to ribociclib versus placebo. CONCLUSIONS: Although exploratory, these results provide insight into alterations associated with the improved response to ribociclib treatment and may inform treatment sequencing in patients with actionable alterations following progression on CDK4/6 inhibitors. Validation of potential biomarkers identified here and development of prospective trials testing their clinical utility are warranted. GOV IDENTIFIERS: NCT01958021, NCT02422615, NCT02278120.

Neratinib + fulvestrant + trastuzumab for HR-positive, HER2-negative, HER2-mutant metastatic breast cancer: outcomes and biomarker analysis from the SUMMIT trial.

BACKGROUND: HER2 mutations are targetable alterations in patients with hormone receptor-positive (HR+) metastatic breast cancer (MBC). In the SUMMIT basket study, patients with HER2-mutant MBC received neratinib monotherapy, neratinib + fulvestrant, or neratinib + fulvestrant + trastuzumab (N + F + T). We report results from 71 patients with HR+, HER2-mutant MBC, including 21 (seven in each arm) from a randomized substudy of fulvestrant versus fulvestrant + trastuzumab (F + T) versus N + F + T. PATIENTS AND METHODS: Patients with HR+ HER2-negative MBC with activating HER2 mutation(s) and prior cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) therapy received N + F + T (oral neratinib 240 mg/day with loperamide prophylaxis, intramuscular fulvestrant 500 mg on days 1, 15, and 29 of cycle 1 then q4w, intravenous trastuzumab 8 mg/kg then 6 mg/kg q3w) or F + T or fulvestrant alone. Those whose disease progressed on F + T or fulvestrant could cross-over to N + F + T. Efficacy endpoints included investigator-assessed objective response rate (ORR), clinical benefit rate (RECIST v1.1), duration of response, and progression-free survival (PFS). Plasma and/or formalin-fixed paraffin-embedded tissue samples were collected at baseline; plasma was collected during and at end of treatment. Extracted DNA was analyzed by next-generation sequencing. RESULTS: ORR for 57 N + F + T-treated patients was 39% [95% confidence interval (CI) 26% to 52%); median PFS was 8.3 months (95% CI 6.0-15.1 months). No responses occurred in fulvestrant- or F + T-treated patients; responses in patients crossing over to N + F + T supported the requirement for neratinib in the triplet. Responses were observed in patients with ductal and lobular histology, 1 or ≥1 HER2 mutations, and co-occurring HER3 mutations. Longitudinal circulating tumor DNA sequencing revealed acquisition of additional HER2 alterations, and mutations in genes including PIK3CA, enabling further precision targeting and possible re-response. CONCLUSIONS: The benefit of N + F + T for HR+ HER2-mutant MBC after progression on CDK4/6is is clinically meaningful and, based on this study, N + F + T has been included in the National Comprehensive Cancer Network treatment guidelines. SUMMIT has improved our understanding of the translational implications of targeting HER2 mutations with neratinib-based therapy.

Ado-trastuzumab emtansine (T-DM1) in patients with HER2-amplified tumors excluding breast and gastric/gastroesophageal junction (GEJ) adenocarcinomas: results from the NCI-MATCH trial (EAY131) subprotocol Q.

