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1 Hypoxia

Evidence supporting the existence of hypoxic tissue areas in solid tumors is derived from data originating from a variety of methods [1]. These include invasive measurements of intratumoral oxygen partial pressures using polarographic needle electrodes (“Eppendorf” microsensor) and histological assays based on the immunodetection of so-called endogenous or exogenous hypoxia markers. In addition, different imaging methods have been developed, which, however, at the present time have not been adopted widely in the clinic. The major cause of tumor hypoxia is an enlargement of the intratumoral diffusion distances of oxygen beyond a critical threshold, which is estimated to be equal to approximately 80 μm at the arterial end of the microvessel. This main origin of continuous or “chronic” hypoxia is modified by other factors, including a reduced oxygen-transport capacity of the blood (anemia) and an increased interstitial fluid pressure, which may lead to a flow stop in microvessels. Besides the phenomenon of continuous tumor hypoxia, one also observes intermittent or “acute” hypoxia, which may be caused by fluctuations in the flux of erythrocytes or by temporary obstructions of tumor capillaries, e.g., by cell aggregates.

Figure 28.1 shows a synopsis of the various mechanisms by which tumor hypoxia may contribute to a more aggressive phenotype and to an increased resistance to therapy. The discovery that hypoxia is one of the most important factors mediating radioresistance can be traced back to the beginning of the twentieth century. We know today that the mechanism behind this observation is a modification of the free radical chemistry under hypoxic conditions, which has also been shown to be important for some forms of chemotherapy and photodynamic therapy [2]. Since the early 1990s, clinical studies indicated that the pathophysiological significance of hypoxia is clearly not limited to this modification of the radiosensitivity of tumor cells [3]. Hypoxia can lead to an increase of the genetic instability of cancer cells both by inducing mutations and by inhibiting DNA repair [4]. Hypoxia may also act as a selective force favoring the emergence of genetically hypoxia-resistant phenotypes. For example, p53-negative, apoptosis-resistant cell populations may emerge after repeated exposures of cells to hypoxia and reoxygenation [5]. Hypoxia has been shown to be important for the maintenance of the stem cell phenotype, and some types of stem cells have been observed to reside in a “hypoxic niche” in vivo [6]. Furthermore, hypoxia can play an important role in the attenuation of an antitumor immune response. For example, macrophages of the pro-tumorigenic M2 phenotype have been found preferentially in hypoxic tumor areas [7]. Consistent with this finding, other reports have demonstrated that hypoxic tumors contain a higher number of macrophages compared to non-hypoxic tumors. Additionally, higher quantities of intratumoral macrophages have been shown to correlate with a poorer patient prognosis [8]. Under hypoxic conditions, an increased expression of the cytokine CCL28 has been detected, which may lead to intratumoral accumulation of immunosuppressive regulatory T cells which express the cognate receptor CXCR10 [9]. Under hypoxic conditions, adenosine may accumulate in the extracellular space and stimulate adenosine receptors (of the A2A and A2B subtypes) on T cells, thereby leading to an inhibition of antitumor T cell responses [10]. Hypoxia has also been shown to be able to trigger the unfolded protein response and autophagy, which may promote tumor growth and resistance to anticancer therapy [11]. It should be mentioned, however, that both processes can also be antitumorigenic, depending on the specific experimental conditions. Although mTOR inhibition is currently being evaluated as a therapeutic strategy, e.g., in malignant gliomas, hypoxia-mediated suppression of mTOR has recently been shown to prevent irreversible cellular senescence, which may attenuate the efficacy of DNA-damaging agents [12]. Arguably, the overall most significant consequence of hypoxia is a large-scale change of the proteome, which is mediated by the activity of several transcription factors, among which the hypoxia-inducible factor 1 (HIF-1) plays the most important role [13].

Fig. 28.1
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Tumor hypoxia is a central driver of malignant progression and resistance to therapy (selection of mechanisms)

