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Chemoresistance is the main obstacle to cancer cure. Contrasting studies focusing on single gene mutations, we hypothesize chemoresistance to be due to inactivation of key pathways affecting cellular mechanisms such as apoptosis, senescence, or DNA repair. In support of this hypothesis, we have previously shown inactivation of either TP53 or its key activators CHK2 and ATM to predict resistance to DNA damaging drugs in breast cancer better than TP53 mutations alone. Further, we hypothesized that redundant pathway(s) may compensate for loss of p53‐pathway signaling and that these are inactivated as well in resistant tumour cells. Here, we assessed genetic alterations of the retinoblastoma gene (RB1) and its key regulators: Cyclin D and E as well as their inhibitors p16 and p27. In an exploratory cohort of 69 patients selected from two prospective studies treated with either doxorubicin monotherapy or 5‐FU and mitomycin for locally advanced breast cancers, we found defects in the pRB‐pathway to be associated with therapy resistance (p‐values ranging from 0.001 to 0.094, depending on the cut‐off value applied to p27 expression levels). Although statistically weaker, we observed confirmatory associations in a validation cohort from another prospective study (n = 107 patients treated with neoadjuvant epirubicin monotherapy; p‐values ranging from 7.0 × 10−4 to 0.001 in the combined data sets). Importantly, inactivation of the p53‐and the pRB‐pathways in concert predicted resistance to therapy more strongly than each of the two pathways assessed individually (exploratory cohort: p‐values ranging from 3.9 × 10−6 to 7.5 × 10−3 depending on cut‐off values applied to ATM and p27 mRNA expression levels). Again, similar findings were confirmed in the validation cohort, with p‐values ranging from 6.0 × 10−7 to 6.5 × 10−5 in the combined data sets. Our findings strongly indicate that concomitant inactivation of the p53‐ and pRB‐ pathways predict resistance towards anthracyclines and mitomycin in breast cancer in vivo.

Transcripts derived from the function as decoys to adsorb miRNAs targeting the tumor suppressor for degradation, and upregulation is known to inhibit growth in preclinical cancer models. Here, 3'UTR transduction influences PTEN, AKT/mTOR signaling, and tumor progression in estrogen receptor (ER)-positive and -negative breast cancer cells. upregulation decreases gene expression in the ER-positive MCF7 and T47D human breast carcinoma cells and accelerates MCF7 tumor growth Of note, transduction significantly decreases ERα () mRNA and protein levels in MCF7 xenografts with a concomitant increase in hsa-miR-26a, a miRNA known to target In the ER-negative MDA-MB-231 and C3HBA breast cancer cells, upregulation of increases gene expression with no influence on hsa-miR-26a,, or ERα expression. While transduction did not influence the growth rate of human MDA-MB-231 xenografts, upregulation profoundly reduces its metastatic propensity. Furthermore, significantly inhibits the growth rate of ER-negative C3HBA murine breast cancer xenografts. transduction had no influence on doxorubicin cytotoxicity in ER-positive MCF7 cells but an increase in doxorubicin sensitivity was observed in the ER-negative MDA-MB-231 cells. In summary, while upregulation decreased transcript levels and stimulated the growth of ER-positive breast cancers, increased transcript levels and inhibited tumor progression was observed in the ER-negative cells. This report highlights the profound biological activity of in breast cancer, which is dictated by the hormone receptor status..

Increased lymphangiogenesis is a common feature of cancer development and progression, yet the influence of impaired lymphangiogenesis on tumor growth is elusive. C3HBA breast cancer and KHT-1 sarcoma cell lines were implanted orthotopically in Chy mice, harboring a heterozygous inactivating mutation of vascular endothelial growth factor receptor-3, resulting in impaired dermal lymphangiogenesis. Accelerated tumor growth was observed in both cancer models in Chy mice, coinciding with reduced peritumoral lymphangiogenesis. An impaired lymphatic washout was observed from the peritumoral area in Chy mice with C3HBA tumors, and the number of macrophages was significantly reduced. While fewer macrophages were detected, the fraction of CD163+ M2 macrophages remained constant, causing a shift towards a higher M2/M1 ratio in Chy mice. No difference in adaptive immune cells was observed between wt and Chy mice. Interestingly, levels of pro- and anti-inflammatory macrophage-associated cytokines were reduced in C3HBA tumors, pointing to an impaired innate immune response. However, IL-6 was profoundly elevated in the C3HBA tumor interstitial fluid, and treatment with the anti-IL-6 receptor antibody tocilizumab inhibited breast cancer growth. Collectively, our data indicate that impaired lymphangiogenesis weakens anti-tumor immunity and favors tumor growth at an early stage of cancer development.