BACKGROUND: The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) is a national precision medicine study incorporating centralized genomic testing to direct refractory cancer patients to molecularly targeted treatment subprotocols. This treatment subprotocol was designed to screen for potential signals of efficacy of ado-trastuzumab emtansine (T-DM1) in HER2-amplified histologies other than breast and gastroesophageal tumors. METHODS: Eligible patients had HER2 amplification at a copy number (CN) >7 based on targeted next-generation sequencing (NGS) with a custom Oncomine AmpliSeq™ (ThermoFisher Scientific) panel. Patients with prior trastuzumab, pertuzumab or T-DM1 treatment were excluded. Patients received T-DM1 at 3.6 mg/kg i.v. every 3 weeks until toxicity or disease progression. Tumor assessments occurred every three cycles. The primary end point was centrally assessed objective response rate (ORR). Exploratory end points included correlating response with HER2 CN by NGS. The impact of co-occurring genomic alterations and PTEN loss by immunohistochemistry were also assessed. RESULTS: Thirty-eight patients were enrolled and 36 included in efficacy analysis. Median prior therapies in the metastatic setting was 3 (range 0-9; unknown in one patient). Median HER2 CN was 17 (range 7-139). Partial responses were observed in two (5.6%) patients: one mucoepidermoid carcinoma of parotid gland and one parotid gland squamous cell cancer. Seventeen patients (47%) had stable disease including 8/10 (80%) with ovarian and uterine carcinomas, with median duration of 4.6 months. The 6-month progression-free survival rate was 23.6% [90% confidence interval 14.2% to 39.2%]. Common toxicities included fatigue, anemia, fever and thrombocytopenia with no new safety signals. There was a trend for tumor shrinkage with higher levels of gene CN as determined by the NGS assay. CONCLUSION: T-DM1 was well tolerated. While this subprotocol did not meet the primary end point for ORR in this heavily pre-treated diverse patient population, clinical activity was seen in salivary gland tumors warranting further study in this tumor type in dedicated trials.

Updated results from MONALEESA-2, a phase III trial of first-line ribociclib plus letrozole versus placebo plus letrozole in hormone receptor-positive, HER2-negative advanced breast cancer.

Background: The phase III MONALEESA-2 study demonstrated significantly prolonged progression-free survival (PFS) and a manageable toxicity profile for first-line ribociclib plus letrozole versus placebo plus letrozole in patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer. Here, we report updated efficacy and safety data, together with exploratory biomarker analyses, from the MONALEESA-2 study. Patients and methods: A total of 668 postmenopausal women with HR+, HER2- recurrent/metastatic breast cancer were randomized (1 : 1; stratified by presence/absence of liver and/or lung metastases) to ribociclib (600 mg/day; 3-weeks-on/1-week-off; 28-day treatment cycles) plus letrozole (2.5 mg/day; continuous) or placebo plus letrozole. The primary end point was locally assessed PFS. The key secondary end point was overall survival (OS). Other secondary end points included overall response rate (ORR) and safety. Biomarker analysis was an exploratory end point. Results: At the time of the second interim analysis, the median duration of follow-up was 26.4 months. Median PFS was 25.3 months [95% confidence interval (CI) 23.0-30.3] for ribociclib plus letrozole and 16.0 months (95% CI 13.4-18.2) for placebo plus letrozole (hazard ratio 0.568; 95% CI 0.457-0.704; log-rank P = 9.63 × 10-8). Ribociclib treatment benefit was maintained irrespective of PIK3CA or TP53 mutation status, total Rb, Ki67, or p16 protein expression, and CDKN2A, CCND1, or ESR1 mRNA levels. Ribociclib benefit was more pronounced in patients with wild-type versus altered receptor tyrosine kinase genes. OS data remain immature, with 116 deaths observed; 50 in the ribociclib arm and 66 in the placebo arm (hazard ratio 0.746; 95% CI 0.517-1.078). The ORR was 42.5% versus 28.7% for all patients treated with ribociclib plus letrozole versus placebo plus letrozole, respectively, and 54.5% versus 38.8%, respectively, for patients with measurable disease. Safety results, after a further 11.1 months of follow-up, were comparable with those reported at the first analysis, with no new or unexpected toxicities observed, and no evidence of cumulative toxicity. Conclusions: The improved efficacy outcomes and manageable tolerability observed with first-line ribociclib plus letrozole are maintained with longer follow-up, relative to letrozole monotherapy. Clinical trials number: NCT01958021.

Growth retardation and tumour inhibition by BRCA1.