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More than 800 direct target genes of HIF-1 are known, and a large number of these have been shown to have a direct pathogenic role within the malignant phenotype (see Fig. 28.2, [13]). HIF-1 is a major trigger of proangiogenic cytokines (e.g., VEGF) in tumor cells. Furthermore, HIF-1 can promote vasculogenesis by the recruitment of CXCR4-positive stem cells from the bone marrow via SDF-1. HIF-1 increases the oxygen-transport capacity of the blood by upregulating EPO. Activation of HIF-1 leads to increased cell motility and invasiveness, mediates the ability to remodel the extracellular matrix, and can confer an augmented metastatic potency. These pivotally important processes may be initiated directly by HIF-1, e.g., via the urokinase-type plasminogen activator and matrix metalloproteinases. Additionally, HIF-1 can transactivate transcription factors (e.g., TWIST) which induce the metastasis-promoting cellular program of epithelial-to-mesenchymal transition [14]. HIF-1 may promote radioresistance by allowing cells to survive in hypoxic areas. Moreover, basal HIF-1 expression, but – interestingly – not hypoxia-induced expression of HIF-1, has been demonstrated to play a role for the expression of genes involved in DNA repair [15]. Target genes of HIF-1 can also mediate chemoresistance, e.g., by induction of the MDR-1 gene. There have also been reports describing a role of HIF-1 in mediating increased genetic instability by decreased homologous recombination repair and reduced mismatch repair [16]. HIF-1 can promote the differentiation of TH17 cells [17], which, depending on the experimental paradigm, have been described to both promote and inhibit the growth of tumors. HIF-1-induced SDF-1 may also contribute to the aforementioned accumulation of macrophages in hypoxic tumors [18]. HIF-1 may stimulate proliferation through the induction of autocrine growth factor loops. A number of publications have described an HIF-1-induced upregulation of telomerase and HIF-1 activated genes which are considered to play a role in the stem cell phenotype. Finally, a central mechanism of HIF-1-mediated maladaptation consists of an extensive metabolic reprogramming which leads to a downregulation of mitochondrial oxidative phosphorylation, e.g., via inhibition of the pyruvate dehydrogenase reaction by PDK-1 and promotion of selective autophagy of mitochondria. Simultaneously, HIF-1 mediates the induction of a glycolytic phenotype by increasing glucose influx (e.g., via GLUT-1), upregulation of key enzymes of glycolysis, and by an increase in the efflux of lactate via the monocarboxylate transporter subtype MCT-4 [13, 19].

Fig. 28.2
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HIF-1 as the central driver of hypoxia-induced transcriptional “maladaptation” in cancer (selection of mechanisms)

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2 Lactate

A substantial part of intratumoral lactate accumulation is the result of HIF-1-mediated metabolic reprogramming. However, comparative analyses of the distribution patterns of hypoxia (as assessed by pimonidazole staining) and locoregional lactate concentrations (analyzed using imaging bioluminescence) have revealed that both parameters are not necessarily co-localized in all cases [20]. Indeed, several HIF-1-independent mechanisms of intratumoral lactate accumulation have been described, e.g., the activation of MYC [21]. Additionally, high lactate levels may also be the consequence of an insufficient waste drainage in poorly vascularized tumor areas. The matter is further complicated by the existence of an intratumoral lactate shuttle between hypoxic (lactate-producing) and normoxic (lactate-consuming) cells [22]. Lactate has been hypothesized to mediate radioresistance by virtue of its antioxidant properties. Lactate also exhibits immunosuppressive properties and promotes cell motility, invasion, and metastasis. Furthermore, lactate may induce angiogenesis, mediate resistance to apoptosis, and may promote a stem cell phenotype. Importantly, lactate can indirectly stabilize HIF-1α and may thus perpetuate the activation of HIF-1 independent of hypoxia [23].

3 Acidosis

HIF-1-induced metabolic reprogramming also contributes to the marked extracellular acidosis often found in malignant tumors by upregulating glycolysis. Nevertheless, direct measurements of intratumoral oxygen and pH levels have revealed unequal distributions of both parameters at the microregional level [24, 25], and glycolysis-deficient cells have been shown to retain the ability to acidify the extracellular environment in vivo [26]. Additional pathogenetic mechanisms yielding an intensified tissue acidosis are based on substantial hydrolysis of ATP (derived from breakdown of substrates other than glucose), glutaminolysis, ketogenesis, and CO2/carbonic acid production [27]. The spectrum of the pathophysiological consequences of intratumoral acidosis includes many processes mentioned for HIF-1 and lactate: acidosis plays a role in mediating radioresistance (e.g., [28]), immune evasion [29], increased cell motility, invasion, metastasis [30, 31], promotion of angiogenesis through VEGF [32], and the stem cell phenotype [33]. Moreover, an acidic extracellular milieu diminishes the effectiveness of basic chemotherapeutic drugs (e.g., doxorubicin, daunorubicin, [34]). Similar to hypoxia and HIF-1, acidosis may contribute to the genetic instability of tumor cells [35] and – similar to hypoxia – is a possible trigger for autophagy [36]. Finally, acidosis has been shown to stabilize HIF-1α independent of hypoxia by nucleolar sequestration of VHL [37].

4 Conclusions

Factors of the microenvironment presented in this report trigger an overlapping range of processes which promote tumor growth and mediate resistance to therapy. The broadest spectrum of these processes is initiated by hypoxia and HIF-1, which are also often at the root of lactate accumulation and intratumoral acidosis. With this in mind, the three factors may be regarded as “siblings.” However, both of the latter factors may also be triggered independently of hypoxia and, importantly, similar pathogenic processes (e.g., radioresistance) may be initiated by all three factors via entirely independent mechanisms (e.g., modification of the spectrum of free radicals generated by radiation vs. scavenging of free radicals). Therefore, the factors discussed here may also act as “accomplices,” depending on the specific triggers for each of them in individual tumors.