PurposePTEN is an important tumor suppressor in breast cancer. Here, we examined the prognostic and predictive value of PTEN and PTEN pseudogene (PTENP1) gene expression in patients with locally advanced breast cancer given neoadjuvant chemotherapy.MethodsThe association between pretreatment PTEN and PTENP1 gene expression, response to neoadjuvant chemotherapy, and recurrence-free and disease-specific survival was assessed in 364 patients with locally advanced breast cancer given doxorubicin, 5-fluorouracil/mitomycin, or epirubicin versus paclitaxel in three phase II prospective studies. Further, protein expression of PTEN or phosphorylated Akt, S6 kinase, and 4EBP1 was assessed in a subgroup of 187 tumors.ResultsNeither PTEN nor PTENP1 gene expression level predicted response to any of the chemotherapy regimens tested (n = 317). Among patients without distant metastases (n = 282), a high pretreatment PTEN mRNA level was associated with inferior relapse-free (RFS; p = 0.001) and disease-specific survival (DSS; p = 0.003). Notably, this association was limited to patients harboring TP53 wild-type tumors (RFS; p = 0.003, DSS; p = 0.009). PTEN mRNA correlated significantly with PTENP1 mRNA levels (r s = 0.456, p < 0.0001) and PTEN protein staining (r s = 0.163, p = 0.036). However, no correlation between PTEN, phosphorylated Akt, S6 kinase or 4EBP1 protein staining, and survival was recorded. Similarly, no correlation between PTENP1 gene expression and survival outcome was observed.ConclusionHigh intratumoral PTEN gene expression was associated with poor prognosis in patients with locally advanced breast cancers harboring wild-type TP53.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-017-4160-5) contains supplementary material, which is available to authorized users.

Anthracyclines are key components of human breast cancer chemotherapy. Here, we explored the role of Akt signaling in anthracycline resistance.The antitumor activity of doxorubicin and Akt inhibitor A-443654 alone or combined was examined in estrogen receptor (ER) positive and negative human breast cancer cell lines. Further, we examined mRNA changes induced by anthracyclines in locally advanced breast cancers biopsied before and after treatment in two clinical trials.Doxorubicin increased Akt phosphorylation in ER positive MCF7 and T47D cell lines, with no effect in ER negative MDA-MB231 breast cancer cells. A-443654 was significantly more cytotoxic in doxorubicin-resistant compared to doxorubicin-naïve MCF7. This difference was not observed in MDA-MB231. Among 24 patients, AKT1 gene expression increased 24 hrs after the initial epirubicin exposure in ER positive tumors responding to therapy (n=6), as compared to ER positive non-responders (n=7) or ER negative tumors (n=11). In contrast, AKT1 mRNA changes after 16 weeks of doxorubicin were unrelated to clinical response and ER status (n=30).In conclusion, rapid Akt activation was observed in ER positive breast cancers which responded to anthracyclines. Increased cytotoxicity of A-443654 in doxorubicin-resistant MCF7 cells indicates a possible role for Akt inhibitors in ER positive breast cancers where chemoresistance evolves.

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Background: While PTEN is frequently downregulated in breast cancer, the mechanism remains obscure. PTEN pseudogene (PTENP1) is reported to function as a decoy adsorbing micro RNAs (miRNAs) targeting PTEN for degradation in prostate cancer cells. A potential role of PTENP1 in PTEN regulation in breast cancer has not been assessed systematically. Method: We transfected murine C3HBA breast cancer cells lacking endogenous PTENP1 with a lentiviral PTENP1 construct to investigate the influence of the pseudogene on PTEN and Akt-mTOR downstream signaling, global gene expression, as well as in vitro and in vivo tumor growth characteristics. Results: Upregulated PTENP1 3´UTR by lentiviral transfection increased PTEN protein levels and reduced cell cycle progression in vitro, but despite this increased Akt phosphorylation was observed. Similarily, PTENP1 transfection decreased tumor growth in C3HBA murine breast cancer in syngeneic, immunocompetent C3H mice, corresponding with upregulated PTEN, but without inhibition of Akt-mTOR signaling. In stead, increased protein levels of p53 and its downstream target activating protein-2 gamma (AP-2γ) was observed as possible tumor suppressors in this setting. Conclusion: In the murine C3HBA breast cancer cell line, PTENP1 transfection was associated with upregulated endogenous PTEN, p53 and AP-2γ protein levels and tumor growth inhibition. Citation Format: Synnøve Yndestad, Eilin Austreid, Kai Ove Skaftnesmo, Per Eystein Lønning, Hans Petter Eikesdal. Introduction of PTEN pseudogene in murine breast cancer upregulates PTEN, p53 and activating protein 2 gamma and delays tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3986. doi:10.1158/1538-7445.AM2015-3986