Inherited mutations in BRCA1 predispose to breast and ovarian cancer, but the role of BRCA1 in sporadic breast and ovarian cancer has previously been elusive. Here, we show that retroviral transfer of the wild-type BRCA1 gene inhibits growth in vitro of all breast and ovarian cancer cell lines tested, but not colon or lung cancer cells or fibroblasts. Mutant BRCA1 has no effect on growth of breast cancer cells; ovarian cancer cell growth is not affected by BRCA1 mutations in the 5' portion of the gene, but is inhibited by 3' BRCA1 mutations. Development of MCF-7 tumours in nude mice is inhibited when MCF-7 cells are transfected with wild-type, but not mutant, BRCA1. Most importantly, among mice with established MCF-7 tumours, peritoneal treatment with a retroviral vector expressing wild-type BRCA1 significantly inhibits tumour growth and increased survival.

Crizotinib in patients with tumors harboring ALK or ROS1 rearrangements in the NCI-MATCH trial.

The NCI-MATCH was designed to characterize the efficacy of targeted therapies in histology-agnostic driver mutation-positive malignancies. Sub-protocols F and G were developed to evaluate the role of crizotinib in rare tumors that harbored either ALK or ROS1 rearrangements. Patients with malignancies that progressed following at least one prior systemic therapy were accrued to the NCI-MATCH for molecular profiling, and those with actionable ALK or ROS1 rearrangements were offered participation in sub-protocols F or G, respectively. There were five patients who enrolled on Arm F (ALK) and four patients on Arm G (ROS1). Few grade 3 or 4 toxicities were noted, including liver test abnormalities, and acute kidney injury. For sub-protocol F (ALK), the response rate was 50% (90% CI 9.8-90.2%) with one complete response among the 4 eligible patients. The median PFS was 3.8 months, and median OS was 4.3 months. For sub-protocol G (ROS1) the response rate was 25% (90% CI 1.3-75.1%). The median PFS was 4.3 months, and median OS 6.2 months. Data from 3 commercial vendors showed that the prevalence of ALK and ROS1 rearrangements in histologies other than non-small cell lung cancer and lymphoma was rare (0.1% and 0.4% respectively). We observed responses to crizotinib which met the primary endpoint for ALK fusions, albeit in a small number of patients. Despite the limited accrual, some of the patients with these oncogenic fusions can respond to crizotinib which may have a therapeutic role in this setting.

Cyclin-dependent kinase inhibitor p27(Kip1) is required for mouse mammary gland morphogenesis and function.

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1-Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1-deficient animals. These hypotheses were investigated in postnatal p27-deficient (p27(-/-)), hemizygous (p27(+/)-), or wild-type (p27(+/+)) mammary glands. Mammary glands from p27(+/)- mice displayed increased ductal branching and proliferation with delayed postlactational involution. In contrast, p27(-/-) mammary glands or wild-type mammary fat pads reconstituted with p27(-/-) epithelium produced the opposite phenotype: hypoplasia, low proliferation, decreased ductal branching, impaired lobuloalveolar differentiation, and inability to lactate. The association of cyclin D1 with Cdk4, the kinase activity of Cdk4 against pRb in vitro, the nuclear localization of cyclin D1, and the stability of cyclin D1 were all severely impaired in p27(-/-) mammary epithelial cells compared with p27(+/+) and p27(+/-) mammary epithelial cells. Therefore, p27 is required for mammary gland development in a dose-dependent fashion and positively regulates cyclin D-Cdk4 function in the mammary gland.

Activated type I TGFbeta receptor kinase enhances the survival of mammary epithelial cells and accelerates tumor progression.

We have examined the effects of transforming growth factor-beta (TGFbeta) signaling on mammary epithelial cell survival. Transgenic mice expressing an active mutant of Alk5 in the mammary gland (MMTV-Alk5(T204D)) exhibited reduced apoptosis in terminal endbuds and during postlactational involution. Transgene-expressing mammary cells contained lower Smad2/3 and higher c-myc levels than controls, high ligand-independent phosphatidylinositol-3 kinase (PI3K) and Akt activities, and were insensitive to TGFbeta-mediated growth arrest. Treatment with a proteasome inhibitor increased Smad2/3 levels and ligand-independent Smad transcriptional reporter activity, as well as reduced both c-myc protein and basal cell proliferation. Treatment with an Alk5 kinase small-molecule inhibitor upregulated Smad2/3 levels, reduced PI3K activity, P-Akt, and c-myc, and inhibited cell survival. Although Alk5(T204D)-expressing mice did not develop mammary tumors, bigenic MMTV-Alk(T204D) x Neu mice developed cancers that were more metastatic than those occurring in MMTV-Neu transgenics. These data suggest that (1) TGFbeta can signal to PI3K/Akt and enhance mammary epithelial cell survival in vivo before cytological or histological evidence of transformation, and (2) TGFbeta signaling can provide epithelial cells with a 'gain-of-function' effect that synergizes with oncogene-induced transformation.

Anti-transforming growth factor (TGF)-beta antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-beta interactions in human breast cancer progression.

TGF-beta effects on angiogenesis, stroma formation, and immune function suggest its possible involvement in tumor progression. This hypothesis was tested using the 2G7 IgG2b, which neutralizes TGF-beta 1, -beta 2, and -beta 3, and the MDA-231 human breast cancer cell line. Inoculation of these cells in athymic mice decreases mouse spleen natural killer (NK) cell activity. Intraperitoneal injections of 2G7 starting 1 d after intraperitoneal inoculation of tumor cells suppressed intraabdominal tumor and lung metastases, whereas the nonneutralizing anti-TGF-beta 12H5 IgG2a had no effect. 2G7 transiently inhibited growth of established MDA-231 subcutaneous tumors. Histologically, both 2G7-treated and control tumors were identical. Intraperitoneal administration of 2G7 resulted in a marked increase in mouse spleen NK cell activity. 2G7 did not inhibit MDA-231 primary tumor or metastases formation, nor did it stimulate NK cell-mediated cytotoxicity in beige NK-deficient nude mice. Finally, serum-free conditioned medium from MDA-231 cells inhibited the NK cell activity of human blood lymphocytes. This inhibition was blocked by the neutralizing anti-TGF-beta 2G7 antibody but not by a nonspecific IgG2. These data support a possible role for tumor cell TGF-beta in the progression of mammary carcinomas by suppressing host immune surveillance.

Blockade of the type I somatomedin receptor inhibits growth of human breast cancer cells in athymic mice.

Insulin and insulin-like growth factors (IGIs) stimulate the growth of human breast cancer cells in vitro. The type I somatomedin receptor (SR) expressed in these cells may mediate the growth effects of these peptides. We have examined the role of this receptor on human breast cancer growth with a monoclonal antibody (alpha-IR-3) that blocks the receptor binding domain and inhibits IGF-I-induced growth. alpha-IR-3 inhibited clonal growth in vitro and blocked the mitogenic effect of exogenous IGF-I in both MCF-7 and MDA-231 breast cancer cell lines. Antibody-induced blockade of the type I SR also inhibited the estrogen-independent MDA-231 cells growing in vivo in nude mice, but growth of the estrogen-dependent MCF-7 cells was unaffected. IGIs are important growth regulators of MDA-231 breast cancer cells. Blockade of this growth stimulatory pathway may provide a new treatment strategy.

Transforming growth factor alpha protection against drug-induced injury to the rat gastric mucosa in vivo.

This study was designed to determine whether transforming growth factor alpha (TGF alpha) protects rat gastric mucosa against ethanol- and aspirin-induced injury. Systemic administration of TGF alpha dose-dependently decreased 100% ethanol-induced gastric mucosal injury; a dose of 50 micrograms/kg delivered intraperitoneally 15 min before ethanol decreased macroscopic mucosal injury by > 90%. At the microscopic level, TGF alpha prevented deep gastric necrotic lesions and reduced disruption of surface epithelium. Pretreatment with orogastric TGF alpha (200 micrograms/kg) only partially (40%) decreased macroscopic ethanol damage. Intraperitoneal administration of TGF alpha at a dose of 10 micrograms/kg, which does not significantly inhibit gastric acid secretion, decreased aspirin-induced macroscopic damage by > 80%. TGF alpha protection does not seem to be mediated by prostaglandin, glutathione, or ornithine decarboxylase-related events, as evidenced by lack of influence of the inhibition of their production. Pretreatment with the sulfhydryl blocking agent N-ethylmaleimide partially abolished (40%) the protective effect of TGF alpha. In addition, systemic administration of TGF alpha resulted in a two-fold increase in tyrosine phosphorylation of phospholipase C-gamma 1 and in a time- and dose-dependent increase in levels of immunoreactive insoluble gastric mucin; these events occurred in a time frame consistent with their participation in the protective effect of TGF alpha.

Synergistic interaction of the Neu proto-oncogene product and transforming growth factor alpha in the mammary epithelium of transgenic mice.

Transgenic mice expressing either the neu proto-oncogene or transforming growth factor (TGF-alpha) in the mammary epithelium develop spontaneous focal mammary tumors that occur after a long latency. Since the epidermal growth factor receptor (EGFR) and Neu are capable of forming heterodimers that are responsive to EGFR ligands such as TGF-alpha, we examined whether coexpression of TGF-alpha and Neu in mammary epithelium could cooperate to accelerate the onset of mammary tumors. To test this hypothesis, we interbred separate transgenic strains harboring either a mouse mammary tumor virus/TGF-alpha or a mouse mammary tumor virus/neu transgene to generate bitransgenic mice that coexpress TGF-alpha and neu in the mammary epithelium. Female mice coexpressing TGF-alpha and neu developed multifocal mammary tumors which arose after a significantly shorter latency period than either parental strain alone. The development of these mammary tumors was correlated with the tyrosine phosphorylation of Neu and the recruitment of c-Src to the Neu complex. Immunoprecipitation and immunoblot analyses with EGFR- and Neu-specific antisera, however, failed to detect physical complexes of these two receptors. Taken together, these observations suggest that Neu and TGF-alpha cooperate in mammary tumorigenesis through a mechanism involving Neu and EGFR transactivation.

Transforming growth factor beta enhances epithelial cell survival via Akt-dependent regulation of FKHRL1.

The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.

Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism.

Transforming growth factor-beta1 (TGF-beta) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-beta are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-beta-induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-beta do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-beta rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160(ROCK), by the expression of dominant-negative mutants, inhibited TGF-beta-mediated EMT. The data suggest that TGF-beta rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.

Reversal of tamoxifen resistance of human breast carcinomas in vivo by neutralizing antibodies to transforming growth factor-beta.

BACKGROUND: Overexpression of transforming growth factor (TGF)-beta has been reported in human breast carcinomas resistant to antiestrogen tamoxifen, but the role of TGF-beta in this resistant phenotype is unclear. We investigated whether inhibition of TGF-beta2, which is overexpressed in LCC2 tamoxifen-resistant human breast cancer cells, could modify antiestrogen resistance. METHODS: TGF-beta2 expression was evaluated in LCC2 cells and tamoxifen-sensitive LCC1 cells by northern blot analysis. Secreted TGF-beta activity was quantified by use of an 125I-TGF-beta competitive radioreceptor assay. Sensitivity to tamoxifen was measured in a soft agarose colony-forming assay and in a xenograft model in nude and beige/nude mice. Natural killer (NK) cell cytotoxicity was measured by 51Cr release from LCC1 and LCC2 cell targets coincubated with human peripheral blood mononuclear cells. Decrease in TGF-beta2 expression in LCC2 cells was achieved by treatment with antisense oligodeoxynucleotides and confirmed by TGF-beta2 immunoblot analysis. RESULTS AND CONCLUSIONS: The proliferative response of LCC2 cells to tamoxifen in vitro was not altered by TGF-beta neutralizing antibodies. However, established LCC2 tumors in nude mice treated with tamoxifen plus TGF-beta antibodies failed to grow, whereas tumors treated with tamoxifen plus a control antibody continued to proliferate. This reversal of tamoxifen resistance by TGF-beta antibodies did not occur in beige/nude mice, which lack NK-cell function, suggesting that immune mechanisms may be involved in the antitumor effects of tamoxifen. Antisense TGF-beta2 oligodeoxynucleotides enhanced the NK sensitivity of LCC2 cells in the presence of tamoxifen. Finally, LCC1 tumors were markedly more sensitive to tamoxifen in NK-active than in NK-deficient mice. IMPLICATIONS: These data suggest that host NK function mediates, in part, the antitumor effect of tamoxifen and that TGF-beta2 may abrogate this mechanism, thus contributing to tamoxifen resistance.

Abrogation of cisplatin-induced programmed cell death in human breast cancer cells by epidermal growth factor antisense RNA.

BACKGROUND: Epidermal growth factor receptor (EGF-R) perturbation by receptor ligand(s), e.g., epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), or receptor-specific antibodies accentuates cisplatin-induced toxicity in tumor cells. This sensitization occurs only in tumor cells with high expression of EGF-R but not in those with low expression of EGF-R. PURPOSE: Therefore, we have studied the role of EGF-R expression on cisplatin-mediated cytotoxicity. METHODS: MDA-468 human breast cancer cells were stably transfected with a p-chloramphenicol acetyl transferase (pact[p]-CAT) vector containing a 4.1-kilobase full-length antisense EGF-R complementary DNA. EGF-R content was assessed by 125I-EGF binding and EGF-R immunoblot assays. Cisplatin sensitivity was evaluated by (a) colony-forming assay in vitro, (b) xenograft growth in nude mice, (c) cell cycle distribution of propidium iodide-labeled DNA, (d) DNA fragmentation in agarose gels, and (e) terminal deoxynucleotidyl transferase (Tdt) fluorescence in situ. Cisplatin uptake was measured by atomic absorption spectroscopy, and the levels of drug-DNA intrastrand adducts were determined by a dissociation-enhanced fluoroimmunoassay that utilizes an antibody against cisplatin-modified DNA. RESULTS: Selected clones (MDA-468/AS-EGFR) exhibited more than 90% loss of both 125I-EGF binding and receptor content determined by western blot analysis, whereas clones transfected with the vector alone (MDA-468/p-CAT) had EGF-R levels similar to those of the parent cells. By use of a colony-forming assay, the 1-hour IC50 (i.e., the concentration of drug required for 1 hour to achieve 50% cell kill) for cisplatin was 2 microM or less for parental and vector-transfected clones (n = 4), whereas it was 25 microM or more for all MDA-468/AS-EGFR clones (n = 3). MDA-468/p-CAT clones exhibited internucleosomal DNA fragmentation, enhanced Tdt-end labeling in situ, and G2 arrest 48 hours after a 1-hour incubation with 3-30 microM cisplatin. Under these conditions, apoptosis and G2 arrest were undetectable in all MDA-468/AS-EGFR clones. An MDA-468 subline selected after long-term treatment with a TGF-alpha-Pseudomonas exotoxin A fusion protein 40 lacked EGF binding and also exhibited cisplatin resistance (1-hour IC50: > 30 microM) compared with parental cells. This EGF-R-dependent difference in cisplatin response was confirmed in a nude mouse xenograft model by use of high- and low-EGF-R-expressing cell clones. Total intracellular drug accumulation after a 1-hour cisplatin exposure, as measured by atomic absorption spectroscopy, was identical in both groups of cells. Intrastrand drug-DNA adducts, however, were statistically higher in high EGF-R expressors than in low-EGF-R-expressing clones. CONCLUSIONS: These data indicate that a critical level of EGF-R signaling, which is amplified in some common human cancers, is necessary for cisplatin-mediated apoptosis in tumor cells and suggest an inhibitory effect of this pathway on the repair of cisplatin-damaged DNA.

Transforming growth factor-beta and breast cancer risk in women with mammary epithelial hyperplasia.

BACKGROUND: Transforming growth factors-beta (TGF-betas) regulate mammary epithelial cell division. Loss of expression of TGF-beta receptor II (TGF-beta-RII) is related to cell proliferation and tumor progression. Breast epithelial hyperplastic lesions lacking atypia (EHLA) are associated with a mild elevation in breast cancer risk. We investigated the expression of TGF-beta-RII in EHLA and the risk of subsequent invasive breast cancer. METHODS: We conducted a nested case-control study of women with biopsy-confirmed EHLA who did not have a history of breast cancer or atypical hyperplasia of the breast. Case patients (n = 54) who subsequently developed invasive breast cancer were matched with control patients (n = 115) who did not. Formalin-fixed, paraffin-embedded sections of breast biopsy specimens of all 169 patients with EHLA were studied by immunohistochemical analysis with antibodies against TGF-beta-RII. All P values are two-sided. RESULTS: Women with breast EHLA and 25%-75% TGF-beta-RII-positive cells or less than 25% TGF-beta-RII-positive cells had odds ratios of invasive breast cancer of 1.98 (95% confidence interval [CI] = 0.95-4.1) or 3.41 (95% CI = 1.2-10.0), respectively (P for trend =.008). These risks are calculated with respect to women with EHLA that had greater than 75% TGF-beta-RII expression. Women with a heterogeneous pattern of TGF-beta-RII expression in their normal breast lobular units and either greater than 75%, 25%-75%, or less than 25% positive cells in their EHLA had odds ratios for breast cancer risk of 0.742 (95% CI = 0.3-1.8), 2.85 (95% CI = 1.1-7.1), or 3.55 (95% CI = 1.0-10.0), respectively (P for trend =.003). These risks are relative to women with a homogeneous pattern of expression in their normal lobular units and greater than 75% positive cells in their EHLA. CONCLUSION: This study indicates that loss of TGF-beta-RII expression in epithelial cells of EHLA is associated with increased risk of invasive breast cancer.

Growth inhibition of human breast cancer cells in vitro with an antibody against the type I somatomedin receptor.

Insulin and insulin-like growth factors (IGFs) stimulate the growth of human breast cancer cells in vitro. The type I somatomedin receptor (SR), expressed in these cells, may mediate the mitogenic effects of these peptides. We have examined the effect of type I SR blockade on human breast cancer growth with a monoclonal antibody (alpha-IR3) that blocks the receptor binding domain. alpha-IR3 inhibited binding of 125I-IGF-I in all breast cancer cell lines tested. Binding affinity of alpha-IR3 was 2 to 5 times higher than that of IGF-I in MDA-231 (Kd 2.1 nM) and MCF-7 cells (Kd 0.6 nM), respectively. In the presence of 10% calf serum, the antibody inhibited anchorage-independent growth of six of seven breast cancer cell lines. This inhibition was reversible with excess IGF-I. In serum-free medium, alpha-IR3 blocked IGF-I-stimulated DNA synthesis in four of four breast cancer cell lines (MCF-7, ZR75-1, MDA-231, and HS578T). However, the antibody did not inhibit basal growth of any of the breast cancer cell lines in serum-free conditions. In three estrogen receptor-positive, estrogen-responsive breast cancer cell lines (MCF-7, ZR75-1, and T47D), type I SR blockade with alpha-IR3 failed to block estrogen-stimulated DNA synthesis or cell proliferation, indicating that secreted IGF activity is not the sole mediator of the growth effects of estrogen. In conclusion, antibody-mediated type I SR blockade does not inhibit basal growth of breast cancer cells under serum-free conditions, arguing against a critical autocrine role of endogenously secreted IGF activity in vitro. However, type I SR blockade inhibits breast cancer cell growth in the presence of serum, suggesting that serum IGFs might be critical endocrine or paracrine regulators of human breast cancer.